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2020 |
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. |
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. |
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. |
2019 |
Trachman, 3rd R J; Autour, A; Jeng, S C Y; Abdolahzadeh, A; Andreoni, A; Cojocaru, R; Garipov, R; Dolgosheina, E V; Knutson, J R; Ryckelynck, M; Unrau, P J; Ferré-D'Amaré, A R Structure and functional reselection of the Mango-III fluorogenic RNA aptamer Article de journal Nat Chem Biol, 15 (5), p. 472-479, 2019, ISBN: 30992561. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN @article{, title = {Structure and functional reselection of the Mango-III fluorogenic RNA aptamer}, author = {3rd R J Trachman and A Autour and S C Y Jeng and A Abdolahzadeh and A Andreoni and R Cojocaru and R Garipov and E V Dolgosheina and J R Knutson and M Ryckelynck and P J Unrau and A R Ferré-D'Amaré}, url = {https://www.ncbi.nlm.nih.gov/pubmed/30992561?dopt=Abstract}, doi = {10.1038/s41589-019-0267-9}, isbn = {30992561}, year = {2019}, date = {2019-01-01}, journal = {Nat Chem Biol}, volume = {15}, number = {5}, pages = {472-479}, abstract = {Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.}, keywords = {RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization. |
Ryckelynck, M Development and engineering of artificial RNAs Article de journal Methods, 161 , p. 1-2, 2019, ISBN: 31195094. Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN @article{, title = {Development and engineering of artificial RNAs}, author = {M Ryckelynck}, url = {https://www.ncbi.nlm.nih.gov/pubmed/31195094?dopt=Abstract}, doi = {10.1016/j.ymeth.2019.06.009}, isbn = {31195094}, year = {2019}, date = {2019-01-01}, journal = {Methods}, volume = {161}, pages = {1-2}, keywords = {RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } |
Autour, A; Bouhedda, F; Cubi, R; Ryckelynck, M Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahigh-throughput screening Article de journal Methods, 161 , p. 46-53, 2019, ISBN: 30902664. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, RYCKELYNCK Aptasensors Fluorogenic biosensors High-throughput screening Light-up aptamer Next generation sequencing RNA, Unité ARN @article{, title = {Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahigh-throughput screening}, author = {A Autour and F Bouhedda and R Cubi and M Ryckelynck}, url = {https://www.ncbi.nlm.nih.gov/pubmed/30902664?dopt=Abstract}, doi = {10.1016/j.ymeth.2019.03.015}, isbn = {30902664}, year = {2019}, date = {2019-01-01}, journal = {Methods}, volume = {161}, pages = {46-53}, abstract = {Biosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission.}, keywords = {RYCKELYNCK, RYCKELYNCK Aptasensors Fluorogenic biosensors High-throughput screening Light-up aptamer Next generation sequencing RNA, Unité ARN}, pubstate = {published}, tppubtype = {article} } Biosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission. |
2018 |
Trachman, 3rd R J; Abdolahzadeh, A; Andreoni, A; Cojocaru, R; Knutson, J R; Ryckelynck, M; Unrau, P J; Ferré-D'Amaré, A R Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness Article de journal Biochemistry, 57 (26), p. 3544-3548, 2018, ISBN: 29768001. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN @article{, title = {Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness}, author = {3rd R J Trachman and A Abdolahzadeh and A Andreoni and R Cojocaru and J R Knutson and M Ryckelynck and P J Unrau and A R Ferré-D'Amaré}, url = {https://www.ncbi.nlm.nih.gov/pubmed/29768001?dopt=Abstract}, doi = {10.1021/acs.biochem.8b00399}, isbn = {29768001}, year = {2018}, date = {2018-01-01}, journal = {Biochemistry}, volume = {57}, number = {26}, pages = {3544-3548}, abstract = {Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ~1 nM affinity and enhances its fluorescence by >1,500-fold was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates over 5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases 18% while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analog studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.}, keywords = {RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ~1 nM affinity and enhances its fluorescence by >1,500-fold was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates over 5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases 18% while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analog studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering. |
