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Wolff, P; Villette, C; Zumsteg, J; Heintz, D; Antoine, L; Chane-Woon-Ming, B; Droogmans, L; Grosjean, H; Westhof, E Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea Article de journal RNA, p. in press, 2020, ISBN: 32994183. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN, WESTHOF, WESTHOF ENNIFAR (hyper)thermophiles Archaea mass spectrometry modifications tRNA @article{, title = {Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea}, author = {P Wolff and C Villette and J Zumsteg and D Heintz and L Antoine and B Chane-Woon-Ming and L Droogmans and H Grosjean and E Westhof}, url = {https://pubmed.ncbi.nlm.nih.gov/32994183/}, doi = {10.1261/rna.077537.120}, isbn = {32994183}, year = {2020}, date = {2020-09-29}, journal = {RNA}, pages = {in press}, abstract = {To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale) and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.}, keywords = {ENNIFAR, Unité ARN, WESTHOF, WESTHOF ENNIFAR (hyper)thermophiles Archaea mass spectrometry modifications tRNA}, pubstate = {published}, tppubtype = {article} } To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale) and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii. |
Westhof, E; Liang, S; Tong, X; Ding, X; Zheng, L; Dai, F Unusual tertiary pairs in eukaryotic tRNA-Ala Article de journal RNA, p. in press, 2020, ISBN: 32737189. Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF, WESTHOF Ala Gly insects mammals tRNA. @article{, title = {Unusual tertiary pairs in eukaryotic tRNA-Ala}, author = {E Westhof and S Liang and X Tong and X Ding and L Zheng and F Dai}, url = {https://pubmed.ncbi.nlm.nih.gov/32737189/}, doi = {10.1261/rna.076299.120}, isbn = {32737189}, year = {2020}, date = {2020-07-31}, journal = {RNA}, pages = {in press}, abstract = {tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb data base (The Lowe Lab, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for Alanine compared to other tRNA species, including tRNAs specific for Glycine. First, besides the well-known G3oU70 base pair in the amino acid stem, there is the frequent occurrence of a second wobble pair at G30oU40, a pair overwhelmingly observed as a Watson-Crick pair throughout phylogeny. Secondly, the tertiary pair R15/Y48 occurs as a purine-purine R15/A48 pair. Finally, the conserved T54/A58 pair maintaining the fold of the T-loop is observed as a purine-purine A54/A58 pair. The R15/A48 and A54/A58 pairs always occur together. The G30oU40 pair occurs alone or together with these other two pairs. The pairing variations are observed to variable extent depending on phylogeny. Among eukaryotes, insects display simultaneously all variations, while mammals present either the G30oU40 pair or both R15/A48 and A54/A58. tRNAs with the anticodon 34A(I)GC36 are the most prone to display all those pair variations in mammals and insects. tRNAs with anticodon Y34GC36 have preferentially G30oU40 only. These unusual pairs are not observed in bacterial, or archaeal tRNAs, probably because of the avoidance of A34-containing anticodons in 4-codon boxes. Among eukaryotes, these unusual pairing features were not observed in fungi and nematodes. These unusual structural features may affect transcription rates (e.g. 54/58) or ribosomal translocation (30/40).}, keywords = {Unité ARN, WESTHOF, WESTHOF Ala Gly insects mammals tRNA.}, pubstate = {published}, tppubtype = {article} } tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb data base (The Lowe Lab, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for Alanine compared to other tRNA species, including tRNAs specific for Glycine. First, besides the well-known G3oU70 base pair in the amino acid stem, there is the frequent occurrence of a second wobble pair at G30oU40, a pair overwhelmingly observed as a Watson-Crick pair throughout phylogeny. Secondly, the tertiary pair R15/Y48 occurs as a purine-purine R15/A48 pair. Finally, the conserved T54/A58 pair maintaining the fold of the T-loop is observed as a purine-purine A54/A58 pair. The R15/A48 and A54/A58 pairs always occur together. The G30oU40 pair occurs alone or together with these other two pairs. The pairing variations are observed to variable extent depending on phylogeny. Among eukaryotes, insects display simultaneously all variations, while mammals present either the G30oU40 pair or both R15/A48 and A54/A58. tRNAs with the anticodon 34A(I)GC36 are the most prone to display all those pair variations in mammals and insects. tRNAs with anticodon Y34GC36 have preferentially G30oU40 only. These unusual pairs are not observed in bacterial, or archaeal tRNAs, probably because of the avoidance of A34-containing anticodons in 4-codon boxes. Among eukaryotes, these unusual pairing features were not observed in fungi and nematodes. These unusual structural features may affect transcription rates (e.g. 54/58) or ribosomal translocation (30/40). |
Wolff, P; Ennifar, E Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes Book Chapter Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 111-118, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006311. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR Native mass spectrometry RNA, Unité ARN @inbook{, title = {Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes}, author = {P Wolff and E Ennifar}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006311}, doi = {10.1007/978-1-0716-0278-2_9}, isbn = {32006311}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {111-118}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.}, keywords = {ENNIFAR, ENNIFAR Native mass spectrometry RNA, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin. |
Werner, S; Schmidt, L; Marchand, V; Kemmer, T; Falschlunger, C; Sednev, M V; Bec, G; Ennifar, E; Höbartner, C; Micura, R; Motorin, Y; Hildebrandt, A; Helm, M Machine Learning of Reverse Transcription Signatures of Variegated Polymerases Allows Mapping and Discrimination of Methylated Purines in Limited Transcriptomes Article de journal Nucleic Acids Res, 48 (7), p. 3734-3746, 2020, ISBN: 32095818. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN @article{, title = {Machine Learning of Reverse Transcription Signatures of Variegated Polymerases Allows Mapping and Discrimination of Methylated Purines in Limited Transcriptomes}, author = {S Werner and L Schmidt and V Marchand and T Kemmer and C Falschlunger and M V Sednev and G Bec and E Ennifar and C Höbartner and R Micura and Y Motorin and A Hildebrandt and M Helm}, url = {https://pubmed.ncbi.nlm.nih.gov/32095818}, doi = {10.1093/nar/gkaa113}, isbn = {32095818}, year = {2020}, date = {2020-01-01}, journal = {Nucleic Acids Res}, volume = {48}, number = {7}, pages = {3734-3746}, abstract = {Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.}, keywords = {ENNIFAR, Unité ARN}, pubstate = {published}, tppubtype = {article} } Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature. |
Vileno, B; Lebars, I Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies Book Chapter Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 217-235, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006317. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR RNA NMR EPR Paramagnetic Spin-label, Unité ARN @inbook{, title = {Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies}, author = {B Vileno and I Lebars}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006317}, doi = {10.1007/978-1-0716-0278-2_15}, isbn = {32006317}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {217-235}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems. Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.}, keywords = {ENNIFAR RNA NMR EPR Paramagnetic Spin-label, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems. Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies. |
Vigouroux, A; Aumont-Nicaise, M; Boussac, A; Marty, L; Bello, Lo L; Legrand, P; Brillet, K; Schalk, I J; Moréra, S A unique ferrous iron binding mode is associated to large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa Article de journal FEBS J, 287 (2), p. 295-309, 2020, ISBN: 31318478. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR Pseudomonas aeruginosa iron pyoverdine siderophore solute-binding protein, Unité ARN @article{, title = {A unique ferrous iron binding mode is associated to large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa}, author = {A Vigouroux and M Aumont-Nicaise and A Boussac and L Marty and L Lo Bello and P Legrand and K Brillet and I J Schalk and S Moréra}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31318478}, doi = {10.1111/febs.15004}, isbn = {31318478}, year = {2020}, date = {2020-01-01}, journal = {FEBS J}, volume = {287}, number = {2}, pages = {295-309}, abstract = {Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Iron release from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore binding protein, respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa. This article is protected by copyright. All rights reserved.}, keywords = {ENNIFAR, ENNIFAR Pseudomonas aeruginosa iron pyoverdine siderophore solute-binding protein, Unité ARN}, pubstate = {published}, tppubtype = {article} } Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Iron release from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore binding protein, respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa. This article is protected by copyright. All rights reserved. |
Trzaska, C; Amand, S; Bailly, C; Leroy, C; Marchand, V; Duvernois-Berthet, E; Saliou, J M; Benhabiles, H; Werkmeister, E; Chassat, T; Guilbert, R; Hannebique, D; Mouray, A; Copin, M C; Moreau, P A; Adriaenssens, E; Kulozik, A; Westhof, E; Tulasne, D; Motorin, Y; Rebuffat, S; Lejeune, F 2,6-Diaminopurine as a Highly Potent Corrector of UGA Nonsense Mutations Article de journal Nat Commun, 11 (1), p. 1509, 2020, ISBN: 32198346. Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF @article{, title = {2,6-Diaminopurine as a Highly Potent Corrector of UGA Nonsense Mutations}, author = {C Trzaska and S Amand and C Bailly and C Leroy and V Marchand and E Duvernois-Berthet and J M Saliou and H Benhabiles and E Werkmeister and T Chassat and R Guilbert and D Hannebique and A Mouray and M C Copin and P A Moreau and E Adriaenssens and A Kulozik and E Westhof and D Tulasne and Y Motorin and S Rebuffat and F Lejeune}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32198346?dopt=Abstract}, doi = {10.1038/s41467-020-15140-z}, isbn = {32198346}, year = {2020}, date = {2020-01-01}, journal = {Nat Commun}, volume = {11}, number = {1}, pages = {1509}, abstract = {Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.}, keywords = {Unité ARN, WESTHOF}, pubstate = {published}, tppubtype = {article} } Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations. |
