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2019
Florentz C, Giege R
History of tRNA research in Strasbourg Journal Article
In: IUBMB Life, vol. 71, no. 8, pp. 1066-1087, 2019, ISBN: 31185141.
Abstract | Links | BibTeX | Tags: FLORENTZ, GIEGE, Strasbourg epistemology evolution genetic code history structural biology transfer RNA translation, Unité ARN
@article{,
title = {History of tRNA research in Strasbourg},
author = {C Florentz and R Giege},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31185141?dopt=Abstract},
doi = {10.1002/iub.2079},
isbn = {31185141},
year = {2019},
date = {2019-01-01},
journal = {IUBMB Life},
volume = {71},
number = {8},
pages = {1066-1087},
abstract = {The tRNA molecules, in addition to translating the genetic code into protein and defining the second genetic code via their aminoacylation by aminoacyl-tRNA synthetases, act in many other cellular functions and dysfunctions. This article, illustrated by personal souvenirs, covers the history of ~60 years tRNA research in Strasbourg. Typical examples point up how the work in Strasbourg was a two-way street, influenced by and at the same time influencing investigators outside of France. All along, research in Strasbourg has nurtured the structural and functional diversity of tRNA. It produced massive sequence and crystallographic data on tRNA and its partners, thereby leading to a deeper physicochemical understanding of tRNA architecture, dynamics, and identity. Moreover, it emphasized the role of nucleoside modifications and in the last two decades, highlighted tRNA idiosyncrasies in plants and organelles, together with cellular and health-focused aspects. The tRNA field benefited from a rich local academic heritage and a strong support by both university and CNRS. Its broad interlinks to the worldwide community of tRNA researchers opens to an exciting future.},
keywords = {FLORENTZ, GIEGE, Strasbourg epistemology evolution genetic code history structural biology transfer RNA translation, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The tRNA molecules, in addition to translating the genetic code into protein and defining the second genetic code via their aminoacylation by aminoacyl-tRNA synthetases, act in many other cellular functions and dysfunctions. This article, illustrated by personal souvenirs, covers the history of ~60 years tRNA research in Strasbourg. Typical examples point up how the work in Strasbourg was a two-way street, influenced by and at the same time influencing investigators outside of France. All along, research in Strasbourg has nurtured the structural and functional diversity of tRNA. It produced massive sequence and crystallographic data on tRNA and its partners, thereby leading to a deeper physicochemical understanding of tRNA architecture, dynamics, and identity. Moreover, it emphasized the role of nucleoside modifications and in the last two decades, highlighted tRNA idiosyncrasies in plants and organelles, together with cellular and health-focused aspects. The tRNA field benefited from a rich local academic heritage and a strong support by both university and CNRS. Its broad interlinks to the worldwide community of tRNA researchers opens to an exciting future.
2018
Jühling T, Duchardt-Ferner E, Bonin S, Wöhnert J, Pütz J, Florentz C, Betat H, Sauter C, Mörl M
Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax Journal Article
In: Nucleic Acids Res, vol. 46, no. 17, pp. 9170-9180, 2018, ISBN: 29986062.
Abstract | Links | BibTeX | Tags: FLORENTZ, FRUGIER, Unité ARN
@article{,
title = {Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax},
author = {T Jühling and E Duchardt-Ferner and S Bonin and J Wöhnert and J Pütz and C Florentz and H Betat and C Sauter and M Mörl},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29986062?dopt=Abstract},
doi = {10.1093/nar/gky593},
isbn = {29986062},
year = {2018},
date = {2018-01-01},
journal = {Nucleic Acids Res},
volume = {46},
number = {17},
pages = {9170-9180},
abstract = {As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.},
keywords = {FLORENTZ, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.
2015
Simon M, Richard E M, Wang X, Shahzad M, Huang V H, Qaiser T A, Potluri P, Mahl S E, Davila A, Nazli S, Hancock S, Yu M, Gargus J, Chang R, Al-Sheqaih N, Newman W G, Abdenur J, Starr A, Hegde R, Dorn T, Busch A, Park E, Wu J F, Schwenzer H, Flierl A, Florentz C, Sissler M, Khan S N, Li R, Guan M X, Friedman T B, Wu D K, Procaccio V, Riazuddin S, Wallace D C, Ahmed Z M, Huang T, Riazuddin S
Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome. Journal Article
In: PLoS Genet, vol. 11, no. 3, pp. e1005097, 2015, ISBN: 25807530.
Abstract | Links | BibTeX | Tags: FLORENTZ, SISSLER, Unité ARN
@article{,
title = {Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome.},
author = {M Simon and E M Richard and X Wang and M Shahzad and V H Huang and T A Qaiser and P Potluri and S E Mahl and A Davila and S Nazli and S Hancock and M Yu and J Gargus and R Chang and N Al-Sheqaih and W G Newman and J Abdenur and A Starr and R Hegde and T Dorn and A Busch and E Park and J F Wu and H Schwenzer and A Flierl and C Florentz and M Sissler and S N Khan and R Li and M X Guan and T B Friedman and D K Wu and V Procaccio and S Riazuddin and D C Wallace and Z M Ahmed and T Huang and S Riazuddin},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25807530?dopt=Abstract},
doi = {10.1371/journal.pgen.1005097. eCollection 2015},
isbn = {25807530},
year = {2015},
date = {2015-01-01},
journal = {PLoS Genet},
volume = {11},
number = {3},
pages = {e1005097},
abstract = {Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.},
keywords = {FLORENTZ, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.
2014
Schwenzer H, Zoll J, Florentz C, Sissler M
Pathogenic Implications of Human Mitochondrial Aminoacyl-tRNA Synthetases. Journal Article
In: Top Curr Chem, vol. 344, pp. 247-292, 2014, ISBN: 23824528.
Abstract | Links | BibTeX | Tags: Aminoacyl-tRNA synthetase Human mitochondrial disorders Pathology-related mutations Respiratory chain defects, FLORENTZ, SISSLER, Unité ARN
@article{,
title = {Pathogenic Implications of Human Mitochondrial Aminoacyl-tRNA Synthetases.},
author = {H Schwenzer and J Zoll and C Florentz and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/pubmed/23824528},
doi = {10.1007/128_2013_457},
isbn = {23824528},
year = {2014},
date = {2014-01-01},
journal = {Top Curr Chem},
volume = {344},
pages = {247-292},
abstract = {Mitochondria are considered as the powerhouse of eukaryotic cells. They host several central metabolic processes fueling the oxidative phosphorylation pathway (OXPHOS) that produces ATP from its precursors ADP and inorganic phosphate Pi (PPi). The respiratory chain complexes responsible for the OXPHOS pathway are formed from complementary sets of protein subunits encoded by the nuclear genome and the mitochondrial genome, respectively. The expression of the mitochondrial genome requires a specific and fully active translation machinery from which aminoacyl-tRNA synthetases (aaRSs) are key actors. Whilst the macromolecules involved in mammalian mitochondrial translation have been under investigation for many years, there has been an explosion of interest in human mitochondrial aaRSs (mt-aaRSs) since the discovery of a large (and growing) number of mutations in these genes that are linked to a variety of neurodegenerative disorders. Herein we will review the present knowledge on mt-aaRSs in terms of their biogenesis, their connection to mitochondrial respiration, i.e., the respiratory chain (RC) complexes, and to the mitochondrial translation machinery. The pathology-related mutations detected so far are described, with special attention given to their impact on mt-aaRSs biogenesis, functioning, and/or subsequent activities. The collected data to date shed light on the diverse routes that are linking primary molecular possible impact of a mutation to its phenotypic expression. It is envisioned that a variety of mechanisms, inside and outside the translation machinery, would play a role on the heterogeneous manifestations of mitochondrial disorders.},
keywords = {Aminoacyl-tRNA synthetase Human mitochondrial disorders Pathology-related mutations Respiratory chain defects, FLORENTZ, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mitochondria are considered as the powerhouse of eukaryotic cells. They host several central metabolic processes fueling the oxidative phosphorylation pathway (OXPHOS) that produces ATP from its precursors ADP and inorganic phosphate Pi (PPi). The respiratory chain complexes responsible for the OXPHOS pathway are formed from complementary sets of protein subunits encoded by the nuclear genome and the mitochondrial genome, respectively. The expression of the mitochondrial genome requires a specific and fully active translation machinery from which aminoacyl-tRNA synthetases (aaRSs) are key actors. Whilst the macromolecules involved in mammalian mitochondrial translation have been under investigation for many years, there has been an explosion of interest in human mitochondrial aaRSs (mt-aaRSs) since the discovery of a large (and growing) number of mutations in these genes that are linked to a variety of neurodegenerative disorders. Herein we will review the present knowledge on mt-aaRSs in terms of their biogenesis, their connection to mitochondrial respiration, i.e., the respiratory chain (RC) complexes, and to the mitochondrial translation machinery. The pathology-related mutations detected so far are described, with special attention given to their impact on mt-aaRSs biogenesis, functioning, and/or subsequent activities. The collected data to date shed light on the diverse routes that are linking primary molecular possible impact of a mutation to its phenotypic expression. It is envisioned that a variety of mechanisms, inside and outside the translation machinery, would play a role on the heterogeneous manifestations of mitochondrial disorders.