Bouhedda, F; Autour, A; Ryckelynck, M Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications Article de journal Int J Mol Sci, 19 (1), p. 44, 2018, ISBN: 29295531. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, RYCKELYNCK WESTHOF RNA biosensing fluorescence fluorogen fluorogenic dye gene expression monitoring in vitro evolution light-up aptamer live-cell imaging, Unité ARN @article{, title = {Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications}, author = {F Bouhedda and A Autour and M Ryckelynck}, url = {https://www.ncbi.nlm.nih.gov/pubmed/29295531?dopt=Abstract}, doi = {10.3390/ijms19010044}, isbn = {29295531}, year = {2018}, date = {2018-01-01}, journal = {Int J Mol Sci}, volume = {19}, number = {1}, pages = {44}, abstract = {An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules.}, keywords = {RYCKELYNCK, RYCKELYNCK WESTHOF RNA biosensing fluorescence fluorogen fluorogenic dye gene expression monitoring in vitro evolution light-up aptamer live-cell imaging, Unité ARN}, pubstate = {published}, tppubtype = {article} } An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules. |
Autour, A; Jeng, S C Y; Cawte, A D; Abdolahzadeh, A; Galli, A; Panchapakesan, SSS.; Rueda, D; Ryckelynck, M; Unrau, P J Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells Article de journal Nat Commun, 9 (1), p. 656, 2018, ISBN: 29440634. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, RYCKELYNCK WESTHOF, Unité ARN @article{, title = {Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells}, author = {A Autour and S C Y Jeng and A D Cawte and A Abdolahzadeh and A Galli and SSS. Panchapakesan and D Rueda and M Ryckelynck and P J Unrau}, url = {https://www.ncbi.nlm.nih.gov/pubmed/29440634?dopt=Abstract}, doi = {10.1038/s41467-018-02993-8}, isbn = {29440634}, year = {2018}, date = {2018-01-01}, journal = {Nat Commun}, volume = {9}, number = {1}, pages = {656}, abstract = {Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.}, keywords = {RYCKELYNCK, RYCKELYNCK WESTHOF, Unité ARN}, pubstate = {published}, tppubtype = {article} } Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells. |
2017 |
Fernandez-Millan, P; Autour, A; Ennifar, E; Westhof, E; Ryckelynck, M Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI Article de journal RNA, 23 (12), p. 1788-1795, 2017, ISBN: 28939697. Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ENNIFAR WESTHOF RYCKELYNCK DFHBI Spinach crystal structure fluorescence fluorogenic RNA aptamer, RYCKELYNCK, Unité ARN, WESTHOF @article{, title = {Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI}, author = {P Fernandez-Millan and A Autour and E Ennifar and E Westhof and M Ryckelynck}, url = {https://www.ncbi.nlm.nih.gov/pubmed/28939697?dopt=Abstract}, doi = {10.1261/rna.063008}, isbn = {28939697}, year = {2017}, date = {2017-01-01}, journal = {RNA}, volume = {23}, number = {12}, pages = {1788-1795}, abstract = {Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach comprising five mutations whom the exact role was not deciphered yet. In this work, we co-crystallized a re-engineered version of iSpinach in complex with the DFHBI and solved the x-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach allows shedding some light on the possible function of the different beneficial mutations carried by iSpinach.}, keywords = {ENNIFAR, ENNIFAR WESTHOF RYCKELYNCK DFHBI Spinach crystal structure fluorescence fluorogenic RNA aptamer, RYCKELYNCK, Unité ARN, WESTHOF}, pubstate = {published}, tppubtype = {article} } Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach comprising five mutations whom the exact role was not deciphered yet. In this work, we co-crystallized a re-engineered version of iSpinach in complex with the DFHBI and solved the x-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach allows shedding some light on the possible function of the different beneficial mutations carried by iSpinach. |
2016 |
Autour, A; Westhof, E; Ryckelynck, M iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications. Article de journal Nucleic Acids Res, 44 (6), p. 2491-2500, 2016, ISBN: 26932363. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN, WESTHOF @article{, title = {iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications.}, author = {A Autour and E Westhof and M Ryckelynck}, url = {http://www.ncbi.nlm.nih.gov/pubmed/26932363?dopt=Abstract}, doi = {10.1093/nar/gkw083}, isbn = {26932363}, year = {2016}, date = {2016-01-01}, journal = {Nucleic Acids Res}, volume = {44}, number = {6}, pages = {2491-2500}, abstract = {Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.}, keywords = {RYCKELYNCK, Unité ARN, WESTHOF}, pubstate = {published}, tppubtype = {article} } Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme. |