Trachman, 3rd R J; Cojocaru, R; Wu, D; Piszczek, G; Ryckelynck, M; Unrau, P J; Ferré-D'Amaré, A R Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair Article de journal Structure, 28 (7), p. 776-785.e773, 2020, ISBN: 32386573. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, RYCKELYNCK RNA aptamer RNA fluorescence RNA structure fluorescent dye structure-guided engineering, Unité ARN @article{, title = {Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair}, author = {3rd R J Trachman and R Cojocaru and D Wu and G Piszczek and M Ryckelynck and P J Unrau and A R Ferré-D'Amaré}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32386573?dopt=Abstract}, doi = {10.1016/j.str.2020.04.007}, isbn = {32386573}, year = {2020}, date = {2020-01-01}, journal = {Structure}, volume = {28}, number = {7}, pages = {776-785.e773}, abstract = {Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.}, keywords = {RYCKELYNCK, RYCKELYNCK RNA aptamer RNA fluorescence RNA structure fluorescent dye structure-guided engineering, Unité ARN}, pubstate = {published}, tppubtype = {article} } Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization. |
Théobald-Dietrich, A; de Wijn, R; Rollet, K; Bluhm, A; Rudinger-Thirion, J; Paulus, C; Lorber, B; Thureau, A; Frugier, M; Sauter, C Structural Analysis of RNA by Small-Angle X-ray Scattering Book Chapter Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 189-215, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006316. Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, FRUGIER IRES Integrative structural biology RNA SEC-SAXS Structure tRNA, Unité ARN @inbook{, title = {Structural Analysis of RNA by Small-Angle X-ray Scattering}, author = {A Théobald-Dietrich and R de Wijn and K Rollet and A Bluhm and J Rudinger-Thirion and C Paulus and B Lorber and A Thureau and M Frugier and C Sauter}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006316}, doi = {10.1007/978-1-0716-0278-2_14}, isbn = {32006316}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {189-215}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules.}, keywords = {ERIANI, FRUGIER, FRUGIER IRES Integrative structural biology RNA SEC-SAXS Structure tRNA, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } Over the past two decades small-angle X-ray scattering (SAXS) has become a popular method to characterize solutions of biomolecules including ribonucleic acid (RNA). In an integrative structural approach, SAXS is complementary to crystallography, NMR, and electron microscopy and provides information about RNA architecture and dynamics. This chapter highlights the practical advantages of combining size-exclusion chromatography and SAXS at synchrotron facilities. It is illustrated by practical case studies of samples ranging from single hairpins and tRNA to a large IRES. The emphasis is also put on sample preparation which is a critical step of SAXS analysis and on optimized protocols for in vitro RNA synthesis ensuring the production of mg amount of pure and homogeneous molecules. |
Simonetti, A; Guca, E; Bochler, A; Kuhn, L; Hashem, Y Structural Insights Into the Mammalian Late-Stage Initiation Complexes Article de journal Cell Rep, 31 (1), p. 107497, 2020, ISBN: 32268096. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN @article{, title = {Structural Insights Into the Mammalian Late-Stage Initiation Complexes}, author = {A Simonetti and E Guca and A Bochler and L Kuhn and Y Hashem}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32268096?dopt=Abstract}, doi = {10.1016/j.celrep.2020.03.061}, isbn = {32268096}, year = {2020}, date = {2020-01-01}, journal = {Cell Rep}, volume = {31}, number = {1}, pages = {107497}, abstract = {In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.}, keywords = {ENNIFAR, Unité ARN}, pubstate = {published}, tppubtype = {article} } In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC. |
Rol-Moreno, J; Kuhn, L; Marzi, S; Simonetti, A Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 329-339, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006323. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR ROMBY Cryo-electron microscopy Mass spectrometry Native ribosome complexes Translation initiation, Unité ARN @inbook{, title = {Grad-cryo-EM: Tool to Isolate Translation Initiation Complexes From Rabbit Reticulocyte Lysate Suitable for Structural Studies}, author = {J Rol-Moreno and L Kuhn and S Marzi and A Simonetti}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006323}, doi = {10.1007/978-1-0716-0278-2_21}, isbn = {32006323}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {329-339}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation. The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.}, keywords = {ENNIFAR ROMBY Cryo-electron microscopy Mass spectrometry Native ribosome complexes Translation initiation, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation. The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts. |
Riley, L G; Rudinger-Thirion, J; Frugier, M; Wilson, M; Luig, M; Alahakoon, T I; Nixon, C Y; Kirk, E P; Roscioli, T; Lunke, S; Stark, Z; Wierenga, K J; Palle, S; Walsh, M; Higgs, E; Arbuckle, S; Thirukeswaran, S; Compton, A G; Thorburn, D R; Christodoulou, J The Expanding LARS2 Phenotypic Spectrum: HLASA, Perrault Syndrome With Leukodystrophy, and Mitochondrial Myopathy Article de journal Hum Mutat, 41 (8), p. 1425-1434, 2020, ISBN: 32442335. Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, FRUGIER Perrault syndrome low frequency hearing loss primary ovarian insufficiency sensorineural hearing loss, Unité ARN @article{, title = {The Expanding LARS2 Phenotypic Spectrum: HLASA, Perrault Syndrome With Leukodystrophy, and Mitochondrial Myopathy}, author = {L G Riley and J Rudinger-Thirion and M Frugier and M Wilson and M Luig and T I Alahakoon and C Y Nixon and E P Kirk and T Roscioli and S Lunke and Z Stark and K J Wierenga and S Palle and M Walsh and E Higgs and S Arbuckle and S Thirukeswaran and A G Compton and D R Thorburn and J Christodoulou}, url = {https://pubmed.ncbi.nlm.nih.gov/32442335/?dopt=Abstract}, doi = {doi: 10.1002/humu.24050}, isbn = {32442335}, year = {2020}, date = {2020-01-01}, journal = {Hum Mutat}, volume = {41}, number = {8}, pages = {1425-1434}, abstract = {Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.(Gly16Ser) and novel, p.(Val82Phe) variant in D-bifunctional protein (HSD17B4). A family was homozygous for mitochondrial leucyl aminocyl tRNA synthetase (mtLeuRS) (LARS2) p.(Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.(Thr522Asn) and a novel variant, p.(Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.(Arg323Gln) and p.(Asn399Ser) variants in Twinkle (C10orf2). A male was homozygous for a novel variant in CLPP, p.(Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome.}, keywords = {ERIANI, FRUGIER, FRUGIER Perrault syndrome low frequency hearing loss primary ovarian insufficiency sensorineural hearing loss, Unité ARN}, pubstate = {published}, tppubtype = {article} } Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.(Gly16Ser) and novel, p.(Val82Phe) variant in D-bifunctional protein (HSD17B4). A family was homozygous for mitochondrial leucyl aminocyl tRNA synthetase (mtLeuRS) (LARS2) p.(Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.(Thr522Asn) and a novel variant, p.(Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.(Arg323Gln) and p.(Asn399Ser) variants in Twinkle (C10orf2). A male was homozygous for a novel variant in CLPP, p.(Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome. |
Pernod, K; Schaeffer, L; Chicher, J; Hok, E; Rick, C; Geslain, R; Eriani, G; Westhof, E; Ryckelynck, M; Martin, F The Nature of the Purine at Position 34 in tRNAs of 4-codon Boxes Is Correlated With Nucleotides at Positions 32 and 38 to Maintain Decoding Fidelity Article de journal Nucleic Acids Res, 48 (11), p. 6170-6183, 2020, ISBN: 32266934. Résumé | Liens | BibTeX | Étiquettes: ERIANI, ERIANI RYCKELYNCK WESTHOF, RYCKELYNCK, Unité ARN, WESTHOF @article{, title = {The Nature of the Purine at Position 34 in tRNAs of 4-codon Boxes Is Correlated With Nucleotides at Positions 32 and 38 to Maintain Decoding Fidelity}, author = {K Pernod and L Schaeffer and J Chicher and E Hok and C Rick and R Geslain and G Eriani and E Westhof and M Ryckelynck and F Martin}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32266934?dopt=Abstract}, doi = {10.1093/nar/gkaa221}, isbn = {32266934}, year = {2020}, date = {2020-01-01}, journal = {Nucleic Acids Res}, volume = {48}, number = {11}, pages = {6170-6183}, abstract = {Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.}, keywords = {ERIANI, ERIANI RYCKELYNCK WESTHOF, RYCKELYNCK, Unité ARN, WESTHOF}, pubstate = {published}, tppubtype = {article} } Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38. |
Orlov, I; Hemmer, C; Ackerer, L; Lorber, B; Ghannam, A; Poignavent, V; Hleibieh, K; Sauter, C; Schmitt-Keichinger, C; Belval, L; Hily, J M; Marmonier, A; Komar, V; Gersch, S; Schellenberger, P; Bron, P; Vigne, E; Muyldermans, S; Lemaire, O; Demangeat, G; Ritzenthaler, C; Klaholz, B P Structural Basis of Nanobody Recognition of Grapevine Fanleaf Virus and of Virus Resistance Loss Article de journal Proc Natl Acad Sci U S A, 117 (20), p. 10848-10855, 2020, ISBN: 32371486. Résumé | Liens | BibTeX | Étiquettes: FRUGIER GFLV nanobody structural biology virus., Unité ARN @article{, title = {Structural Basis of Nanobody Recognition of Grapevine Fanleaf Virus and of Virus Resistance Loss}, author = {I Orlov and C Hemmer and L Ackerer and B Lorber and A Ghannam and V Poignavent and K Hleibieh and C Sauter and C Schmitt-Keichinger and L Belval and J M Hily and A Marmonier and V Komar and S Gersch and P Schellenberger and P Bron and E Vigne and S Muyldermans and O Lemaire and G Demangeat and C Ritzenthaler and B P Klaholz}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32371486?dopt=Abstract}, doi = {10.1073/pnas.1913681117}, isbn = {32371486}, year = {2020}, date = {2020-01-01}, journal = {Proc Natl Acad Sci U S A}, volume = {117}, number = {20}, pages = {10848-10855}, abstract = {Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.}, keywords = {FRUGIER GFLV nanobody structural biology virus., Unité ARN}, pubstate = {published}, tppubtype = {article} } Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss. |