Schwenzer H, Scheper G C, Zorn N, Moulinier L, Gaudry A, Leize E, Martin F, Florentz C, Poch O, Sissler M
Released selective pressure on a structural domain gives new insights on the functional relaxation of mitochondrial aspartyl-tRNA synthetase. Journal Article
In: Biochimie, vol. 100, pp. 18-26, 2014, ISBN: 24120687.
Abstract | Links | BibTeX | Tags: Aminoacyl-tRNA synthetase Bioinformatics MTS Mitochondria Molecular biology Translation aaRS aminoacyl-tRNA synthetase (specificity is indicated by the name of the amino acid abbreviated in a three-letter code, e.g. AspRS stands for aspartyl-tRNA synthetase) mitochondrial mitochondrial targeting sequence mt, ERIANI, FLORENTZ, SISSLER, transferred to the cognate tRNA, Unité ARN
@article{,
title = {Released selective pressure on a structural domain gives new insights on the functional relaxation of mitochondrial aspartyl-tRNA synthetase.},
author = {H Schwenzer and G C Scheper and N Zorn and L Moulinier and A Gaudry and E Leize and F Martin and C Florentz and O Poch and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/pubmed/24120687},
doi = {10.1016/j.biochi.2013.09.027},
isbn = {24120687},
year = {2014},
date = {2014-01-01},
journal = {Biochimie},
volume = {100},
pages = {18-26},
abstract = {Mammalian mitochondrial aminoacyl-tRNA synthetases are nuclear-encoded enzymes that are essential for mitochondrial protein synthesis. Due to an endosymbiotic origin of the mitochondria, many of them share structural domains with homologous bacterial enzymes of same specificity. This is also the case for human mitochondrial aspartyl-tRNA synthetase (AspRS) that shares the so-called bacterial insertion domain with bacterial homologs. The function of this domain in the mitochondrial proteins is unclear. Here, we show by bioinformatic analyses that the sequences coding for the bacterial insertion domain are less conserved in opisthokont and protist than in bacteria and viridiplantae. The divergence suggests a loss of evolutionary pressure on this domain for non-plant mitochondrial AspRSs. This discovery is further connected with the herein described occurrence of alternatively spliced transcripts of the mRNAs coding for some mammalian mitochondrial AspRSs. Interestingly, the spliced transcripts alternately lack one of the four exons that code for the bacterial insertion domain. Although we showed that the human alternative transcript is present in all tested tissues; co-exists with the full-length form, possesses 5'- and 3'-UTRs, a poly-A tail and is bound to polysomes, we were unable to detect the corresponding protein. The relaxed selective pressure combined with the occurrence of alternative splicing, involving a single structural sub-domain, favors the hypothesis of the loss of function of this domain for AspRSs of mitochondrial location. This evolutionary divergence is in line with other characteristics, established for the human mt-AspRS, that indicate a functional relaxation of non-viridiplantae mt-AspRSs when compared to bacterial and plant ones, despite their common ancestry.},
keywords = {Aminoacyl-tRNA synthetase Bioinformatics MTS Mitochondria Molecular biology Translation aaRS aminoacyl-tRNA synthetase (specificity is indicated by the name of the amino acid abbreviated in a three-letter code, e.g. AspRS stands for aspartyl-tRNA synthetase) mitochondrial mitochondrial targeting sequence mt, ERIANI, FLORENTZ, SISSLER, transferred to the cognate tRNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mammalian mitochondrial aminoacyl-tRNA synthetases are nuclear-encoded enzymes that are essential for mitochondrial protein synthesis. Due to an endosymbiotic origin of the mitochondria, many of them share structural domains with homologous bacterial enzymes of same specificity. This is also the case for human mitochondrial aspartyl-tRNA synthetase (AspRS) that shares the so-called bacterial insertion domain with bacterial homologs. The function of this domain in the mitochondrial proteins is unclear. Here, we show by bioinformatic analyses that the sequences coding for the bacterial insertion domain are less conserved in opisthokont and protist than in bacteria and viridiplantae. The divergence suggests a loss of evolutionary pressure on this domain for non-plant mitochondrial AspRSs. This discovery is further connected with the herein described occurrence of alternatively spliced transcripts of the mRNAs coding for some mammalian mitochondrial AspRSs. Interestingly, the spliced transcripts alternately lack one of the four exons that code for the bacterial insertion domain. Although we showed that the human alternative transcript is present in all tested tissues; co-exists with the full-length form, possesses 5'- and 3'-UTRs, a poly-A tail and is bound to polysomes, we were unable to detect the corresponding protein. The relaxed selective pressure combined with the occurrence of alternative splicing, involving a single structural sub-domain, favors the hypothesis of the loss of function of this domain for AspRSs of mitochondrial location. This evolutionary divergence is in line with other characteristics, established for the human mt-AspRS, that indicate a functional relaxation of non-viridiplantae mt-AspRSs when compared to bacterial and plant ones, despite their common ancestry.
2012
Juhling F, Putz J, Florentz C, Stadler P F
Armless mitochondrial tRNAs in enoplea (nematoda). Journal Article
In: RNA Biol, vol. 9, no. 9, pp. 1161 - 1166, 2012, ISBN: 23018779.
Abstract | Links | BibTeX | Tags: Enoplea Mermithidae Mitochondrial tRNA D-loop T-loop, FLORENTZ, Unité ARN
@article{,
title = {Armless mitochondrial tRNAs in enoplea (nematoda).},
author = {F Juhling and J Putz and C Florentz and P F Stadler},
url = {http://www.ncbi.nlm.nih.gov/pubmed/23018779?dopt=Abstract},
doi = {10.4161/rna.21630},
isbn = {23018779},
year = {2012},
date = {2012-01-01},
journal = {RNA Biol},
volume = {9},
number = {9},
pages = {1161 - 1166},
abstract = {The mitochondrial genome of metazoan animal typically encodes 22 tRNAs. Nematode mt-tRNAs normally lack the T-stem and instead feature a replacement loop. In the class Enoplea, putative mt-tRNAs that are even further reduced have been predicted to lack both the T- and the D-arm. Here we investigate these tRNA candidates in detail. Three lines of computational evidence support that they are indeed minimal functional mt-tRNAs: (1) the high level of conservation of both sequence and secondary structure, (2) the perfect preservation of the anticodons, and (3) the persistence of these sequence elements throughout several genome rearrangements that place them between different flanking genes.},
keywords = {Enoplea Mermithidae Mitochondrial tRNA D-loop T-loop, FLORENTZ, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The mitochondrial genome of metazoan animal typically encodes 22 tRNAs. Nematode mt-tRNAs normally lack the T-stem and instead feature a replacement loop. In the class Enoplea, putative mt-tRNAs that are even further reduced have been predicted to lack both the T- and the D-arm. Here we investigate these tRNA candidates in detail. Three lines of computational evidence support that they are indeed minimal functional mt-tRNAs: (1) the high level of conservation of both sequence and secondary structure, (2) the perfect preservation of the anticodons, and (3) the persistence of these sequence elements throughout several genome rearrangements that place them between different flanking genes.