Matsumura, S; Kun, A; Ryckelynck, M; Coldren, F; Szilagyi, A; Jossinet, F; Rick, C; Nghe, P; Szathmary, E; Griffiths, A D Transient compartmentalization of RNA replicators prevents extinction due to parasites. Article de journal Science, 354 (6317), p. 1293-1296, 2016, ISBN: 27940874. Résumé | Liens | BibTeX | Étiquettes: JOSSINET RYCKELYNCK WESTHOF, RYCKELYNCK, Unité ARN @article{, title = {Transient compartmentalization of RNA replicators prevents extinction due to parasites.}, author = {S Matsumura and A Kun and M Ryckelynck and F Coldren and A Szilagyi and F Jossinet and C Rick and P Nghe and E Szathmary and A D Griffiths}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27940874?dopt=Abstract}, doi = {10.1126/science.aag1582}, isbn = {27940874}, year = {2016}, date = {2016-01-01}, journal = {Science}, volume = {354}, number = {6317}, pages = {1293-1296}, abstract = {The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.}, keywords = {JOSSINET RYCKELYNCK WESTHOF, RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold. |
2015 |
Woronoff, G; Ryckelynck, M; Wessel, J; Schicke, O; Griffiths, A; P., Soumillion Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets. Article de journal Chembiochem, 16 (9), p. 1343-1349, 2015. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN, WESTHOF droplet-based microfluidics enzyme catalysis in vitro translation penicillin acylase protein expression @article{, title = {Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets.}, author = {G Woronoff and M Ryckelynck and J Wessel and O Schicke and A Griffiths and Soumillion P.}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25914325?dopt=Abstract}, doi = {10.1002/cbic.201500087}, year = {2015}, date = {2015-01-01}, journal = {Chembiochem}, volume = {16}, number = {9}, pages = {1343-1349}, abstract = {There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects}, keywords = {RYCKELYNCK, Unité ARN, WESTHOF droplet-based microfluidics enzyme catalysis in vitro translation penicillin acylase protein expression}, pubstate = {published}, tppubtype = {article} } There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects |
Ryckelynck, M; Baudrey, S; Rick, C; Marin, A; Coldren, F; Westhof, E; Griffiths, A D Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions. Article de journal RNA, 21 (3), p. 458-469, 2015, ISBN: 25605963. Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN, WESTHOF, WESTHOF RNA droplet-based microfluidics high-throughput screening in vitro evolution ribozymes @article{, title = {Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions.}, author = {M Ryckelynck and S Baudrey and C Rick and A Marin and F Coldren and E Westhof and A D Griffiths}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25605963?dopt=Abstract}, doi = {10.1261/rna.048033.114}, isbn = {25605963}, year = {2015}, date = {2015-01-01}, journal = {RNA}, volume = {21}, number = {3}, pages = {458-469}, abstract = {In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, kss cat, ∼28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.}, keywords = {RYCKELYNCK, Unité ARN, WESTHOF, WESTHOF RNA droplet-based microfluidics high-throughput screening in vitro evolution ribozymes}, pubstate = {published}, tppubtype = {article} } In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, kss cat, ∼28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction. |
2008 |
Ryckelynck, M; Paulus, C A; Frugier, M Post-Translational Modifications Guard Yeast from Misaspartylation Article de journal Biochemistry, 47 (47), p. 12476-12482, 2008, ISBN: 18956891, (1520-4995 (Electronic) 0006-2960 (Linking) Journal article). Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, RYCKELYNCK, Unité ARN @article{, title = {Post-Translational Modifications Guard Yeast from Misaspartylation}, author = {M Ryckelynck and C A Paulus and M Frugier}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18956885}, isbn = {18956891}, year = {2008}, date = {2008-01-01}, journal = {Biochemistry}, volume = {47}, number = {47}, pages = {12476-12482}, abstract = {Yeast aspartyl-tRNA synthetase (AspRS) is downregulated at the post-transcriptional level. This complex retro-inhibition mechanism causes the cell to equilibrate cellular concentrations of tRNA (Asp), AspRS, and its encoding mRNA. This strategy hinders AspRS accumulation to keep misacylation of heterologous tRNAs under control. Here, the AspRS concentration was increased artificially in vivo but did not generate tRNA (Asn) and/or tRNA (Glu) misaspartylation or the logical consecutive post-translational stress. This work allowed the detection of an additional subtle cellular lock capable of blocking AspRS toxicity. This study revealed the presence of post-translational modifications in the N-terminal extension of AspRS. We hypothesize that by neutralizing the lysine-rich motif contained in this domain, the cell mobilizes an additional strategy that considerably reduces the probability of the enzyme binding and aspartylating noncognate tRNAs and thus harming its own translation.