Quang, Nguyen N; Goudey, S; Ségéral, E; Mohammad, A; Lemoine, S; Blugeon, C; Versapuech, M; Paillart, J C; Berlioz-Torrent, C; Emiliani, S; Gallois-Montbrun, S Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection Article de journal Retrovirology, 17 (1), p. 25, 2020, ISBN: 32807178. Résumé | Liens | BibTeX | Étiquettes: MARQUET PAILLART HIV RNA Alternative splicing Viral transcriptome ONT long-read sequencing, PAILLART, Unité ARN @article{, title = {Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection}, author = {N Nguyen Quang and S Goudey and E Ségéral and A Mohammad and S Lemoine and C Blugeon and M Versapuech and J C Paillart and C Berlioz-Torrent and S Emiliani and S Gallois-Montbrun}, url = {https://pubmed.ncbi.nlm.nih.gov/32807178/}, doi = {10.1186/s12977-020-00533-1}, isbn = {32807178}, year = {2020}, date = {2020-01-01}, journal = {Retrovirology}, volume = {17}, number = {1}, pages = {25}, abstract = {Background Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells. Results ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build ﾓsplice treesﾔ, a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. Conclusion ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells. Background}, keywords = {MARQUET PAILLART HIV RNA Alternative splicing Viral transcriptome ONT long-read sequencing, PAILLART, Unité ARN}, pubstate = {published}, tppubtype = {article} } Background Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells. Results ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build ﾓsplice treesﾔ, a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. Conclusion ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells. Background |
Miao, Z; Tidu, A; Eriani, G; Martin, F Secondary structure of the SARS-CoV-2 5'-UTR Article de journal RNA Biol, p. in press, 2020, ISBN: 32965173. Résumé | Liens | BibTeX | Étiquettes: ERIANI, ERIANI 5ʹ-UTR SARS-CoV-2 probing secondary structure, Unité ARN @article{, title = {Secondary structure of the SARS-CoV-2 5'-UTR}, author = {Z Miao and A Tidu and G Eriani and F Martin}, url = {https://pubmed.ncbi.nlm.nih.gov/32965173/}, doi = {10.1080/15476286.2020.1814556}, isbn = {32965173}, year = {2020}, date = {2020-01-01}, journal = {RNA Biol}, pages = {in press}, abstract = {The SARS-CoV-2, a positive-sense single-stranded RNA Coronavirus, is a global threat to human health. Thus, understanding its life cycle mechanistically would be important to facilitate the design of antiviral drugs. A key aspect of viral progression is the synthesis of viral proteins by the ribosome of the human host. In Coronaviruses, this process is regulated by the viral 5' and 3' untranslated regions (UTRs), but the precise regulatory mechanism has not yet been well understood. In particular, the 5'-UTR of the viral genome is most likely involved in translation initiation of viral proteins. Here, we performed inline probing and RNase V1 probing to establish a model of the secondary structure of SARS-CoV-2 5'-UTR. We found that the 5'-UTR contains stable structures including a very stable four-way junction close to the AUG start codon. Sequence alignment analysis of SARS-CoV-2 variants 5'-UTRs revealed a highly conserved structure with few co-variations that confirmed our secondary structure model based on probing experiments.}, keywords = {ERIANI, ERIANI 5ʹ-UTR SARS-CoV-2 probing secondary structure, Unité ARN}, pubstate = {published}, tppubtype = {article} } The SARS-CoV-2, a positive-sense single-stranded RNA Coronavirus, is a global threat to human health. Thus, understanding its life cycle mechanistically would be important to facilitate the design of antiviral drugs. A key aspect of viral progression is the synthesis of viral proteins by the ribosome of the human host. In Coronaviruses, this process is regulated by the viral 5' and 3' untranslated regions (UTRs), but the precise regulatory mechanism has not yet been well understood. In particular, the 5'-UTR of the viral genome is most likely involved in translation initiation of viral proteins. Here, we performed inline probing and RNase V1 probing to establish a model of the secondary structure of SARS-CoV-2 5'-UTR. We found that the 5'-UTR contains stable structures including a very stable four-way junction close to the AUG start codon. Sequence alignment analysis of SARS-CoV-2 variants 5'-UTRs revealed a highly conserved structure with few co-variations that confirmed our secondary structure model based on probing experiments. |
Miao, Z; Adamiak, R W; Antczak, M; Boniecki, M J; Bujnicki, J M; Chen, S J; Cheng, C Y; Cheng, Y; Chou, F C; Das, R; Dokholyan, N V; Ding, F; Geniesse, C; Jiang, Y; Joshi, A; Krokhotin, A; Magnus, M; Mailhot, O; Major, F; Mann, T H; Piatkowski, P; Pluta, R; Popenda, M; Sarzynska, J; Sun, L; Szachniuk, M; Tian, S; Wang, J; Wang, J; Watkins, A M; Wiedemann, J; Xiao, Y; Xu, X; Yesselman, J D; Zhang, D; Zhang, Y; Zhang, Z; Zhao, C; Zhao, P; Zhou, Y; Zok, T; Zyla, A; Ren, A; Batey, R T; Golden, B L; Huang, L; Lilley, D M; Liu, Y; Patel, D J; Westhof, E RNA-Puzzles Round IV: 3D Structure Predictions of Four Ribozymes and Two Aptamers Article de journal RNA, 26 (8), p. 982-995, 2020, ISBN: 32371455. Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF, WESTHOF RNA structure aptamer modeing prediction ribozyme @article{, title = {RNA-Puzzles Round IV: 3D Structure Predictions of Four Ribozymes and Two Aptamers}, author = {Z Miao and R W Adamiak and M Antczak and M J Boniecki and J M Bujnicki and S J Chen and C Y Cheng and Y Cheng and F C Chou and R Das and N V Dokholyan and F Ding and C Geniesse and Y Jiang and A Joshi and A Krokhotin and M Magnus and O Mailhot and F Major and T H Mann and P Piatkowski and R Pluta and M Popenda and J Sarzynska and L Sun and M Szachniuk and S Tian and J Wang and J Wang and A M Watkins and J Wiedemann and Y Xiao and X Xu and J D Yesselman and D Zhang and Y Zhang and Z Zhang and C Zhao and P Zhao and Y Zhou and T Zok and A Zyla and A Ren and R T Batey and B L Golden and L Huang and D M Lilley and Y Liu and D J Patel and E Westhof}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32371455?dopt=Abstract}, doi = {10.1261/rna.075341.120}, isbn = {32371455}, year = {2020}, date = {2020-01-01}, journal = {RNA}, volume = {26}, number = {8}, pages = {982-995}, abstract = {RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of six RNA sequences: four structures of nucleolytic ribozymes and two of riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss a) the comparison between the automated web server and human experts; b) the prediction of coaxial stacking; c) the prediction of structural details and ligand binding; d) the development of novel prediction methods; and e) the potential improvements to be made. It is illustrated that correct coaxial stacking and tertiary contacts are key for the prediction of RNA architecture, while ligand binding modes can be only predicted with low resolution and accurate ligand binding prediction still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.}, keywords = {Unité ARN, WESTHOF, WESTHOF RNA structure aptamer modeing prediction ribozyme}, pubstate = {published}, tppubtype = {article} } RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of six RNA sequences: four structures of nucleolytic ribozymes and two of riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss a) the comparison between the automated web server and human experts; b) the prediction of coaxial stacking; c) the prediction of structural details and ligand binding; d) the development of novel prediction methods; and e) the potential improvements to be made. It is illustrated that correct coaxial stacking and tertiary contacts are key for the prediction of RNA architecture, while ligand binding modes can be only predicted with low resolution and accurate ligand binding prediction still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools. |
Menendez-Gil, P; Caballero, C J; Catalan-Moreno, A; Irurzun, N; Barrio-Hernandez, I; Caldelari, I; Toledo-Arana, A Differential evolution in 3'UTRs leads to specific gene expression in Staphylococcus Article de journal Nucleic Acids Res, 48 (5), p. 2544-2563, 2020, ISBN: 32016395. Résumé | Liens | BibTeX | Étiquettes: ROMBY, Unité ARN @article{, title = {Differential evolution in 3'UTRs leads to specific gene expression in Staphylococcus}, author = {P Menendez-Gil and C J Caballero and A Catalan-Moreno and N Irurzun and I Barrio-Hernandez and I Caldelari and A Toledo-Arana}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32016395}, doi = {10.1093/nar/gkaa047}, isbn = {32016395}, year = {2020}, date = {2020-01-01}, journal = {Nucleic Acids Res}, volume = {48}, number = {5}, pages = {2544-2563}, abstract = {The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity.}, keywords = {ROMBY, Unité ARN}, pubstate = {published}, tppubtype = {article} } The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity. |
Magnus, M; Antczak, M; Zok, T; Wiedemann, J; Lukasiak, P; Cao, Y; Bujnicki, J M; Westhof, E; Szachniuk, M; Miao, Z RNA-Puzzles toolkit: a computational resource of RNA 3D structure benchmark datasets, structure manipulation, and evaluation tools Article de journal Nucleic Acids Res, 48 (2), p. 576-588, 2020, ISBN: 31799609. Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF @article{, title = {RNA-Puzzles toolkit: a computational resource of RNA 3D structure benchmark datasets, structure manipulation, and evaluation tools}, author = {M Magnus and M Antczak and T Zok and J Wiedemann and P Lukasiak and Y Cao and J M Bujnicki and E Westhof and M Szachniuk and Z Miao}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31799609}, doi = {10.1093/nar/gkz1108.}, isbn = {31799609}, year = {2020}, date = {2020-01-01}, journal = {Nucleic Acids Res}, volume = {48}, number = {2}, pages = {576-588}, abstract = {Significant improvements have been made in the efficiency and accuracy of RNA 3D structure prediction methods during the succeeding challenges of RNA-Puzzles, a community-wide effort on the assessment of blind prediction of RNA tertiary structures. The RNA-Puzzles contest has shown, among others, that the development and validation of computational methods for RNA fold prediction strongly depend on the benchmark datasets and the structure comparison algorithms. Yet, there has been no systematic benchmark set or decoy structures available for the 3D structure prediction of RNA, hindering the standardization of comparative tests in the modeling of RNA structure. Furthermore, there has not been a unified set of tools that allows deep and complete RNA structure analysis, and at the same time, that is easy to use. Here, we present RNA-Puzzles toolkit, a computational resource including (i) decoy sets generated by different RNA 3D structure prediction methods (raw, for-evaluation and standardized datasets), (ii) 3D structure normalization, analysis, manipulation, visualization tools (RNA_format, RNA_normalizer, rna-tools) and (iii) 3D structure comparison metric tools (RNAQUA, MCQ4Structures). This resource provides a full list of computational tools as well as a standard RNA 3D structure prediction assessment protocol for the community.}, keywords = {Unité ARN, WESTHOF}, pubstate = {published}, tppubtype = {article} } Significant improvements have been made in the efficiency and accuracy of RNA 3D structure prediction methods during the succeeding challenges of RNA-Puzzles, a community-wide effort on the assessment of blind prediction of RNA tertiary structures. The RNA-Puzzles contest has shown, among others, that the development and validation of computational methods for RNA fold prediction strongly depend on the benchmark datasets and the structure comparison algorithms. Yet, there has been no systematic benchmark set or decoy structures available for the 3D structure prediction of RNA, hindering the standardization of comparative tests in the modeling of RNA structure. Furthermore, there has not been a unified set of tools that allows deep and complete RNA structure analysis, and at the same time, that is easy to use. Here, we present RNA-Puzzles toolkit, a computational resource including (i) decoy sets generated by different RNA 3D structure prediction methods (raw, for-evaluation and standardized datasets), (ii) 3D structure normalization, analysis, manipulation, visualization tools (RNA_format, RNA_normalizer, rna-tools) and (iii) 3D structure comparison metric tools (RNAQUA, MCQ4Structures). This resource provides a full list of computational tools as well as a standard RNA 3D structure prediction assessment protocol for the community. |