Juhling F, Putz J, Bernt M, Donath A, Middendorf M, Florentz C, Stadler P F
In: Nucleic Acids Res, vol. 40, no. 7, pp. 2833-2845, 2012, ISBN: 22139921, (First published online: December 1, 2011).
Abstract | Links | BibTeX | Tags: FLORENTZ, Unité ARN
@article{,
title = {Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements.},
author = {F Juhling and J Putz and M Bernt and A Donath and M Middendorf and C Florentz and P F Stadler},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22139921},
doi = {10.1093/nar/gkr1131},
isbn = {22139921},
year = {2012},
date = {2012-01-01},
journal = {Nucleic Acids Res},
volume = {40},
number = {7},
pages = {2833-2845},
abstract = {Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit 'bizarre' secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading 'pseudogenes', even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.},
note = {First published online: December 1, 2011},
keywords = {FLORENTZ, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit 'bizarre' secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading 'pseudogenes', even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.
Giege R, Juhling F, Putz J, Stadler P, Sauter C, Florentz C
Structure of transfer RNAs: similarity and variability Journal Article
In: Wiley Interdiscip Rev RNA, vol. 3, no. 1, pp. 37-61, 2012, ISBN: 21957054.
Abstract | Links | BibTeX | Tags: FLORENTZ, FRUGIER, Unité ARN
@article{,
title = {Structure of transfer RNAs: similarity and variability},
author = {R Giege and F Juhling and J Putz and P Stadler and C Sauter and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/pubmed/21957054?dopt=Abstrac},
doi = {10.1002/wrna.103},
isbn = {21957054},
year = {2012},
date = {2012-01-01},
journal = {Wiley Interdiscip Rev RNA},
volume = {3},
number = {1},
pages = {37-61},
abstract = {Transfer RNAs (tRNAs) are ancient molecules whose origin goes back to the beginning of life on Earth. Key partners in the ribosome-translation machinery, tRNAs read genetic information on messenger RNA and deliver codon specified amino acids attached to their distal 3′-extremity for peptide bond synthesis on the ribosome. In addition to this universal function, tRNAs participate in a wealth of other biological processes and undergo intricate maturation events. Our understanding of tRNA biology has been mainly phenomenological, but ongoing progress in structural biology is giving a robust physico-chemical basis that explains many facets of tRNA functions. Advanced sequence analysis of tRNA genes and their RNA transcripts have uncovered rules that underly tRNA 2D folding and 3D L-shaped architecture, as well as provided clues about their evolution. The increasing number of X-ray structures of free, protein- and ribosome-bound tRNA, reveal structural details accounting for the identity of the 22 tRNA families (one for each proteinogenic amino acid) and for the multifunctionality of a given family. Importantly, the structural role of post-transcriptional tRNA modifications is being deciphered. On the other hand, the plasticity of tRNA structure during function has been illustrated using a variety of technical approaches that allow dynamical insights. The large range of structural properties not only allows tRNAs to be the key actors of translation, but also sustain a diversity of unrelated functions from which only a few have already been pinpointed. Many surprises can still be expected. WIREs RNA 2012, 3:3761. doi: 10.1002/wrna.103},
keywords = {FLORENTZ, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Transfer RNAs (tRNAs) are ancient molecules whose origin goes back to the beginning of life on Earth. Key partners in the ribosome-translation machinery, tRNAs read genetic information on messenger RNA and deliver codon specified amino acids attached to their distal 3′-extremity for peptide bond synthesis on the ribosome. In addition to this universal function, tRNAs participate in a wealth of other biological processes and undergo intricate maturation events. Our understanding of tRNA biology has been mainly phenomenological, but ongoing progress in structural biology is giving a robust physico-chemical basis that explains many facets of tRNA functions. Advanced sequence analysis of tRNA genes and their RNA transcripts have uncovered rules that underly tRNA 2D folding and 3D L-shaped architecture, as well as provided clues about their evolution. The increasing number of X-ray structures of free, protein- and ribosome-bound tRNA, reveal structural details accounting for the identity of the 22 tRNA families (one for each proteinogenic amino acid) and for the multifunctionality of a given family. Importantly, the structural role of post-transcriptional tRNA modifications is being deciphered. On the other hand, the plasticity of tRNA structure during function has been illustrated using a variety of technical approaches that allow dynamical insights. The large range of structural properties not only allows tRNAs to be the key actors of translation, but also sustain a diversity of unrelated functions from which only a few have already been pinpointed. Many surprises can still be expected. WIREs RNA 2012, 3:3761. doi: 10.1002/wrna.103
Gaudry A, Lorber B, Neuenfeldt A, Sauter C, Florentz C, Sissler M
Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein. Journal Article
In: Protein Eng Des Sel, vol. 25, no. 9, pp. 473-481, 2012, ISBN: 22871419.
Abstract | Links | BibTeX | Tags: FLORENTZ, FRUGIER, SISSLER, Unité ARN
@article{,
title = {Re-designed N-terminus enhances expression, solubility and crystallizability of mitochondrial protein.},
author = {A Gaudry and B Lorber and A Neuenfeldt and C Sauter and C Florentz and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22871419?report=&dispmax=200&tool=PubCrawler_2.23},
isbn = {22871419},
year = {2012},
date = {2012-01-01},
journal = {Protein Eng Des Sel},
volume = {25},
number = {9},
pages = {473-481},
abstract = {Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5'-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences.},
keywords = {FLORENTZ, FRUGIER, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5'-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences.
Fender A, Gaudry A, Jühling F, Sissler M, Florentz C
Adaptation of aminoacylation identity rules to mammalian mitochondria. Journal Article
In: Biochimie, vol. 94, no. 5, pp. 1090-1097, 2012, ISBN: 22402012, (Available online 1 March 2012).
Abstract | Links | BibTeX | Tags: FLORENTZ, Identity elements Mitochondrial tRNA Cross-aminoacylation Structural plasticity, SISSLER, Unité ARN
@article{,
title = {Adaptation of aminoacylation identity rules to mammalian mitochondria.},
author = {A Fender and A Gaudry and F Jühling and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/pubmed/22402012?dopt=Abstract},
doi = {10.1016/j.biochi.2012.02.030},
isbn = {22402012},
year = {2012},
date = {2012-01-01},
journal = {Biochimie},
volume = {94},
number = {5},
pages = {1090-1097},
abstract = {Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA(Asp) variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.},
note = {Available online 1 March 2012},
keywords = {FLORENTZ, Identity elements Mitochondrial tRNA Cross-aminoacylation Structural plasticity, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA(Asp) variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.
2011
Schellenberger P, Sauter C, Lorber B, Bron P, Trapani S, Bergdoll M, Marmonier A, Schmitt-Kelchinger C, Lemaire O, Demangeat G, Ritzenthaler C
Structural insights into viral determinants of nematode mediated Grapevine fanleaf virus transmission. Journal Article
In: PLoS Pathog, vol. 7, no. 5, pp. e1002034, 2011, ISBN: 21625570.
Abstract | Links | BibTeX | Tags: FLORENTZ, FRUGIER, Unité ARN
@article{,
title = {Structural insights into viral determinants of nematode mediated Grapevine fanleaf virus transmission.},
author = {P Schellenberger and C Sauter and B Lorber and P Bron and S Trapani and M Bergdoll and A Marmonier and C Schmitt-Kelchinger and O Lemaire and G Demangeat and C Ritzenthaler},
url = {http://www.ncbi.nlm.nih.gov/pubmed/21625570},
doi = {10.1371/journal.ppat.1002034},
isbn = {21625570},
year = {2011},
date = {2011-01-01},
journal = {PLoS Pathog},
volume = {7},
number = {5},
pages = {e1002034},
abstract = {Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.},
keywords = {FLORENTZ, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.
