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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 Journal Article RNA, pp. in press, 2020, ISBN: 32994183. Abstract | Links | BibTeX | Tags: 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 Journal Article RNA, pp. in press, 2020, ISBN: 32737189. Abstract | Links | BibTeX | Tags: 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). |
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 Journal Article Front Microbiol, pp. in press, 2020. Links | BibTeX | Tags: 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} } |
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 , pp. 101-110, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006310. Abstract | Links | BibTeX | Tags: 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. |
Bernacchi, S Dynamic Light Scattering Analysis on RNA Associated to Proteins Book Chapter Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , pp. 31-39, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006306. Abstract | Links | BibTeX | Tags: 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. |
Bernacchi, S; Ennifar, E Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, 2113 , pp. 237-250, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006318. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article Nat Chem Biol, 16 (1), pp. 69-76, 2020, ISBN: 31636432. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article Biophys J, 119 (2), pp. 419-433, 2020, ISBN: 32574557. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article RNA, 26 (9), pp. 1184-1197, 2020, ISBN: 32430362. Abstract | Links | BibTeX | Tags: 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 |
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 Journal Article Proc Natl Acad Sci U S A, 117 (35), pp. 21288-21298, 2020, ISBN: 32817544. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article Crystals, 10 (2), pp. 65, 2020. Abstract | Links | BibTeX | Tags: 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. |
Desgranges, E; Caldelari, I; Marzi, S; Lalaouna, D Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs Journal Article Biochim Biophys Acta Gene Regul Mech, 1863 (3), pp. 194506, 2020, ISBN: 32068131. Abstract | Links | BibTeX | Tags: 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. |
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) Journal Article Angew Chem Int Ed Engl, 59 (17), pp. 6881-6886, 2020, ISBN: 31999864. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article Mol Microbiol, 113 (3), pp. 603-612, 2020, ISBN: 31705780. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article EMBO J, pp. in press, 2020, ISBN: 32812257. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article Semin Cell Dev Biol, pp. in press, 2020, ISBN: 32847707. Abstract | Links | BibTeX | Tags: 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. |
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 Gut, 69 (2), pp. 380-392, 2020, ISBN: 31076402. Abstract | Links | BibTeX | Tags: 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. |
Injarabian, L; Scherlinger, M; Devin, A; Ransac, S; Lykkesfeldt, J; Marteyn, B S Ascorbate maintains a low plasma oxygen level Journal Article Sci Rep, 10 (1), pp. 10659, 2020, ISBN: 32606354. Abstract | Links | BibTeX | Tags: 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. |
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 Journal Article J Virol, 94 (20), pp. e01035-01020, 2020, ISBN: 32727879. Abstract | Links | BibTeX | Tags: 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 immun |