Lopez, P; Girardi, E; Mounce, B C; Weiss, A; Chane-Woon-Ming, B; Messmer, M; Kaukinen, P; Kopp, A; Bortolamiol-Becet, D; Fendri, A; Vignuzzi, M; Brino, L; Pfeffer, S High-throughput Fluorescence-Based Screen Identifies the Neuronal microRNA miR-124 as a Positive Regulator of Alphavirus Infection Article de journal J Virol, 94 (9), p. e02145-02119, 2020, ISBN: 32102877. Résumé | Liens | BibTeX | Étiquettes: PFEFFER, Unité ARN @article{, title = {High-throughput Fluorescence-Based Screen Identifies the Neuronal microRNA miR-124 as a Positive Regulator of Alphavirus Infection}, author = {P Lopez and E Girardi and B C Mounce and A Weiss and B Chane-Woon-Ming and M Messmer and P Kaukinen and A Kopp and D Bortolamiol-Becet and A Fendri and M Vignuzzi and L Brino and S Pfeffer}, url = {https://pubmed.ncbi.nlm.nih.gov/32102877}, doi = {10.1128/JVI.02145-19}, isbn = {32102877}, year = {2020}, date = {2020-01-01}, journal = {J Virol}, volume = {94}, number = {9}, pages = {e02145-02119}, abstract = {Micro (mi)RNAs are small regulatory RNAs, which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified sixteen miRNAs showing a positive effect on Sindbis virus (SINV) expressing GFP, among which a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 or silent mutations to disrupt this binding site in the viral RNA abolished the positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved for other alphaviruses as its inhibition reduces chikungunya virus (CHIKV) viral production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.IMPORTANCEArthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arboviruses infection, neurological diseases like meningitis or encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified the neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphaviruses infection.}, keywords = {PFEFFER, Unité ARN}, pubstate = {published}, tppubtype = {article} } Micro (mi)RNAs are small regulatory RNAs, which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified sixteen miRNAs showing a positive effect on Sindbis virus (SINV) expressing GFP, among which a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 or silent mutations to disrupt this binding site in the viral RNA abolished the positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved for other alphaviruses as its inhibition reduces chikungunya virus (CHIKV) viral production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.IMPORTANCEArthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arboviruses infection, neurological diseases like meningitis or encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified the neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphaviruses infection. |
Lechner, A; Wolff, P; Leize-Wagner, E; François, Y N Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry Article de journal Anal Chem, 92 (10), p. 7363-7370, 2020, ISBN: 32343557. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN @article{, title = {Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry}, author = {A Lechner and P Wolff and E Leize-Wagner and Y N François}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32343557?dopt=Abstract}, doi = {10.1021/acs.analchem.0c01345}, isbn = {32343557}, year = {2020}, date = {2020-01-01}, journal = {Anal Chem}, volume = {92}, number = {10}, pages = {7363-7370}, abstract = {Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.}, keywords = {ENNIFAR, Unité ARN}, pubstate = {published}, tppubtype = {article} } Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved. |
Kruse, H; Mrazikova, K; D'Ascenzo, L; Sponer, J; Auffinger, P Short but Weak! The Z-DNA lone-pair···π Conundrum Challenges Standard Carbon Van Der Waals Radii Article de journal Angew Chem Int Ed Engl, p. in press, 2020, ISBN: 32516461. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR lp···pi interactions molecular recognition non-covalent interactions van der Waals radii Z-DNA, Unité ARN @article{, title = {Short but Weak! The Z-DNA lone-pair···π Conundrum Challenges Standard Carbon Van Der Waals Radii}, author = {H Kruse and K Mrazikova and L D'Ascenzo and J Sponer and P Auffinger}, url = {https://pubmed.ncbi.nlm.nih.gov/32516461/}, doi = {10.1002/anie.202004201}, isbn = {32516461}, year = {2020}, date = {2020-01-01}, journal = {Angew Chem Int Ed Engl}, pages = {in press}, abstract = {Current interest for lone-pair···π (lp···π) non-covalent interactions is gaining momentum in biochemistry and (supramolecular)chemistry. However, the physico-chemical origin of the exceptionally short (≈2.8 Å) oxygen to nucleobase plane distances observed in prototypical Z-DNA CpG steps remains unclear. Accurate quantum mechanical calculations including SAPT2+3 interaction energy decompositions demonstrate that lp···π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatics interactions combined with stereochemical effects imposed by the structural context. They also suggest that the carbon van der Waals (vdW) radii originally derived for sp3carbons should not be used for the smaller sp2 atoms. Using a more adapted carbon vdW radius results in lp···π contacts being no longer of the sub-vdW type. Overall, our findings challenge the whole lp···π concept that refers to elusive orbital interactions that cannot explain short contact distances.}, keywords = {ENNIFAR lp···pi interactions molecular recognition non-covalent interactions van der Waals radii Z-DNA, Unité ARN}, pubstate = {published}, tppubtype = {article} } Current interest for lone-pair···π (lp···π) non-covalent interactions is gaining momentum in biochemistry and (supramolecular)chemistry. However, the physico-chemical origin of the exceptionally short (≈2.8 Å) oxygen to nucleobase plane distances observed in prototypical Z-DNA CpG steps remains unclear. Accurate quantum mechanical calculations including SAPT2+3 interaction energy decompositions demonstrate that lp···π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatics interactions combined with stereochemical effects imposed by the structural context. They also suggest that the carbon van der Waals (vdW) radii originally derived for sp3carbons should not be used for the smaller sp2 atoms. Using a more adapted carbon vdW radius results in lp···π contacts being no longer of the sub-vdW type. Overall, our findings challenge the whole lp···π concept that refers to elusive orbital interactions that cannot explain short contact distances. |
Kanja, M; Cappy, P; Levy, N; Oladosu, O; Schmidt, S; Rossolillo, P; Winter, F; Gasser, R; Moog, C; Ruff, M; Negroni, M; Lener, D NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases Article de journal J Virol, 94 (20), p. e01035-01020, 2020, ISBN: 32727879. Résumé | Liens | BibTeX | Étiquettes: NEGRONI, NEGRONI evolution human immunodeficiency virus integrase phylogenetic groups, Unité ARN @article{, title = {NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases}, author = {M Kanja and P Cappy and N Levy and O Oladosu and S Schmidt and P Rossolillo and F Winter and R Gasser and C Moog and M Ruff and M Negroni and D Lener}, url = {https://pubmed.ncbi.nlm.nih.gov/32727879/}, doi = {10.1128/JVI.01035-20}, isbn = {32727879}, year = {2020}, date = {2020-01-01}, journal = {J Virol}, volume = {94}, number = {20}, pages = {e01035-01020}, abstract = {Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3' processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K's could be permutated or additional K's could be inserted in the motif, generally without affecting integration per se Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.}, keywords = {NEGRONI, NEGRONI evolution human immunodeficiency virus integrase phylogenetic groups, Unité ARN}, pubstate = {published}, tppubtype = {article} } Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3' processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K's could be permutated or additional K's could be inserted in the motif, generally without affecting integration per se Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein. |
Injarabian, L; Scherlinger, M; Devin, A; Ransac, S; Lykkesfeldt, J; Marteyn, B S Ascorbate maintains a low plasma oxygen level Article de journal Sci Rep, 10 (1), p. 10659, 2020, ISBN: 32606354. Résumé | Liens | BibTeX | Étiquettes: MARTEYN, Unité ARN @article{, title = {Ascorbate maintains a low plasma oxygen level}, author = {L Injarabian and M Scherlinger and A Devin and S Ransac and J Lykkesfeldt and B S Marteyn}, url = {https://pubmed.ncbi.nlm.nih.gov/32606354/}, doi = {10.1038/s41598-020-67778-w}, isbn = {32606354}, year = {2020}, date = {2020-01-01}, journal = {Sci Rep}, volume = {10}, number = {1}, pages = {10659}, abstract = {In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 μM in fresh plasma vs < 2 μM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 μM), compared to control (Ascorbateplasma > 15 μM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions' efficacy.}, keywords = {MARTEYN, Unité ARN}, pubstate = {published}, tppubtype = {article} } In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 μM in fresh plasma vs < 2 μM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 μM), compared to control (Ascorbateplasma > 15 μM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions' efficacy. |
Herzog, K; Bandiera, S; Pernot, S; Fauvelle, C; Jühling, F; Weiss, A; Bull, A; Durand, S C; Chane-Woon-Ming, B; Pfeffer, S; Mercey, M; Lerat, H; Meunier, J C; Raffelsberger, W; Brino, L; Baumert, T F; Zeisel, M B Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity Article de journal Gut, 69 (2), p. 380-392, 2020, ISBN: 31076402. Résumé | Liens | BibTeX | Étiquettes: PFEFFER, PFEFFER HCV hepatitis C hepatocyte molecular mechanisms, Unité ARN @article{, title = {Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity}, author = {K Herzog and S Bandiera and S Pernot and C Fauvelle and F Jühling and A Weiss and A Bull and S C Durand and B Chane-Woon-Ming and S Pfeffer and M Mercey and H Lerat and J C Meunier and W Raffelsberger and L Brino and T F Baumert and M B Zeisel}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31076402?dopt=Abstract}, doi = {10.1136/gutjnl-2018-317423}, isbn = {31076402}, year = {2020}, date = {2020-01-01}, journal = {Gut}, volume = {69}, number = {2}, pages = {380-392}, abstract = {OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.}, keywords = {PFEFFER, PFEFFER HCV hepatitis C hepatocyte molecular mechanisms, Unité ARN}, pubstate = {published}, tppubtype = {article} } OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis. |