Schellenberger P, Demangeat G, Lemaire O, Ritzenthaler C, Bergdoll M, Olieric V, Sauter C, Lorber B
Strategies for the crystallization of viruses: Using phase diagrams and gels to produce 3D crystals of Grapevine fanleaf virus Journal Article
In: J Struct Biol, vol. 174, no. 2, pp. 344-351, 2011, ISSN: 1095-8657 (Electronic) 1047-8477 (Linking), (Schellenberger, Pascale Demangeat, Gerard Lemaire, Olivier Ritzenthaler, Christophe Bergdoll, Marc Olieric, Vincent Sauter, Claude Lorber, Bernard United States Journal of structural biology J Struct Biol. 2011 May;174(2):344-51. Epub 2011 Feb 23.).
Abstract | Links | BibTeX | Tags: FLORENTZ, FRUGIER, Unité ARN
@article{,
title = {Strategies for the crystallization of viruses: Using phase diagrams and gels to produce 3D crystals of Grapevine fanleaf virus},
author = {P Schellenberger and G Demangeat and O Lemaire and C Ritzenthaler and M Bergdoll and V Olieric and C Sauter and B Lorber},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21352920},
doi = {10.1016/j.jsb.2011.02.007},
issn = {1095-8657 (Electronic) 1047-8477 (Linking)},
year = {2011},
date = {2011-01-01},
journal = {J Struct Biol},
volume = {174},
number = {2},
pages = {344-351},
abstract = {The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly(297)Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7A. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3A by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies.},
note = {Schellenberger, Pascale
Demangeat, Gerard
Lemaire, Olivier
Ritzenthaler, Christophe
Bergdoll, Marc
Olieric, Vincent
Sauter, Claude
Lorber, Bernard
United States
Journal of structural biology
J Struct Biol. 2011 May;174(2):344-51. Epub 2011 Feb 23.},
keywords = {FLORENTZ, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly(297)Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7A. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3A by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies.
Messmer M, Florentz C, Schwenzer H, Scheper G C, van der Knaap M S, Marechal-Drouard L, Sissler M
A human pathology-related mutation prevents import of an aminoacyl-tRNA synthetase into mitochondria Journal Article
In: Biochem J, vol. 433, no. 3, pp. 441-446, 2011, ISSN: 1470-8728 (Electronic) 0264-6021 (Linking), (Messmer, Marie Florentz, Catherine Schwenzer, Hagen Scheper, Gert C van der Knaap, Marjo S Marechal-Drouard, Laurence Sissler, Marie Research Support, Non-U.S. Gov't England The Biochemical journal Biochem J. 2011 Jan 14;433(3):441-6.).
Abstract | Links | BibTeX | Tags: Aspartate-tRNA Ligase/*genetics/*metabolism Cell Line Humans Leukoencephalopathies/etiology/genetics Mitochondria/*metabolism *Mutation, FLORENTZ, Missense Protein Transport, SISSLER, Unité ARN
@article{,
title = {A human pathology-related mutation prevents import of an aminoacyl-tRNA synthetase into mitochondria},
author = {M Messmer and C Florentz and H Schwenzer and G C Scheper and M S van der Knaap and L Marechal-Drouard and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21121901},
doi = {10.1042/BJ20101902},
issn = {1470-8728 (Electronic) 0264-6021 (Linking)},
year = {2011},
date = {2011-01-01},
journal = {Biochem J},
volume = {433},
number = {3},
pages = {441-446},
abstract = {Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser to Gly mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.},
note = {Messmer, Marie
Florentz, Catherine
Schwenzer, Hagen
Scheper, Gert C
van der Knaap, Marjo S
Marechal-Drouard, Laurence
Sissler, Marie
Research Support, Non-U.S. Gov't
England
The Biochemical journal
Biochem J. 2011 Jan 14;433(3):441-6.},
keywords = {Aspartate-tRNA Ligase/*genetics/*metabolism Cell Line Humans Leukoencephalopathies/etiology/genetics Mitochondria/*metabolism *Mutation, FLORENTZ, Missense Protein Transport, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser to Gly mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.
2009
Juhling F, Morl M, Hartmann R K, Sprinzl M, Stadler P F, Putz J
tRNAdb 2009: compilation of tRNA sequences and tRNA genes Journal Article
In: Nucleic Acids Res, vol. 37, no. Database issue, pp. D159-162, 2009, ISBN: 18957446, (1362-4962 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: FLORENTZ, Unité ARN
@article{,
title = {tRNAdb 2009: compilation of tRNA sequences and tRNA genes},
author = {F Juhling and M Morl and R K Hartmann and M Sprinzl and P F Stadler and J Putz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18957446},
isbn = {18957446},
year = {2009},
date = {2009-01-01},
journal = {Nucleic Acids Res},
volume = {37},
number = {Database issue},
pages = {D159-162},
abstract = {One of the first specialized collections of nucleic acid sequences in life sciences was the 'compilation of tRNA sequences and sequences of tRNA genes' (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs.},
note = {1362-4962 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {FLORENTZ, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
One of the first specialized collections of nucleic acid sequences in life sciences was the 'compilation of tRNA sequences and sequences of tRNA genes' (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs.
Messmer M, Putz J, Suzuki T, Sauter C, Sissler M, Florentz C
Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny Journal Article
In: Nucleic Acids Res, vol. 37, no. 20, pp. 6881-6895, 2009, ISBN: 19767615, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Asp/*chemistry/*metabolism Transcription, Base Sequence Databases, FLORENTZ, FRUGIER, Genetic, Nucleic Acid Humans Molecular Sequence Data Nucleic Acid Conformation Phylogeny RNA/*chemistry/*metabolism RNA, SISSLER, Transfer, Unité ARN
@article{,
title = {Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny},
author = {M Messmer and J Putz and T Suzuki and C Sauter and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19767615},
isbn = {19767615},
year = {2009},
date = {2009-01-01},
journal = {Nucleic Acids Res},
volume = {37},
number = {20},
pages = {6881-6895},
abstract = {Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA(Asp) in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA(Asp) including predominantly the cloverleaf. On the contrary, the native tRNA(Asp) folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis-Westhof interactions, the tertiary network core building rules apply to all tRNA(Asp) from mammalian mitochondria.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Asp/*chemistry/*metabolism Transcription, Base Sequence Databases, FLORENTZ, FRUGIER, Genetic, Nucleic Acid Humans Molecular Sequence Data Nucleic Acid Conformation Phylogeny RNA/*chemistry/*metabolism RNA, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA(Asp) in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA(Asp) including predominantly the cloverleaf. On the contrary, the native tRNA(Asp) folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis-Westhof interactions, the tertiary network core building rules apply to all tRNA(Asp) from mammalian mitochondria.
Messmer M, Gaudry A, Sissler M, Florentz C
Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNA(Asp) Journal Article
In: RNA, vol. 15, no. 8, pp. 1462-1468, 2009, ISBN: 19535463, (1469-9001 (Electronic) Letter Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Asp/*chemistry/*genetics/metabolism Transfer RNA Aminoacylation/genetics, Binding Sites/genetics Humans Kinetics Mitochondrial Myopathies/genetics/metabolism/pathology Models, FLORENTZ, Missense Nucleic Acid Conformation RNA/*chemistry/*genetics/metabolism RNA, Molecular Mutation, SISSLER, Transfer, Unité ARN
@article{,
title = {Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNA(Asp)},
author = {M Messmer and A Gaudry and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19535463},
isbn = {19535463},
year = {2009},
date = {2009-01-01},
journal = {RNA},
volume = {15},
number = {8},
pages = {1462-1468},
abstract = {More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA(Asp) to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA(Asp) with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.},
note = {1469-9001 (Electronic)
Letter
Research Support, Non-U.S. Gov't},
keywords = {Asp/*chemistry/*genetics/metabolism Transfer RNA Aminoacylation/genetics, Binding Sites/genetics Humans Kinetics Mitochondrial Myopathies/genetics/metabolism/pathology Models, FLORENTZ, Missense Nucleic Acid Conformation RNA/*chemistry/*genetics/metabolism RNA, Molecular Mutation, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA(Asp) to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA(Asp) with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.