}, note = {1520-4995 (Electronic) 0006-2960 (Linking) Journal article}, keywords = {ERIANI, FRUGIER, RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } Yeast aspartyl-tRNA synthetase (AspRS) is downregulated at the post-transcriptional level. This complex retro-inhibition mechanism causes the cell to equilibrate cellular concentrations of tRNA (Asp), AspRS, and its encoding mRNA. This strategy hinders AspRS accumulation to keep misacylation of heterologous tRNAs under control. Here, the AspRS concentration was increased artificially in vivo but did not generate tRNA (Asn) and/or tRNA (Glu) misaspartylation or the logical consecutive post-translational stress. This work allowed the detection of an additional subtle cellular lock capable of blocking AspRS toxicity. This study revealed the presence of post-translational modifications in the N-terminal extension of AspRS. We hypothesize that by neutralizing the lysine-rich motif contained in this domain, the cell mobilizes an additional strategy that considerably reduces the probability of the enzyme binding and aspartylating noncognate tRNAs and thus harming its own translation. |
2005 |
Ryckelynck, M; Masquida, B; Giege, R; Frugier, M An intricate RNA structure with two tRNA-derived motifs directs complex formation between yeast aspartyl-tRNA synthetase and its mRNA Article de journal J Mol Biol, 354 (3), p. 614-629, 2005, ISBN: 16257416, (0022-2836 (Print) Journal Article). Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, Messenger/*chemistry/*metabolism RNA, Molecular Molecular Sequence Data Nucleic Acid Conformation Protein Structure, Non-U.S. Gov't Saccharomyces cerevisiae/*enzymology/*genetics Sequence Alignment Sequence Homology, Nucleic Acid Solubility, RYCKELYNCK, Tertiary RNA, Transfer/*chemistry/*metabolism Research Support, Unité ARN, WESTHOF GIEGE FRUGIER Aspartate-tRNA Ligase/genetics/*metabolism Base Sequence DNA Footprinting Gene Deletion Models @article{, title = {An intricate RNA structure with two tRNA-derived motifs directs complex formation between yeast aspartyl-tRNA synthetase and its mRNA}, author = {M Ryckelynck and B Masquida and R Giege and M Frugier}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16257416}, isbn = {16257416}, year = {2005}, date = {2005-01-01}, journal = {J Mol Biol}, volume = {354}, number = {3}, pages = {614-629}, abstract = {Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase.}, note = {0022-2836 (Print) Journal Article}, keywords = {ERIANI, FRUGIER, Messenger/*chemistry/*metabolism RNA, Molecular Molecular Sequence Data Nucleic Acid Conformation Protein Structure, Non-U.S. Gov't Saccharomyces cerevisiae/*enzymology/*genetics Sequence Alignment Sequence Homology, Nucleic Acid Solubility, RYCKELYNCK, Tertiary RNA, Transfer/*chemistry/*metabolism Research Support, Unité ARN, WESTHOF GIEGE FRUGIER Aspartate-tRNA Ligase/genetics/*metabolism Base Sequence DNA Footprinting Gene Deletion Models}, pubstate = {published}, tppubtype = {article} } Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase. |
Ryckelynck, M; Giege, R; Frugier, M tRNAs and tRNA mimics as cornerstones of aminoacyl-tRNA synthetase regulations Article de journal Biochimie, 87 (9-10), p. 835-845, 2005, ISBN: 15925436, (0300-9084 Journal article). Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, GIEGE FRUGIER, RYCKELYNCK, Unité ARN @article{, title = {tRNAs and tRNA mimics as cornerstones of aminoacyl-tRNA synthetase regulations}, author = {M Ryckelynck and R Giege and M Frugier}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15925436}, isbn = {15925436}, year = {2005}, date = {2005-01-01}, journal = {Biochimie}, volume = {87}, number = {9-10}, pages = {835-845}, abstract = {Structural plasticity of transfer RNA (tRNA) molecules is essential for interactions with their biological partners in aminoacylation reactions and during ribosome-dependent protein synthesis. This holds true when tRNAs are recruited for other functions than translation. Here we review regulation pathways where tRNAs and tRNA mimics play a pivotal role. We further discuss the importance of the identity signals used in aminoacylation that are also required to specify regulatory mechanisms. Such mechanisms are diverse and intervene in transcription, splicing and translation. Altogether, the review highlights the many manners architectural features of tRNA were selected by evolution to control biological key processes.}, note = {0300-9084 Journal article}, keywords = {ERIANI, FRUGIER, GIEGE FRUGIER, RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } Structural plasticity of transfer RNA (tRNA) molecules is essential for interactions with their biological partners in aminoacylation reactions and during ribosome-dependent protein synthesis. This holds true when tRNAs are recruited for other functions than translation. Here we review regulation pathways where tRNAs and tRNA mimics play a pivotal role. We further discuss the importance of the identity signals used in aminoacylation that are also required to specify regulatory mechanisms. Such mechanisms are diverse and intervene in transcription, splicing and translation. Altogether, the review highlights the many manners architectural features of tRNA were selected by evolution to control biological key processes. |