Girardi, E; Pfeffer, S; Baumert, T F; Majzoub, K Roadblocks and fast tracks: How RNA binding proteins affect the viral RNA journey in the cell Article de journal Semin Cell Dev Biol, p. in press, 2020, ISBN: 32847707. Résumé | Liens | BibTeX | Étiquettes: PFEFFER, PFEFFER Innate Immunity RNA biology RNA viruses Technology Viral RNA sensing Viral Translation Virology., Unité ARN @article{, title = {Roadblocks and fast tracks: How RNA binding proteins affect the viral RNA journey in the cell}, author = {E Girardi and S Pfeffer and T F Baumert and K Majzoub}, url = {https://pubmed.ncbi.nlm.nih.gov/32847707/}, doi = {10.1016/j.semcdb.2020.08.006}, isbn = {32847707}, year = {2020}, date = {2020-01-01}, journal = {Semin Cell Dev Biol}, pages = {in press}, abstract = {As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.}, keywords = {PFEFFER, PFEFFER Innate Immunity RNA biology RNA viruses Technology Viral RNA sensing Viral Translation Virology., Unité ARN}, pubstate = {published}, tppubtype = {article} } As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats. |
Ghosh, S; Guimaraes, J C; Lanzafame, M; Schmidt, A; Syed, A P; Dimitriades, B; Börsch, A; Ghosh, S; Mittal, N; Montavon, T; Correia, A L; Danner, J; Meister, G; Terracciano, L M; Pfeffer, S; Piscuoglio, S; Zavolan, M Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation Article de journal EMBO J, p. in press, 2020, ISBN: 32812257. Résumé | Liens | BibTeX | Étiquettes: PFEFFER, PFEFFER Argonaute 1 breast cancer endogenous dsRNA interferon response translation readthrough, Unité ARN @article{, title = {Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation}, author = {S Ghosh and J C Guimaraes and M Lanzafame and A Schmidt and A P Syed and B Dimitriades and A Börsch and S Ghosh and N Mittal and T Montavon and A L Correia and J Danner and G Meister and L M Terracciano and S Pfeffer and S Piscuoglio and M Zavolan}, url = {https://pubmed.ncbi.nlm.nih.gov/32812257/}, doi = {10.15252/embj.2019103922}, isbn = {32812257}, year = {2020}, date = {2020-01-01}, journal = {EMBO J}, pages = {in press}, abstract = {Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.}, keywords = {PFEFFER, PFEFFER Argonaute 1 breast cancer endogenous dsRNA interferon response translation readthrough, Unité ARN}, pubstate = {published}, tppubtype = {article} } Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth. |
Georg, J; Lalaouna, D; Hou, S; Lott, S C; Caldelari, I; Marzi, S; Hess, W R; Romby, P The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs Article de journal Mol Microbiol, 113 (3), p. 603-612, 2020, ISBN: 31705780. Résumé | Liens | BibTeX | Étiquettes: ROMBY, ROMBY Staphylococcus aureus CopraRNA MAPS post-transcriptional regulation sRNAs, Unité ARN @article{, title = {The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs}, author = {J Georg and D Lalaouna and S Hou and S C Lott and I Caldelari and S Marzi and W R Hess and P Romby}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31705780}, doi = {10.1111/mmi.14420}, isbn = {31705780}, year = {2020}, date = {2020-01-01}, journal = {Mol Microbiol}, volume = {113}, number = {3}, pages = {603-612}, abstract = {Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic miRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNA's targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more detail. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable.}, keywords = {ROMBY, ROMBY Staphylococcus aureus CopraRNA MAPS post-transcriptional regulation sRNAs, Unité ARN}, pubstate = {published}, tppubtype = {article} } Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic miRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNA's targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more detail. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable. |
Gasser, C; Delazer, I; Neuner, E; Pascher, K; Brillet, K; Klotz, S; Trixl, L; Himmelstoß, M; Ennifar, E; Rieder, D; Lusser, A; Micura, R Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL) Article de journal Angew Chem Int Ed Engl, 59 (17), p. 6881-6886, 2020, ISBN: 31999864. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR RNA sequencing RNA structures gene expression nucleoside modifications oligonucleotides, Unité ARN @article{, title = {Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL)}, author = {C Gasser and I Delazer and E Neuner and K Pascher and K Brillet and S Klotz and L Trixl and M Himmelstoß and E Ennifar and D Rieder and A Lusser and R Micura}, url = {https://pubmed.ncbi.nlm.nih.gov/31999864/}, doi = {10.1002/anie.201916272}, isbn = {31999864}, year = {2020}, date = {2020-01-01}, journal = {Angew Chem Int Ed Engl}, volume = {59}, number = {17}, pages = {6881-6886}, abstract = {Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.}, keywords = {ENNIFAR, ENNIFAR RNA sequencing RNA structures gene expression nucleoside modifications oligonucleotides, Unité ARN}, pubstate = {published}, tppubtype = {article} } Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision. |
Desgranges, E; Caldelari, I; Marzi, S; Lalaouna, D Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs Article de journal Biochim Biophys Acta Gene Regul Mech, 1863 (3), p. 194506, 2020, ISBN: 32068131. Résumé | Liens | BibTeX | Étiquettes: ROMBY Bacteria Post-transcriptional regulation RNA sequencing Regulatory network Small regulatory RNA Targetome, Unité ARN @article{, title = {Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs}, author = {E Desgranges and I Caldelari and S Marzi and D Lalaouna}, url = {https://www.ncbi.nlm.nih.gov/pubmed/32068131}, doi = {10.1016/j.bbagrm.2020.194506}, isbn = {32068131}, year = {2020}, date = {2020-01-01}, journal = {Biochim Biophys Acta Gene Regul Mech}, volume = {1863}, number = {3}, pages = {194506}, abstract = {Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.}, keywords = {ROMBY Bacteria Post-transcriptional regulation RNA sequencing Regulatory network Small regulatory RNA Targetome, Unité ARN}, pubstate = {published}, tppubtype = {article} } Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila. |
de Wijn, R; Rollet, K; Engilberge, S; McEwen, A G; Hennig, O; Betat, H; Mörl, M; Riobé, F; Maury, O; Girard, E; Bénas, P; Lorber, B; Sauter, C Monitoring the Production of High Diffraction-Quality Crystals of Two Enzymes in Real Time Using In Situ Dynamic Light Scattering Article de journal Crystals, 10 (2), p. 65, 2020. Résumé | Liens | BibTeX | Étiquettes: FRUGIER enzyme crystallization dynamic light scattering nucleation nucleant Tb-Xo4 crystallophore microcrystals nanocrystals X-ray diffraction XtalController, Unité ARN @article{, title = {Monitoring the Production of High Diffraction-Quality Crystals of Two Enzymes in Real Time Using In Situ Dynamic Light Scattering}, author = {R de Wijn and K Rollet and S Engilberge and A G McEwen and O Hennig and H Betat and M Mörl and F Riobé and O Maury and E Girard and P Bénas and B Lorber and C Sauter}, url = {https://www.mdpi.com/2073-4352/10/2/65}, doi = {10.3390/cryst10020065}, year = {2020}, date = {2020-01-01}, journal = {Crystals}, volume = {10}, number = {2}, pages = {65}, abstract = {The reproducible preparation of well-diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus, and the lysozyme from hen egg white in the presence of a synthetic chemical nucleant. We were able to (i) detect early nucleation events and (ii) drive the crystallization system (through cycles of dissolution/crystallization) toward growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction.}, keywords = {FRUGIER enzyme crystallization dynamic light scattering nucleation nucleant Tb-Xo4 crystallophore microcrystals nanocrystals X-ray diffraction XtalController, Unité ARN}, pubstate = {published}, tppubtype = {article} } The reproducible preparation of well-diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus, and the lysozyme from hen egg white in the presence of a synthetic chemical nucleant. We were able to (i) detect early nucleation events and (ii) drive the crystallization system (through cycles of dissolution/crystallization) toward growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction. |
Chernorudskiy, A; Varone, E; Colombo, S F; Fumagalli, S; Cagnotto, A; Cattaneo, A; Briens, M; Baltzinger, M; Kuhn, L; Bachi, A; Berardi, A; Salmona, M; Musco, G; Borgese, N; Lescure, A; Zito, E Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity Article de journal Proc Natl Acad Sci U S A, 117 (35), p. 21288-21298, 2020, ISBN: 32817544. Résumé | Liens | BibTeX | Étiquettes: LESCURE, LESCURE calcium sensor SEPN1 endoplasmic reticulum stress of the endoplasmic reticulum, Unité ARN @article{, title = {Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity}, author = {A Chernorudskiy and E Varone and S F Colombo and S Fumagalli and A Cagnotto and A Cattaneo and M Briens and M Baltzinger and L Kuhn and A Bachi and A Berardi and M Salmona and G Musco and N Borgese and A Lescure and E Zito}, url = {https://pubmed.ncbi.nlm.nih.gov/32817544/}, doi = {10.1073/pnas.2003847117}, isbn = {32817544}, year = {2020}, date = {2020-01-01}, journal = {Proc Natl Acad Sci U S A}, volume = {117}, number = {35}, pages = {21288-21298}, abstract = {The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.}, keywords = {LESCURE, LESCURE calcium sensor SEPN1 endoplasmic reticulum stress of the endoplasmic reticulum, Unité ARN}, pubstate = {published}, tppubtype = {article} } The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores. |