Messmer M, Blais S P, Balg C, Chenevert R, Grenier L, Lague P, Sauter C, Sissler M, Giege R, Lapointe J, Florentz C
Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetase by adenylate analogs Journal Article
In: Biochimie, vol. 91, no. 5, pp. 596-603, 2009, ISBN: 19254750, (1638-6183 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Adenosine Monophosphate/*analogs & derivatives/*pharmacology Animals Aspartate-tRNA Ligase/*antagonists & inhibitors/*chemistry/metabolism Catalytic Domain Cattle Humans Mitochondria/*drug effects/*enzymology Molecular Structure Structure-Activity Relationship, FLORENTZ, SISSLER, Unité ARN
@article{,
title = {Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetase by adenylate analogs},
author = {M Messmer and S P Blais and C Balg and R Chenevert and L Grenier and P Lague and C Sauter and M Sissler and R Giege and J Lapointe and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19254750},
isbn = {19254750},
year = {2009},
date = {2009-01-01},
journal = {Biochimie},
volume = {91},
number = {5},
pages = {596-603},
abstract = {Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. L-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K(i) values in the micromolar range (4-27 microM for human mt- and mammalian cyt-AspRSs). 5'-O-[N-(L-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.},
note = {1638-6183 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Adenosine Monophosphate/*analogs & derivatives/*pharmacology Animals Aspartate-tRNA Ligase/*antagonists & inhibitors/*chemistry/metabolism Catalytic Domain Cattle Humans Mitochondria/*drug effects/*enzymology Molecular Structure Structure-Activity Relationship, FLORENTZ, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. L-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K(i) values in the micromolar range (4-27 microM for human mt- and mammalian cyt-AspRSs). 5'-O-[N-(L-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.
Florentz C
Organisation et expression des genomes des organites Book Chapter
In: Weil, J H (Ed.): Biochemie Generale 11e edition, pp. 619-624, Dunod, Paris, 2009.
Links | BibTeX | Tags: FLORENTZ, Unité ARN
@inbook{,
title = {Organisation et expression des genomes des organites},
author = {C Florentz},
editor = {J H Weil},
url = {http://www.unitheque.com/Livre/dunod/Sciences_sup/Biochimie_generale-31587.html},
year = {2009},
date = {2009-01-01},
booktitle = {Biochemie Generale 11e edition},
pages = {619-624},
publisher = {Dunod},
address = {Paris},
keywords = {FLORENTZ, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
2008
Sissler M, Lorber B, Messmer M, Schaller A, Putz J, Florentz C
Handling mammalian mitochondrial tRNAs and aminoacyl-tRNA synthetases for functional and structural characterization Journal Article
In: Methods, vol. 44, no. 2, pp. 176-189, 2008, ISBN: 18241799, (1046-2023 (Print) Journal article).
Abstract | Links | BibTeX | Tags: FLORENTZ, FLORENTZ GIEGE, SISSLER, Unité ARN
@article{,
title = {Handling mammalian mitochondrial tRNAs and aminoacyl-tRNA synthetases for functional and structural characterization},
author = {M Sissler and B Lorber and M Messmer and A Schaller and J Putz and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18241799},
isbn = {18241799},
year = {2008},
date = {2008-01-01},
journal = {Methods},
volume = {44},
number = {2},
pages = {176-189},
abstract = {The mammalian mitochondrial (mt) genome codes for only 13 proteins, which are essential components in the process of oxidative phosphorylation of ADP into ATP. Synthesis of these proteins relies on a proper mt translation machinery. While 22 tRNAs and 2 rRNAs are also coded by the mt genome, all other factors including the set of aminoacyl-tRNA synthetases (aaRSs) are encoded in the nucleus and imported. Investigation of mammalian mt aminoacylation systems (and mt translation in general) gains more and more interest not only in regard of evolutionary considerations but also with respect to the growing number of diseases linked to mutations in the genes of either mt-tRNAs, synthetases or other factors. Here we report on methodological approaches for biochemical, functional, and structural characterization of human/mammalian mt-tRNAs and aaRSs. Procedures for preparation of native and in vitro transcribed tRNAs are accompanied by recommendations for specific handling of tRNAs incline to structural instability and chemical fragility. Large-scale preparation of mg amounts of highly soluble recombinant synthetases is a prerequisite for structural investigations that requires particular optimizations. Successful examples leading to crystallization of four mt-aaRSs and high-resolution structures are recalled and limitations discussed. Finally, the need for and the state-of-the-art in setting up an in vitro mt translation system are emphasized. Biochemical characterization of a subset of mammalian aminoacylation systems has already revealed a number of unprecedented peculiarities of interest for the study of evolution and forensic research. Further efforts in this field will certainly be rewarded by many exciting discoveries.},
note = {1046-2023 (Print)
Journal article},
keywords = {FLORENTZ, FLORENTZ GIEGE, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The mammalian mitochondrial (mt) genome codes for only 13 proteins, which are essential components in the process of oxidative phosphorylation of ADP into ATP. Synthesis of these proteins relies on a proper mt translation machinery. While 22 tRNAs and 2 rRNAs are also coded by the mt genome, all other factors including the set of aminoacyl-tRNA synthetases (aaRSs) are encoded in the nucleus and imported. Investigation of mammalian mt aminoacylation systems (and mt translation in general) gains more and more interest not only in regard of evolutionary considerations but also with respect to the growing number of diseases linked to mutations in the genes of either mt-tRNAs, synthetases or other factors. Here we report on methodological approaches for biochemical, functional, and structural characterization of human/mammalian mt-tRNAs and aaRSs. Procedures for preparation of native and in vitro transcribed tRNAs are accompanied by recommendations for specific handling of tRNAs incline to structural instability and chemical fragility. Large-scale preparation of mg amounts of highly soluble recombinant synthetases is a prerequisite for structural investigations that requires particular optimizations. Successful examples leading to crystallization of four mt-aaRSs and high-resolution structures are recalled and limitations discussed. Finally, the need for and the state-of-the-art in setting up an in vitro mt translation system are emphasized. Biochemical characterization of a subset of mammalian aminoacylation systems has already revealed a number of unprecedented peculiarities of interest for the study of evolution and forensic research. Further efforts in this field will certainly be rewarded by many exciting discoveries.
Bonnefond L, Florentz C, Giege R, Rudinger-Thirion J
Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture Journal Article
In: RNA, vol. 14, no. 4, pp. 641-648, 2008, ISBN: 18268021, (1469-9001 (Electronic) In Vitro Journal Article Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: Base Sequence Humans Mitochondrial Diseases/genetics/metabolism Models, FLORENTZ, FRUGIER, Molecular Molecular Sequence Data Nucleic Acid Conformation *Point Mutation RNA/*chemistry/*genetics/metabolism RNA Stability RNA, Transfer, Tyr/*chemistry/*genetics/metabolism *Transfer RNA Aminoacylation, Unité ARN
@article{,
title = {Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture},
author = {L Bonnefond and C Florentz and R Giege and J Rudinger-Thirion},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18268021},
isbn = {18268021},
year = {2008},
date = {2008-01-01},
journal = {RNA},
volume = {14},
number = {4},
pages = {641-648},
abstract = {A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA(Tyr) molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA(Tyr) tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA(Tyr) molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA(Tyr) via nonclassical Watson-Crick G15-C48 and G13-A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA(Tyr) life cycle.},
note = {1469-9001 (Electronic)
In Vitro
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {Base Sequence Humans Mitochondrial Diseases/genetics/metabolism Models, FLORENTZ, FRUGIER, Molecular Molecular Sequence Data Nucleic Acid Conformation *Point Mutation RNA/*chemistry/*genetics/metabolism RNA Stability RNA, Transfer, Tyr/*chemistry/*genetics/metabolism *Transfer RNA Aminoacylation, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA(Tyr) molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA(Tyr) tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA(Tyr) molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA(Tyr) via nonclassical Watson-Crick G15-C48 and G13-A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA(Tyr) life cycle.