Frugier, M; Ryckelynck, M; Giege, R tRNA-balanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway Article de journal EMBO Rep, 6 (9), p. 860-865, 2005, ISBN: 16113655, (1469-221x Journal article). Résumé | Liens | BibTeX | Étiquettes: ERIANI, FRUGIER, GIEGE FRUGIER, RYCKELYNCK, Unité ARN @article{, title = {tRNA-balanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway}, author = {M Frugier and M Ryckelynck and R Giege}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16113655}, isbn = {16113655}, year = {2005}, date = {2005-01-01}, journal = {EMBO Rep}, volume = {6}, number = {9}, pages = {860-865}, abstract = {Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation.}, note = {1469-221x Journal article}, keywords = {ERIANI, FRUGIER, GIEGE FRUGIER, RYCKELYNCK, Unité ARN}, pubstate = {published}, tppubtype = {article} } Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation. |
2003 |
Ryckelynck, M; Giege, R; Frugier, M Yeast tRNA(Asp) charging accuracy is threatened by the N-terminal extension of aspartyl-tRNA synthetase Article de journal J Biol Chem, 278 (11), p. 9683-9690, 2003, ISBN: 12486031, (0021-9258 Journal Article). Résumé | Liens | BibTeX | Étiquettes: Amino Acid Motifs Aspartate-tRNA Ligase/*metabolism Aspartic Acid/chemistry Base Sequence Codon Escherichia coli/metabolism Kinetics Molecular Sequence Data Nucleic Acid Conformation Nucleic Acids/chemistry Protein Structure, Asp/*chemistry Support, ERIANI, FRUGIER, Messenger/metabolism RNA, Non-U.S. Gov't Yeasts/metabolism, RYCKELYNCK, Secondary Protein Structure, Tertiary RNA, Transfer, Unité ARN @article{, title = {Yeast tRNA(Asp) charging accuracy is threatened by the N-terminal extension of aspartyl-tRNA synthetase}, author = {M Ryckelynck and R Giege and M Frugier}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12486031}, isbn = {12486031}, year = {2003}, date = {2003-01-01}, journal = {J Biol Chem}, volume = {278}, number = {11}, pages = {9683-9690}, abstract = {This study evaluates the role of the N-terminal extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation. The presence of an RNA-binding motif in this extension, conserved in eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific tRNA binding properties to this enzyme. Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast. Using in vitro transcripts mutated at identity positions, it is demonstrated that the extension grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging. Yeast tRNA(Glu(UUC)) and tRNA(Asn(GUU)) were identified as the most easily mischarged tRNA species. Both have a G at the discriminator position, and their anticodon differs only by one change from the GUC aspartate anticodon.}, note = {0021-9258 Journal Article}, keywords = {Amino Acid Motifs Aspartate-tRNA Ligase/*metabolism Aspartic Acid/chemistry Base Sequence Codon Escherichia coli/metabolism Kinetics Molecular Sequence Data Nucleic Acid Conformation Nucleic Acids/chemistry Protein Structure, Asp/*chemistry Support, ERIANI, FRUGIER, Messenger/metabolism RNA, Non-U.S. Gov't Yeasts/metabolism, RYCKELYNCK, Secondary Protein Structure, Tertiary RNA, Transfer, Unité ARN}, pubstate = {published}, tppubtype = {article} } This study evaluates the role of the N-terminal extension from yeast aspartyl-tRNA synthetase in tRNA aspartylation. The presence of an RNA-binding motif in this extension, conserved in eukaryotic class IIb aminoacyl-tRNA synthetases, provides nonspecific tRNA binding properties to this enzyme. Here, it is assumed that the additional contacts the 70 amino acid-long appendix of aspartyl-tRNA synthetase makes with tRNA could be important in expression of aspartate identity in yeast. Using in vitro transcripts mutated at identity positions, it is demonstrated that the extension grants better aminoacylation efficiency but reduced specificity to the synthetase, increasing considerably the risk of noncognate tRNA mischarging. Yeast tRNA(Glu(UUC)) and tRNA(Asn(GUU)) were identified as the most easily mischarged tRNA species. Both have a G at the discriminator position, and their anticodon differs only by one change from the GUC aspartate anticodon. |