Brillet, K; Martinez-Zapien, D; Bec, G; Ennifar, E; Dock-Bregeon, A C; Lebars, I Different views of the dynamic landscape covered by the 5'-hairpin of the 7SK small nuclear RNA Article de journal RNA, 26 (9), p. 1184-1197, 2020, ISBN: 32430362. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR 7SK HEXIM RNA Tat structure, Unité ARN @article{, title = {Different views of the dynamic landscape covered by the 5'-hairpin of the 7SK small nuclear RNA}, author = {K Brillet and D Martinez-Zapien and G Bec and E Ennifar and A C Dock-Bregeon and I Lebars}, url = {https://pubmed.ncbi.nlm.nih.gov/32430362/}, doi = {10.1261/rna.074955.120}, isbn = {32430362}, year = {2020}, date = {2020-01-01}, journal = {RNA}, volume = {26}, number = {9}, pages = {1184-1197}, abstract = {The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairpin of the 7SKsnRNA. This hairpin (HP1), comprising the signature sequence of the 7SKsnRNA, has been the subject of three independent structural studies aiming at identifying the structural features that could drive the recognition by the two proteins, both depending on Arginine Rich Motifs (ARM). Interestingly, four distinct structures were determined. In an attempt to provide a comprehensive view of the structure-function relationship of this versatile RNA, we present here a structural analysis of the models, highlighting how HP1 is able to adopt distinct conformations with significant impact on the compactness of the molecule. Since these models are solved under different conditions by NMR and crystallography, the impact of the buffer composition on the conformational variation was investigated by complementary biophysical approaches. Finally, using Isothermal Titration Calorimetry, we determined the thermodynamic signatures of the Tat-ARM and HEXIM-ARM peptide interactions with the RNA, showing that they are associated with distinct binding}, keywords = {ENNIFAR, ENNIFAR 7SK HEXIM RNA Tat structure, Unité ARN}, pubstate = {published}, tppubtype = {article} } The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairpin of the 7SKsnRNA. This hairpin (HP1), comprising the signature sequence of the 7SKsnRNA, has been the subject of three independent structural studies aiming at identifying the structural features that could drive the recognition by the two proteins, both depending on Arginine Rich Motifs (ARM). Interestingly, four distinct structures were determined. In an attempt to provide a comprehensive view of the structure-function relationship of this versatile RNA, we present here a structural analysis of the models, highlighting how HP1 is able to adopt distinct conformations with significant impact on the compactness of the molecule. Since these models are solved under different conditions by NMR and crystallography, the impact of the buffer composition on the conformational variation was investigated by complementary biophysical approaches. Finally, using Isothermal Titration Calorimetry, we determined the thermodynamic signatures of the Tat-ARM and HEXIM-ARM peptide interactions with the RNA, showing that they are associated with distinct binding |
Boutant, E; Bonzi, J; Anton, H; Nasim, Bin M; Cathagne, R; Real, E; Dujardin, D; Carl, P; Didier, P; Paillart, J C; Marquet, R; Mely, Y; de Rocquigny, H; Bernacchi, S Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane Article de journal Biophys J, 119 (2), p. 419-433, 2020, ISBN: 32574557. Résumé | Liens | BibTeX | Étiquettes: MARQUET, MARQUET PAILLART, PAILLART, Unité ARN @article{, title = {Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane}, author = {E Boutant and J Bonzi and H Anton and M Bin Nasim and R Cathagne and E Real and D Dujardin and P Carl and P Didier and J C Paillart and R Marquet and Y Mely and H de Rocquigny and S Bernacchi}, url = {https://pubmed.ncbi.nlm.nih.gov/32574557/}, doi = {10.1016/j.bpj.2020.05.035}, isbn = {32574557}, year = {2020}, date = {2020-01-01}, journal = {Biophys J}, volume = {119}, number = {2}, pages = {419-433}, abstract = {The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.}, keywords = {MARQUET, MARQUET PAILLART, PAILLART, Unité ARN}, pubstate = {published}, tppubtype = {article} } The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol. |
Bouhedda, F; Fam, K T; Collot, M; Autour, A; Marzi, S; Klymchenko, A; Ryckelynck, M A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells Article de journal Nat Chem Biol, 16 (1), p. 69-76, 2020, ISBN: 31636432. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN @article{, title = {A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells}, author = {F Bouhedda and K T Fam and M Collot and A Autour and S Marzi and A Klymchenko and M Ryckelynck}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31636432}, doi = {https://doi.org/10.1038/s41589-019-0381-8}, isbn = {31636432}, year = {2020}, date = {2020-01-01}, journal = {Nat Chem Biol}, volume = {16}, number = {1}, pages = {69-76}, abstract = {Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.}, keywords = {RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes. |
Bernacchi, S; Ennifar, E Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 237-250, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006318. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR MARQUET PAILLART HIV-1 Viral RNA Aminoglycosides Dimerization initiation site ITC Thermodynamics RNAdrug interaction, Unité ARN @inbook{, title = {Analysis of the HIV-1 Genomic RNA Dimerization Initiation Site Binding to Aminoglycoside Antibiotics Using Isothermal Titration Calorimetry}, author = {S Bernacchi and E Ennifar}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006318}, doi = {10.1007/978-1-0716-0278-2_16}, isbn = {32006318}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {237-250}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity.}, keywords = {ENNIFAR, ENNIFAR MARQUET PAILLART HIV-1 Viral RNA Aminoglycosides Dimerization initiation site ITC Thermodynamics RNAdrug interaction, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } Isothermal titration calorimetry (ITC) provides a sensitive, powerful, and accurate tool to suitably analyze the thermodynamic of RNA binding events. This approach does not require any modification or labeling of the system under analysis and is performed in solution. ITC is a very convenient technique that provides an accurate determination of binding parameters, as well as a complete thermodynamic profile of the molecular interactions. Here we show how this approach can be used to characterize the interactions between the dimerization initiation site (DIS) RNA localized within the HIV-1 viral genome and aminoglycoside antibiotics. Our ITC study showed that the 4,5-disubstituted 2-desoxystreptamine (2-DOS) aminoglycosides can bind the DIS with a nanomolar affinity and a high specificity. |
Bernacchi, S Dynamic Light Scattering Analysis on RNA Associated to Proteins Book Chapter Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 31-39, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006306. Résumé | Liens | BibTeX | Étiquettes: MARQUET PAILLART Dynamic light scattering Protein-RNA interactions Hydrodynamic radius Diffusion coefficient Pr55Gag precursor Viral genomic RNA Viral spliced RNA Viral assembly, Unité ARN @inbook{, title = {Dynamic Light Scattering Analysis on RNA Associated to Proteins}, author = {S Bernacchi}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006306}, doi = {10.1007/978-1-0716-0278-2_4}, isbn = {32006306}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {31-39}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {Dynamic light scattering represents an accurate, robust, and reliable technique to analyze molecule size in solution and monitor their interactions in real time. Here, we describe how to analyze by DLS an RNA-protein interaction. In our frame, we studied complexes formed between RNA fragments derived from the genome of HIV-1 in association with the viral precursor Pr55Gag. These interactions are crucial for the specific selection of the viral genomic RNA (gRNA) from the bulk of the viral spliced and cellular RNAs. This chapter displays how DLS allows to characterize the interactions that regulate the early steps of viral assembly.}, keywords = {MARQUET PAILLART Dynamic light scattering Protein-RNA interactions Hydrodynamic radius Diffusion coefficient Pr55Gag precursor Viral genomic RNA Viral spliced RNA Viral assembly, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } Dynamic light scattering represents an accurate, robust, and reliable technique to analyze molecule size in solution and monitor their interactions in real time. Here, we describe how to analyze by DLS an RNA-protein interaction. In our frame, we studied complexes formed between RNA fragments derived from the genome of HIV-1 in association with the viral precursor Pr55Gag. These interactions are crucial for the specific selection of the viral genomic RNA (gRNA) from the bulk of the viral spliced and cellular RNAs. This chapter displays how DLS allows to characterize the interactions that regulate the early steps of viral assembly. |
Antoine, L; Wolff, P Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry Book Chapter Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , p. 101-110, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006310. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR ROMBY 2D Gel isolation Nano-LC-MS/MS Posttranscriptional tRNA modifications, Unité ARN @inbook{, title = {Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry}, author = {L Antoine and P Wolff}, editor = {V Arluison and F Wien}, url = {https://pubmed.ncbi.nlm.nih.gov/32006310}, doi = {10.1007/978-1-0716-0278-2_8}, isbn = {32006310}, year = {2020}, date = {2020-01-01}, booktitle = {RNA Spectroscopy: Methods and Protocols}, volume = {2113}, pages = {101-110}, publisher = {Springer Protocols, Humana Press}, address = {New York, NY}, series = {Methods in Molecular Biology}, abstract = {RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.}, keywords = {ENNIFAR, ENNIFAR ROMBY 2D Gel isolation Nano-LC-MS/MS Posttranscriptional tRNA modifications, Unité ARN}, pubstate = {published}, tppubtype = {inbook} } RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications. |
Ali, L M; Pitchai, F N; Vivet-Boudou, V; Chameettachal, A; Jabeen, A; Pillai, V N; Mustafa, F; Marquet, R; Rizvi, T A Role of purine-rich regions in Mason-Pfizer monkey virus (MPMV) Genomic RNA Packaging and propagation Article de journal Front Microbiol, p. in press, 2020. Liens | BibTeX | Étiquettes: MARQUET, MARQUET PAILLART, Unité ARN @article{, title = {Role of purine-rich regions in Mason-Pfizer monkey virus (MPMV) Genomic RNA Packaging and propagation}, author = {L M Ali and F N Pitchai and V Vivet-Boudou and A Chameettachal and A Jabeen and V N Pillai and F Mustafa and R Marquet and T A Rizvi}, url = {none}, year = {2020}, date = {2020-01-01}, journal = {Front Microbiol}, pages = {in press}, keywords = {MARQUET, MARQUET PAILLART, Unité ARN}, pubstate = {published}, tppubtype = {article} } |
2019 |