2007
Scheper G C, van der Klok T, van Andel R J, van Berkel C G, Sissler M, Smet J, Muravina T I, Serkov S V, Uziel G, Bugiani M, Schiffmann R, Krageloh-Mann I, Smeitink J A, Florentz C, Coster R Van, Pronk J C, van der Knaap M S
Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation Journal Article
In: Nat Genet, vol. 39, no. 4, pp. 534-539, 2007, ISBN: 17384640, (1061-4036 (Print) Journal Article).
Abstract | Links | BibTeX | Tags: FLORENTZ, SISSLER, Unité ARN
@article{,
title = {Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation},
author = {G C Scheper and T van der Klok and R J van Andel and C G van Berkel and M Sissler and J Smet and T I Muravina and S V Serkov and G Uziel and M Bugiani and R Schiffmann and I Krageloh-Mann and J A Smeitink and C Florentz and R Van Coster and J C Pronk and M S van der Knaap},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17384640},
isbn = {17384640},
year = {2007},
date = {2007-01-01},
journal = {Nat Genet},
volume = {39},
number = {4},
pages = {534-539},
abstract = {Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.},
note = {1061-4036 (Print)
Journal Article},
keywords = {FLORENTZ, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.
2006
Fender A, Sauter C, Messmer M, Putz J, Giege R, Florentz C, Sissler M
Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system Journal Article
In: J Biol Chem, vol. 281, no. 23, pp. 15980-15986, 2006, ISBN: 16597625, (0021-9258 (Print) Journal Article).
Abstract | Links | BibTeX | Tags: Acylation Base Sequence Humans Kinetics Mutagenesis Nucleic Acid Conformation Plasmids RNA | Non-U.S. Gov't, Asp/chemistry/genetics/*metabolism Research Support, FLORENTZ, FRUGIER, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN
@article{,
title = {Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system},
author = {A Fender and C Sauter and M Messmer and J Putz and R Giege and C Florentz and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16597625},
isbn = {16597625},
year = {2006},
date = {2006-01-01},
journal = {J Biol Chem},
volume = {281},
number = {23},
pages = {15980-15986},
abstract = {In mammalian mitochondria the translational machinery is of dual origin with tRNAs encoded by a simplified and rapidly evolving mitochondrial (mt) genome and aminoacyl-tRNA synthetases (aaRS) coded by the nuclear genome, and imported. Mt-tRNAs are atypical with biased sequences, size variations in loops and stems, and absence of residues forming classical tertiary interactions, whereas synthetases appear typical. This raises questions about identity elements in mt-tRNAs and adaptation of their cognate mt-aaRSs. We have explored here the human mt-aspartate system in which a prokaryotic-type AspRS, highly similar to the Escherichia coli enzyme, recognizes a bizarre tRNA(Asp). Analysis of human mt-tRNA(Asp) transcripts confirms the identity role of the GUC anticodon as in other aspartylation systems but reveals the non-involvement of position 73. This position is otherwise known as the site of a universally conserved major aspartate identity element, G73, also known as a primordial identity signal. In mt-tRNA(Asp), position 73 can be occupied by any of the four nucleotides without affecting aspartylation. Sequence alignments of various AspRSs allowed placing Gly-269 at a position occupied by Asp-220, the residue contacting G73 in the crystallographic structure of E. coli AspRS-tRNA(Asp) complex. Replacing this glycine by an aspartate renders human mt-AspRS more discriminative to G73. Restriction in the aspartylation identity set, driven by a rapid mutagenic rate of the mt-genome, suggests a reverse evolution of the mt-tRNA(Asp) identity elements in regard to its bacterial ancestor.},
note = {0021-9258 (Print)
Journal Article},
keywords = {Acylation Base Sequence Humans Kinetics Mutagenesis Nucleic Acid Conformation Plasmids RNA | Non-U.S. Gov't, Asp/chemistry/genetics/*metabolism Research Support, FLORENTZ, FRUGIER, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In mammalian mitochondria the translational machinery is of dual origin with tRNAs encoded by a simplified and rapidly evolving mitochondrial (mt) genome and aminoacyl-tRNA synthetases (aaRS) coded by the nuclear genome, and imported. Mt-tRNAs are atypical with biased sequences, size variations in loops and stems, and absence of residues forming classical tertiary interactions, whereas synthetases appear typical. This raises questions about identity elements in mt-tRNAs and adaptation of their cognate mt-aaRSs. We have explored here the human mt-aspartate system in which a prokaryotic-type AspRS, highly similar to the Escherichia coli enzyme, recognizes a bizarre tRNA(Asp). Analysis of human mt-tRNA(Asp) transcripts confirms the identity role of the GUC anticodon as in other aspartylation systems but reveals the non-involvement of position 73. This position is otherwise known as the site of a universally conserved major aspartate identity element, G73, also known as a primordial identity signal. In mt-tRNA(Asp), position 73 can be occupied by any of the four nucleotides without affecting aspartylation. Sequence alignments of various AspRSs allowed placing Gly-269 at a position occupied by Asp-220, the residue contacting G73 in the crystallographic structure of E. coli AspRS-tRNA(Asp) complex. Replacing this glycine by an aspartate renders human mt-AspRS more discriminative to G73. Restriction in the aspartylation identity set, driven by a rapid mutagenic rate of the mt-genome, suggests a reverse evolution of the mt-tRNA(Asp) identity elements in regard to its bacterial ancestor.
2005
Sissler M, Putz J, Fasiolo F, Florentz C
Mitochondrial aminoacyl-tRNA synthetases Book Chapter
In: Ibba, M; Francklyn, C; Cusak, S (Ed.): The Aminoacyl-tRNA Synthetases, Landes Bioscience, 2005.
Abstract | Links | BibTeX | Tags: FLORENTZ, FLORENTZ FASIOLO, SISSLER, Unité ARN
@inbook{,
title = {Mitochondrial aminoacyl-tRNA synthetases},
author = {M Sissler and J Putz and F Fasiolo and C Florentz},
editor = {M Ibba and C Francklyn and S Cusak},
url = {http://www.ncbi.nlm.nih.gov/books/NBK6033},
year = {2005},
date = {2005-01-01},
booktitle = {The Aminoacyl-tRNA Synthetases},
publisher = {Landes Bioscience},
abstract = {Mitochondria and chloroplasts have their own genomes that encode a small number of proteins whose synthesis depends on translation machineries of multiple origin. Whereas tRNAs, rRNAs and some ribosomal proteins are often encoded by the organellar genome, all other factors and in particular aminoacyl-tRNA synthetases (aaRSs) are nuclear encoded, synthesized in the cytosol and imported. Thus, two to three sets of aaRSs coexist in eukaryotic cells, namely cytosolic, mitochondrial and chloroplastic versions. Here, the diversity in the structural and functional properties of organellar aaRSs is illustrated by mammalian mitochondrial aaRSs (size, oligomeric structure, efficiency of aminoacylation, cross reactions, identity sets). Additionally, means by which nuclear genes encode cytosolic, mitochondrial and chloroplastic aaRSs are reviewed on the basis of database exploration on fully sequenced (although not completely annotated) genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorabditis elegans, Drosophila melanogaster and Arabidopsis thaliana.},
keywords = {FLORENTZ, FLORENTZ FASIOLO, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Mitochondria and chloroplasts have their own genomes that encode a small number of proteins whose synthesis depends on translation machineries of multiple origin. Whereas tRNAs, rRNAs and some ribosomal proteins are often encoded by the organellar genome, all other factors and in particular aminoacyl-tRNA synthetases (aaRSs) are nuclear encoded, synthesized in the cytosol and imported. Thus, two to three sets of aaRSs coexist in eukaryotic cells, namely cytosolic, mitochondrial and chloroplastic versions. Here, the diversity in the structural and functional properties of organellar aaRSs is illustrated by mammalian mitochondrial aaRSs (size, oligomeric structure, efficiency of aminoacylation, cross reactions, identity sets). Additionally, means by which nuclear genes encode cytosolic, mitochondrial and chloroplastic aaRSs are reviewed on the basis of database exploration on fully sequenced (although not completely annotated) genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorabditis elegans, Drosophila melanogaster and Arabidopsis thaliana.
Florentz C
Participation a la mise à jour de l'ouvrage Book Chapter
In: Weil, J H (Ed.): Biochimie Generale, 10e edition, Dunod, 2005.
Links | BibTeX | Tags: FLORENTZ, Unité ARN
@inbook{,
title = {Participation a la mise à jour de l'ouvrage},
author = {C Florentz},
editor = {J H Weil},
url = {none},
year = {2005},
date = {2005-01-01},
booktitle = {Biochimie Generale, 10e edition},
publisher = {Dunod},
keywords = {FLORENTZ, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Bonnefond L, Fender A, Rudinger-Thirion J, Giege R, Florentz C, Sissler M
Toward the Full Set of Human Mitochondrial Aminoacyl-tRNA Synthetases: Characterization of AspRS and TyrRS Journal Article
In: Biochemistry, vol. 44, no. 12, pp. 4805-4816, 2005, ISBN: 15779907, (0006-2960 Journal Article).
Abstract | Links | BibTeX | Tags: FLORENTZ, FRUGIER, SISSLER, Unité ARN
@article{,
title = {Toward the Full Set of Human Mitochondrial Aminoacyl-tRNA Synthetases: Characterization of AspRS and TyrRS},
author = {L Bonnefond and A Fender and J Rudinger-Thirion and R Giege and C Florentz and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15779907},
isbn = {15779907},
year = {2005},
date = {2005-01-01},
journal = {Biochemistry},
volume = {44},
number = {12},
pages = {4805-4816},
abstract = {The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.},
note = {0006-2960
Journal Article},
keywords = {FLORENTZ, FRUGIER, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.
2004
Sohm B, Sissler M, Park H, King M P, Florentz C
Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase Journal Article
In: J Mol Biol, vol. 339, no. 1, pp. 17-29, 2004, ISBN: 15123417, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Cultured, FLORENTZ, FLORENTZ *Acylation Base Sequence Comparative Study Human Kinetics Leucine/metabolism Leucine-tRNA Ligase/genetics/*metabolism Mitochondria/*metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation Osteosarcoma/metabolism RNA/*genetics/metabolism RNA, Genetic/*genetics Tumor Cells, Leu/genetics/*metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Support, P.H.S. Transcription, SISSLER, Transfer, U.S. Gov't, Unité ARN
@article{,
title = {Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase},
author = {B Sohm and M Sissler and H Park and M P King and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15123417},
isbn = {15123417},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {339},
number = {1},
pages = {17-29},
abstract = {Accuracy of protein synthesis depends on specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases. Rules governing these processes have been established for numerous prokaryotic and eukaryotic cytoplasmic systems, but only limited information is available for human mitochondrial systems. It has been shown that the in vitro transcribed human mitochondrial tRNA(Leu(UUR)) does not fold into the expected cloverleaf, but is however aminoacylated by the human mitochondrial leucyl-tRNA synthetase. Here, the role of the structure of the amino acid acceptor branch and the anticodon branch of tRNA(Leu(UUR)) in recognition by leucyl-tRNA synthetase was investigated. The kinetic parameters for aminoacylation of wild-type and mutant tRNA(Leu(UUR)) transcripts and of native tRNA(Leu(UUR)) were determined. Solution structure probing was performed in the presence or in the absence of leucyl-tRNA synthetase and correlated with the aminoacylation kinetics for each tRNA. Replacement of mismatches in either the anticodon-stem or D-stem that are present in the wild-type tRNA(Leu(UUR)) by G-C base-pairs is sufficient to induce (i) cloverleaf folding, (ii) improved aminoacylation efficiency, and (iii) interactions with the synthetase that are similar to those with the native tRNA(Leu(UUR)). Leucyl-tRNA synthetase contacts tRNA(Leu(UUR)) in the amino acid acceptor stem, the anticodon stem, and the D-loop, which is unprecedented for a leucine aminoacylation system.},
note = {0022-2836
Journal Article},
keywords = {Cultured, FLORENTZ, FLORENTZ *Acylation Base Sequence Comparative Study Human Kinetics Leucine/metabolism Leucine-tRNA Ligase/genetics/*metabolism Mitochondria/*metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation Osteosarcoma/metabolism RNA/*genetics/metabolism RNA, Genetic/*genetics Tumor Cells, Leu/genetics/*metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Support, P.H.S. Transcription, SISSLER, Transfer, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Accuracy of protein synthesis depends on specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases. Rules governing these processes have been established for numerous prokaryotic and eukaryotic cytoplasmic systems, but only limited information is available for human mitochondrial systems. It has been shown that the in vitro transcribed human mitochondrial tRNA(Leu(UUR)) does not fold into the expected cloverleaf, but is however aminoacylated by the human mitochondrial leucyl-tRNA synthetase. Here, the role of the structure of the amino acid acceptor branch and the anticodon branch of tRNA(Leu(UUR)) in recognition by leucyl-tRNA synthetase was investigated. The kinetic parameters for aminoacylation of wild-type and mutant tRNA(Leu(UUR)) transcripts and of native tRNA(Leu(UUR)) were determined. Solution structure probing was performed in the presence or in the absence of leucyl-tRNA synthetase and correlated with the aminoacylation kinetics for each tRNA. Replacement of mismatches in either the anticodon-stem or D-stem that are present in the wild-type tRNA(Leu(UUR)) by G-C base-pairs is sufficient to induce (i) cloverleaf folding, (ii) improved aminoacylation efficiency, and (iii) interactions with the synthetase that are similar to those with the native tRNA(Leu(UUR)). Leucyl-tRNA synthetase contacts tRNA(Leu(UUR)) in the amino acid acceptor stem, the anticodon stem, and the D-loop, which is unprecedented for a leucine aminoacylation system.
Sissler M, Helm M, Frugier M, Giege R, Florentz C
Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants Journal Article
In: RNA, vol. 10, no. 5, pp. 841-853, 2004, ISBN: 15100439, (1355-8382 Journal Article).
Abstract | Links | BibTeX | Tags: ERIANI, FLORENTZ, FLORENTZ GIEGE Acylation Aminoacyltransferases/*genetics Human MERRF Syndrome/genetics Mitochondria/*genetics Mitochondrial Diseases/*genetics Mutation Nucleic Acid Conformation RNA, FRUGIER, Lys/*genetics Sequence Analysis, Non-U.S. Gov't Variation (Genetics), RNA Support, SISSLER, Transfer, Unité ARN
@article{,
title = {Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants},
author = {M Sissler and M Helm and M Frugier and R Giege and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15100439},
isbn = {15100439},
year = {2004},
date = {2004-01-01},
journal = {RNA},
volume = {10},
number = {5},
pages = {841-853},
abstract = {In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C.A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.},
note = {1355-8382
Journal Article},
keywords = {ERIANI, FLORENTZ, FLORENTZ GIEGE Acylation Aminoacyltransferases/*genetics Human MERRF Syndrome/genetics Mitochondria/*genetics Mitochondrial Diseases/*genetics Mutation Nucleic Acid Conformation RNA, FRUGIER, Lys/*genetics Sequence Analysis, Non-U.S. Gov't Variation (Genetics), RNA Support, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C.A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.
Levinger L, Oestreich I, Florentz C, Morl M
A pathogenesis-associated mutation in human mitochondrial tRNALeu(UUR) leads to reduced 3'-end processing and CCA addition Journal Article
In: J Mol Biol, vol. 337, no. 3, pp. 535-544, 2004, ISBN: 15019775, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: FLORENTZ, FLORENTZ Human Kinetics Mitochondrial Diseases/*genetics Nucleic Acid Conformation *Point Mutation RNA/*genetics/physiology *RNA 3' End Processing RNA Nucleotidyltransferases/metabolism RNA, Leu/*genetics/physiology Support, Non-U.S. Gov't Support, P.H.S., Transfer, U.S. Gov't, Unité ARN
@article{,
title = {A pathogenesis-associated mutation in human mitochondrial tRNALeu(UUR) leads to reduced 3'-end processing and CCA addition},
author = {L Levinger and I Oestreich and C Florentz and M Morl},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15019775},
isbn = {15019775},
year = {2004},
date = {2004-01-01},
journal = {J Mol Biol},
volume = {337},
number = {3},
pages = {535-544},
abstract = {Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.},
note = {0022-2836
Journal Article},
keywords = {FLORENTZ, FLORENTZ Human Kinetics Mitochondrial Diseases/*genetics Nucleic Acid Conformation *Point Mutation RNA/*genetics/physiology *RNA 3' End Processing RNA Nucleotidyltransferases/metabolism RNA, Leu/*genetics/physiology Support, Non-U.S. Gov't Support, P.H.S., Transfer, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.