Vicens, Q; Coumoul, X; Souciet, J L A forum where french-speaking faculty can exchange research on teaching Article de journal Biochem Mol Biol Educ, 47 (5), p. 599-606, 2019, ISBN: 31141270. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR ducational specialist faculty development instructional change, Unité ARN @article{, title = {A forum where french-speaking faculty can exchange research on teaching}, author = {Q Vicens and X Coumoul and J L Souciet}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31141270?dopt=Abstract}, doi = {10.1002/bmb.21258}, isbn = {31141270}, year = {2019}, date = {2019-01-01}, journal = {Biochem Mol Biol Educ}, volume = {47}, number = {5}, pages = {599-606}, abstract = {In order to help promote instructional change at French-speaking universities in Europe, we initiated a series of 1-day events centered on learning innovations. Since 2015, these events have been taking place every 6 months at the Université Paris Descartes, with the moral support of three learned scientific societies, the French Academy of Sciences, and sponsoring by leaders in textbook editing and classroom technologies. Each event gathers ~ 40 participants (faculty members, postdocs, and educational specialists) from four countries (Belgium, France, Luxemburg, Switzerland) and invitees, who share their active learning practices, flipped classroom variations representing the most popular strategy. Their experience revealed that faculty who invest themselves in revamping teaching are still isolated at their institutions, although institutional and national support have now been gaining momentum. In particular, the role of educational specialists (known as ingénieurs pédagogiques in France) is key to help faculty move away from lecturing. Overall, our event series illustrates that a hands-off approach is effective to foster a cross-border community of committed academics in a context where the process of changing the way we teach at universities is still in its infancy.}, keywords = {ENNIFAR ducational specialist faculty development instructional change, Unité ARN}, pubstate = {published}, tppubtype = {article} } In order to help promote instructional change at French-speaking universities in Europe, we initiated a series of 1-day events centered on learning innovations. Since 2015, these events have been taking place every 6 months at the Université Paris Descartes, with the moral support of three learned scientific societies, the French Academy of Sciences, and sponsoring by leaders in textbook editing and classroom technologies. Each event gathers ~ 40 participants (faculty members, postdocs, and educational specialists) from four countries (Belgium, France, Luxemburg, Switzerland) and invitees, who share their active learning practices, flipped classroom variations representing the most popular strategy. Their experience revealed that faculty who invest themselves in revamping teaching are still isolated at their institutions, although institutional and national support have now been gaining momentum. In particular, the role of educational specialists (known as ingénieurs pédagogiques in France) is key to help faculty move away from lecturing. Overall, our event series illustrates that a hands-off approach is effective to foster a cross-border community of committed academics in a context where the process of changing the way we teach at universities is still in its infancy. |
van der Knaap, M S; Bugiani, M; Mendes, M I; Riley, L G; Smith, D E C; Rudinger-Thirion, J; Frugier, M; Breur, M; Crawford, J; van Gaalen, J; Schouten, M; Willems, M; Waisfisz, Q; Mau-Them, F T; Rodenburg, R J; Taft, R J; Keren, B; Christodoulou, J; Depienne, C; Simons, C; Salomons, G S; Mochel, F Biallelic variants in LARS2 and KARS cause deafness and (ovario)leukodystrophy Article de journal Neurology, 92 (11), p. e1225-e1237, 2019, ISBN: 30737337. Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, Unité ARN @article{, title = {Biallelic variants in \textit{LARS2} and \textit{KARS} cause deafness and (ovario)leukodystrophy}, author = {M S van der Knaap and M Bugiani and M I Mendes and L G Riley and D E C Smith and J Rudinger-Thirion and M Frugier and M Breur and J Crawford and J van Gaalen and M Schouten and M Willems and Q Waisfisz and F T Mau-Them and R J Rodenburg and R J Taft and B Keren and J Christodoulou and C Depienne and C Simons and G S Salomons and F Mochel}, url = {https://www.ncbi.nlm.nih.gov/pubmed/30737337}, doi = {10.1212/WNL.0000000000007098}, isbn = {30737337}, year = {2019}, date = {2019-01-01}, journal = {Neurology}, volume = {92}, number = {11}, pages = {e1225-e1237}, abstract = {OBJECTIVE: To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively. METHODS: We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts. RESULTS: Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity. CONCLUSION: This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies.}, keywords = {ERIANI, FRUGIER, Unité ARN}, pubstate = {published}, tppubtype = {article} } OBJECTIVE: To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively. METHODS: We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts. RESULTS: Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity. CONCLUSION: This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies. |
Pineiro, A; Munoz, E; Sabin, J; Costas, M; Bastos, M; Velazquez-Campoy, A; Garrido, P F; Dumas, P; Ennifar, E; Garcia-Rio, L; Rial, J; Pérez, D; Fraga, P; Rodriguez, A; Cotelo, C AFFINImeter: A software to analyze molecular recognition processes from experimental data Article de journal Anal Biochem, 5 (577), p. 117-134, 2019, ISBN: 30849378. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR Affinity Analysis software Isothermal titration calorimetry Kinetics Molecular recognition Thermodynamics, Unité ARN @article{, title = {AFFINImeter: A software to analyze molecular recognition processes from experimental data}, author = {A Pineiro and E Munoz and J Sabin and M Costas and M Bastos and A Velazquez-Campoy and P F Garrido and P Dumas and E Ennifar and L Garcia-Rio and J Rial and D Pérez and P Fraga and A Rodriguez and C Cotelo}, url = {https://www.ncbi.nlm.nih.gov/pubmed/30849378?dopt=Abstract}, doi = {10.1016/j.ab.2019.02.031}, isbn = {30849378}, year = {2019}, date = {2019-01-01}, journal = {Anal Biochem}, volume = {5}, number = {577}, pages = {117-134}, abstract = {The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software. Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of surpramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided.}, keywords = {ENNIFAR, ENNIFAR Affinity Analysis software Isothermal titration calorimetry Kinetics Molecular recognition Thermodynamics, Unité ARN}, pubstate = {published}, tppubtype = {article} } The comprehension of molecular recognition phenomena demands the understanding of the energetic and kinetic processes involved. General equations valid for the thermodynamic analysis of any observable that is assessed as a function of the concentration of the involved compounds are described, together with their implementation in the AFFINImeter software. Here, a maximum of three different molecular species that can interact with each other to form an enormous variety of surpramolecular complexes are considered. The corrections currently employed to take into account the effects of dilution, volume displacement, concentration errors and those due to external factors, especially in the case of ITC measurements, are included. The methods used to fit the model parameters to the experimental data, and to generate the uncertainties are described in detail. A simulation tool and the so called kinITC analysis to get kinetic information from calorimetric experiments are also presented. An example of how to take advantage of the AFFINImeter software for the global multi-temperature analysis of a system exhibiting cooperative 1:2 interactions is presented and the results are compared with data previously published. Some useful recommendations for the analysis of experiments aimed at studying molecular interactions are provided. |
Lopez, P; Girardi, E; Pfeffer, S [Importance of cellular microRNAs in the regulation of viral infections] Article de journal Med Sci (Paris), 35 (8-9), p. 667-673, 2019, ISBN: 31532379. Résumé | Liens | BibTeX | Étiquettes: PFEFFER, Unité ARN @article{, title = {[Importance of cellular microRNAs in the regulation of viral infections]}, author = {P Lopez and E Girardi and S Pfeffer}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31532379}, doi = {10.1051/medsci/2019130}, isbn = {31532379}, year = {2019}, date = {2019-01-01}, journal = {Med Sci (Paris)}, volume = {35}, number = {8-9}, pages = {667-673}, abstract = {Viruses are obligatory intracellular parasites that rely on a wide range of cellular factors to successfully accomplish their infectious cycle. Among those, micro (mi)RNAs have recently emerged as important modulators of viral infections. These small regulatory molecules act as repressors of gene expression. During infection, miRNAs can function by targeting either cellular or viral RNAs. In this review, we will recapitulate what has been reported to date on this interplay between cellular miRNAs and viruses and the effect on the infection. Furthermore, we will briefly discuss the possibilities of interfering with the infection through the modulation of this pathway to develop novel antiviral therapies.}, keywords = {PFEFFER, Unité ARN}, pubstate = {published}, tppubtype = {article} } Viruses are obligatory intracellular parasites that rely on a wide range of cellular factors to successfully accomplish their infectious cycle. Among those, micro (mi)RNAs have recently emerged as important modulators of viral infections. These small regulatory molecules act as repressors of gene expression. During infection, miRNAs can function by targeting either cellular or viral RNAs. In this review, we will recapitulate what has been reported to date on this interplay between cellular miRNAs and viruses and the effect on the infection. Furthermore, we will briefly discuss the possibilities of interfering with the infection through the modulation of this pathway to develop novel antiviral therapies. |
Kruse, H; Sponer, J; Auffinger, P Comment on "Evaluating Unexpectedly Short Non-covalent Distances in X-ray Crystal Structures of Proteins with Electronic Structure Analysis" Article de journal J Chem Inf Model, 59 (9), p. 3605-3608, 2019, ISBN: 31424937. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN @article{, title = {Comment on "Evaluating Unexpectedly Short Non-covalent Distances in X-ray Crystal Structures of Proteins with Electronic Structure Analysis"}, author = {H Kruse and J Sponer and P Auffinger}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31424937?dopt=Abstract}, doi = {10.1021/acs.jcim.9b00473}, isbn = {31424937}, year = {2019}, date = {2019-01-01}, journal = {J Chem Inf Model}, volume = {59}, number = {9}, pages = {3605-3608}, abstract = {1 Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, CZ-61265 Brno, Czech Republic. 2 Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg 67084, France.}, keywords = {ENNIFAR, Unité ARN}, pubstate = {published}, tppubtype = {article} } 1 Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, CZ-61265 Brno, Czech Republic. 2 Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg 67084, France. |
Janvier, A; Despons, L; Schaeffer, L; Tidu, A; Martin, F; Eriani, G A tRNA-mimic Strategy to Explore the Role of G34 of tRNAGly in Translation and Codon Frameshifting Article de journal Int J Mol Sci, 20 (16), p. 3911, 2019, ISBN: 31405256. Résumé | Liens | BibTeX | Étiquettes: ERIANI, ERIANI LESCURE IRES element frameshifting genetic code glycine codon tRNA translation, Unité ARN @article{, title = {A tRNA-mimic Strategy to Explore the Role of G34 of tRNA^{Gly} in Translation and Codon Frameshifting}, author = {A Janvier and L Despons and L Schaeffer and A Tidu and F Martin and G Eriani}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31405256?dopt=Abstract}, doi = {10.3390/ijms20163911}, isbn = {31405256}, year = {2019}, date = {2019-01-01}, journal = {Int J Mol Sci}, volume = {20}, number = {16}, pages = {3911}, abstract = {Decoding of the 61 sense codons of the genetic code requires a variable number of tRNAs that establish codon-anticodon interactions. Thanks to the wobble base pairing at the third codon position, less than 61 different tRNA isoacceptors are needed to decode the whole set of codons. On the tRNA, a subtle distribution of nucleoside modifications shapes the anticodon loop structure and participates to accurate decoding and reading frame maintenance. Interestingly, although the 61 anticodons should exist in tRNAs, a strict absence of some tRNAs decoders is found in several codon families. For instance, in Eukaryotes, G34-containing tRNAs translating 3-, 4- and 6-codon boxes are absent. This includes tRNA specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr, and Val. tRNAGly is the only exception for which in the three kingdoms, a G34-containing tRNA exists to decode C3 and U3-ending codons. To understand why G34-tRNAGly exists, we analysed at the genome wide level the codon distribution in codon +1 relative to the four GGN Gly codons. When considering codon GGU, a bias was found towards an unusual high usage of codons starting with a G whatever the amino acid at +1 codon. It is expected that GGU codons are decoded by G34-containing tRNAGly, decoding also GGC codons. Translation studies revealed that the presence of a G at the first position of the downstream codon reduces the +1 frameshift by stabilizing the G34U3 wobble interaction. This result partially explains why G34-containing tRNAGly exists in Eukaryotes whereas all the other G34-containing tRNAs for multiple codon boxes are absent.}, keywords = {ERIANI, ERIANI LESCURE IRES element frameshifting genetic code glycine codon tRNA translation, Unité ARN}, pubstate = {published}, tppubtype = {article} } Decoding of the 61 sense codons of the genetic code requires a variable number of tRNAs that establish codon-anticodon interactions. Thanks to the wobble base pairing at the third codon position, less than 61 different tRNA isoacceptors are needed to decode the whole set of codons. On the tRNA, a subtle distribution of nucleoside modifications shapes the anticodon loop structure and participates to accurate decoding and reading frame maintenance. Interestingly, although the 61 anticodons should exist in tRNAs, a strict absence of some tRNAs decoders is found in several codon families. For instance, in Eukaryotes, G34-containing tRNAs translating 3-, 4- and 6-codon boxes are absent. This includes tRNA specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr, and Val. tRNAGly is the only exception for which in the three kingdoms, a G34-containing tRNA exists to decode C3 and U3-ending codons. To understand why G34-tRNAGly exists, we analysed at the genome wide level the codon distribution in codon +1 relative to the four GGN Gly codons. When considering codon GGU, a bias was found towards an unusual high usage of codons starting with a G whatever the amino acid at +1 codon. It is expected that GGU codons are decoded by G34-containing tRNAGly, decoding also GGC codons. Translation studies revealed that the presence of a G at the first position of the downstream codon reduces the +1 frameshift by stabilizing the G34U3 wobble interaction. This result partially explains why G34-containing tRNAGly exists in Eukaryotes whereas all the other G34-containing tRNAs for multiple codon boxes are absent. |
Jackson, C B; Huemer, M; Bolognini, R; Martin, F; Szinnai, G; Donner, B C; Richter, U; Battersby, B J; Nuoffer, J M; Suomalainen, A; Schaller, A Hum Mol Genet, 28 (4), p. 639-649, 2019, ISBN: 30358850. Résumé | Liens | BibTeX | Étiquettes: ERIANI, Unité ARN @article{, title = {A variant in MRPS14 (uS14m) causes perinatal hypertrophic cardiomyopathy with neonatal lactic acidosis, growth retardation, dysmorphic features and neurological involvement}, author = {C B Jackson and M Huemer and R Bolognini and F Martin and G Szinnai and B C Donner and U Richter and B J Battersby and J M Nuoffer and A Suomalainen and A Schaller}, url = {https://www.ncbi.nlm.nih.gov/pubmed/30358850}, doi = {10.1093/hmg/ddy374}, isbn = {30358850}, year = {2019}, date = {2019-01-01}, journal = {Hum Mol Genet}, volume = {28}, number = {4}, pages = {639-649}, abstract = {Dysfunction of mitochondrial translation is increasingly an important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts mitochondrial translation was impaired and ectopic expression of a wild type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel. Collectively, our data demonstrates pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly.}, keywords = {ERIANI, Unité ARN}, pubstate = {published}, tppubtype = {article} } Dysfunction of mitochondrial translation is increasingly an important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts mitochondrial translation was impaired and ectopic expression of a wild type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel. Collectively, our data demonstrates pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly. |
Grassart, A; Malardé, V; Gobaa, S; Sartori-Rupp, A; Kerns, J; Karalis, K; Marteyn, B; Sansonetti, P; Sauvonnet, N Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection Article de journal Cell Host Microbe, 26 (3), p. 435-444.e434, 2019, ISBN: 31492657. Résumé | Liens | BibTeX | Étiquettes: MARTEYN, MARTEYN Gut-on-Chip Intestine-Chip Organ-on-Chip enterocyte host-pathogen interactions intestine microengineering peristalsis shear stress stretching, Unité ARN @article{, title = {Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection}, author = {A Grassart and V Malardé and S Gobaa and A Sartori-Rupp and J Kerns and K Karalis and B Marteyn and P Sansonetti and N Sauvonnet}, url = {https://pubmed.ncbi.nlm.nih.gov/31492657-bioengineered-human-organ-on-chip-reveals-intestinal-microenvironment-and-mechanical-forces-impacting-shigella-infection/?from_term=Grassart+marteyn&from_pos=1}, doi = {10.1016/j.chom.2019.08.007}, isbn = {31492657}, year = {2019}, date = {2019-01-01}, journal = {Cell Host Microbe}, volume = {26}, number = {3}, pages = {435-444.e434}, abstract = {Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens.}, keywords = {MARTEYN, MARTEYN Gut-on-Chip Intestine-Chip Organ-on-Chip enterocyte host-pathogen interactions intestine microengineering peristalsis shear stress stretching, Unité ARN}, pubstate = {published}, tppubtype = {article} } Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens. |
Desgranges, E; Bronesky, D; Corvaglia, A; François, P; Caballero, C; Prado, L; Toledo-Arana, A; Lasa, I; Moreau, K; Vandenesch, F; Marzi, S; Romby, P; Caldelari, I [RsaI, a multifaceted regulatory RNA, modulates the metabolism of the opportunistic pathogen Staphylococcus aureus] Article de journal Med Sci (Paris), 35 (12), p. 1221-1223, 2019, ISBN: 31903946. Liens | BibTeX | Étiquettes: ROMBY, Unité ARN @article{, title = {[RsaI, a multifaceted regulatory RNA, modulates the metabolism of the opportunistic pathogen Staphylococcus aureus]}, author = {E Desgranges and D Bronesky and A Corvaglia and P François and C Caballero and L Prado and A Toledo-Arana and I Lasa and K Moreau and F Vandenesch and S Marzi and P Romby and I Caldelari}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31903946?dopt=Abstract}, doi = {10.1051/medsci/2019235}, isbn = {31903946}, year = {2019}, date = {2019-01-01}, journal = {Med Sci (Paris)}, volume = {35}, number = {12}, pages = {1221-1223}, keywords = {ROMBY, Unité ARN}, pubstate = {published}, tppubtype = {article} } |
de Wijn, R; Hennig, O; Roche, J; Engilberge, S; Rollet, K; Fernandez-Millan, P; Brillet, K; Betat, H; Mörl, M; Roussel, A; Girard, E; Mueller-Dieckmann, C; Fox, G C; Olieric, V; Gavira, J A; Lorber, B; Sauter, C A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography Article de journal IUCrJ, 6 (Pt 3), p. 454-464, 2019, ISBN: 31098026. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, FRUGIER, FRUGIER ENNIFAR ChipX3 counter-diffusion crystallization ligand soaking macromolecule microfluidics protein structure room temperature seeding serial crystallography trace fluorescent labeling, Unité ARN @article{, title = {A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography}, author = {R de Wijn and O Hennig and J Roche and S Engilberge and K Rollet and P Fernandez-Millan and K Brillet and H Betat and M Mörl and A Roussel and E Girard and C Mueller-Dieckmann and G C Fox and V Olieric and J A Gavira and B Lorber and C Sauter}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31098026?dopt=Abstract}, doi = {10.1107/S2052252519003622}, isbn = {31098026}, year = {2019}, date = {2019-01-01}, journal = {IUCrJ}, volume = {6}, number = {Pt 3}, pages = {454-464}, abstract = {Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.}, keywords = {ENNIFAR, FRUGIER, FRUGIER ENNIFAR ChipX3 counter-diffusion crystallization ligand soaking macromolecule microfluidics protein structure room temperature seeding serial crystallography trace fluorescent labeling, Unité ARN}, pubstate = {published}, tppubtype = {article} } Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept. |
Bronesky, D; Desgranges, E; Corvaglia, A; François, P; Caballero, C J; Prado, L; Toledo-Arana, A; Lasa, I; Moreau, K; Vandenesch, F; Marzi, S; Romby, P; Caldelari, I A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus Article de journal EMBO J, 38 (6), p. e99363, 2019, ISBN: 30760492. Résumé | Liens | BibTeX | Étiquettes: ROMBY, ROMBY sRNA carbon metabolism catabolite control protein A pathogenic bacteria regulatory RNAs translational regulation, Unité ARN @article{, title = {A multifaceted small RNA modulates gene expression upon glucose limitation in \textit{Staphylococcus aureus}}, author = {D Bronesky and E Desgranges and A Corvaglia and P François and C J Caballero and L Prado and A Toledo-Arana and I Lasa and K Moreau and F Vandenesch and S Marzi and P Romby and I Caldelari}, url = {https://www.ncbi.nlm.nih.gov/pubmed/30760492}, doi = {10.15252/embj.201899363}, isbn = {30760492}, year = {2019}, date = {2019-01-01}, journal = {EMBO J}, volume = {38}, number = {6}, pages = {e99363}, abstract = {Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.}, keywords = {ROMBY, ROMBY sRNA carbon metabolism catabolite control protein A pathogenic bacteria regulatory RNAs translational regulation, Unité ARN}, pubstate = {published}, tppubtype = {article} } Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested. |