Levinger L, Mörl M, Florentz C
Mitochondrial tRNA 3' end metabolism and human disease Journal Article
In: Nucleic Acids Res, vol. 32, no. 18, pp. 5430-5441, 2004, ISBN: 15477393, (1362-4962 Journal Article Review Review, Tutorial).
Abstract | Links | BibTeX | Tags: FLORENTZ, Non-U.S. Gov't Support, P.H.S., Transfer/chemistry/*genetics/*metabolism Support, U.S. Gov't, Unité ARN
@article{,
title = {Mitochondrial tRNA 3' end metabolism and human disease},
author = {L Levinger and M Mörl and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15477393},
isbn = {15477393},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {18},
pages = {5430-5441},
abstract = {Over 150 mutations in the mitochondrial genome have been shown to be associated with human disease. Remarkably, two-thirds of them are found in tRNA genes, which constitute only one-tenth of the mitochondrial genome. A total of 22 tRNAs punctuate the genome and are produced together with 11 mRNAs and 2 rRNAs from long polycistronic primary transcripts with almost no spacers. Pre-tRNAs thus require precise endonucleolytic excision. Furthermore, the CCA triplet which forms the 3' end of all tRNAs is not encoded, but must be synthesized by the CCA-adding enzyme after 3' end cleavage. Amino acid attachment to the CCA of mature tRNA is performed by aminoacyl-tRNA synthetases, which, like the preceding processing enzymes, are nuclear-encoded and imported into mitochondria. Here, we critically review the effectiveness and reliability of evidence obtained from reactions with in vitro transcripts that pathogenesis-associated mutant mitochondrial tRNAs can lead to deficiencies in tRNA 3' end metabolism (3' end cleavage, CCA addition and aminoacylation) toward an understanding of molecular mechanisms underlying human tRNA disorders. These defects probably contribute, individually and cumulatively, to the progression of human mitochondrial diseases.},
note = {1362-4962
Journal Article
Review
Review, Tutorial},
keywords = {FLORENTZ, Non-U.S. Gov't Support, P.H.S., Transfer/chemistry/*genetics/*metabolism Support, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Over 150 mutations in the mitochondrial genome have been shown to be associated with human disease. Remarkably, two-thirds of them are found in tRNA genes, which constitute only one-tenth of the mitochondrial genome. A total of 22 tRNAs punctuate the genome and are produced together with 11 mRNAs and 2 rRNAs from long polycistronic primary transcripts with almost no spacers. Pre-tRNAs thus require precise endonucleolytic excision. Furthermore, the CCA triplet which forms the 3' end of all tRNAs is not encoded, but must be synthesized by the CCA-adding enzyme after 3' end cleavage. Amino acid attachment to the CCA of mature tRNA is performed by aminoacyl-tRNA synthetases, which, like the preceding processing enzymes, are nuclear-encoded and imported into mitochondria. Here, we critically review the effectiveness and reliability of evidence obtained from reactions with in vitro transcripts that pathogenesis-associated mutant mitochondrial tRNAs can lead to deficiencies in tRNA 3' end metabolism (3' end cleavage, CCA addition and aminoacylation) toward an understanding of molecular mechanisms underlying human tRNA disorders. These defects probably contribute, individually and cumulatively, to the progression of human mitochondrial diseases.
Fender A, Sissler M, Florentz C, Giege R
Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems Journal Article
In: Biochimie, vol. 86, no. 1, pp. 21-29, 2004, ISBN: 14987797, (0300-9084 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acyl/genetics/*metabolism Saccharomyces cerevisiae Substrate Specificity/genetics/physiology Support, Chemical Molecular Sequence Data Mutation Nucleic Acid Conformation Protein Binding/physiology RNA, FLORENTZ, GIEGE FLORENTZ Amino Acid Activation/physiology Amino Acyl-tRNA Ligases/*metabolism Anticodon Bacterial Proteins/metabolism Base Sequence Cloning, Molecular Computer Simulation Escherichia coli Models, Non-U.S. Gov't Thermus thermophilus, SISSLER, Transfer, Transfer/genetics/*metabolism RNA, Unité ARN
@article{,
title = {Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems},
author = {A Fender and M Sissler and C Florentz and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14987797},
isbn = {14987797},
year = {2004},
date = {2004-01-01},
journal = {Biochimie},
volume = {86},
number = {1},
pages = {21-29},
abstract = {The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.},
note = {0300-9084
Journal Article},
keywords = {Amino Acyl/genetics/*metabolism Saccharomyces cerevisiae Substrate Specificity/genetics/physiology Support, Chemical Molecular Sequence Data Mutation Nucleic Acid Conformation Protein Binding/physiology RNA, FLORENTZ, GIEGE FLORENTZ Amino Acid Activation/physiology Amino Acyl-tRNA Ligases/*metabolism Anticodon Bacterial Proteins/metabolism Base Sequence Cloning, Molecular Computer Simulation Escherichia coli Models, Non-U.S. Gov't Thermus thermophilus, SISSLER, Transfer, Transfer/genetics/*metabolism RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.
Fender A, Geslain R, Eriani G, Giege R, Sissler M, Florentz C
A yeast arginine specific tRNA is a remnant aspartate acceptor Journal Article
In: Nucleic Acids Res, vol. 32, no. 17, pp. 5076-5086, 2004, ISBN: 15452274, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Arg/*chemistry/genetics/metabolism RNA, Asp/*chemistry/genetics/metabolism Saccharomyces cerevisiae/*genetics Sequence Alignment Support, ERIANI, ERIANI FLORENTZ GIEGE Aspartic Acid/metabolism Base Sequence *Evolution, FLORENTZ, Fungal/*chemistry/genetics/metabolism RNA, Molecular Molecular Sequence Data Point Mutation RNA, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN
@article{,
title = {A yeast arginine specific tRNA is a remnant aspartate acceptor},
author = {A Fender and R Geslain and G Eriani and R Giege and M Sissler and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15452274},
isbn = {15452274},
year = {2004},
date = {2004-01-01},
journal = {Nucleic Acids Res},
volume = {32},
number = {17},
pages = {5076-5086},
abstract = {High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes.},
note = {1362-4962
Journal Article},
keywords = {Arg/*chemistry/genetics/metabolism RNA, Asp/*chemistry/genetics/metabolism Saccharomyces cerevisiae/*genetics Sequence Alignment Support, ERIANI, ERIANI FLORENTZ GIEGE Aspartic Acid/metabolism Base Sequence *Evolution, FLORENTZ, Fungal/*chemistry/genetics/metabolism RNA, Molecular Molecular Sequence Data Point Mutation RNA, Non-U.S. Gov't, SISSLER, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes.
Barends S, Rudinger-Thirion J, Florentz C, Giege R, Pleij C W, Kraal B
tRNA-like structure regulates translation of Brome mosaic virus RNA Journal Article
In: J Virol, vol. 78, no. 8, pp. 4003-4010, 2004, ISBN: 15047816, (0022-538x Journal Article).
Abstract | Links | BibTeX | Tags: ase Sequence Bromovirus/*genetics/metabolism Genetic Complementation Test Genome, FLORENTZ, FRUGIER, Genetic Triticum/virology Tyrosine/chemistry Tyrosine-tRNA Ligase/chemistry/genetics/metabolism Viral Proteins/chemistry/genetics, Non-U.S. Gov't Translation, Transfer/chemistry/genetics RNA, Unité ARN, Viral Molecular Sequence Data Nucleic Acid Conformation RNA, Viral/*chemistry/*genetics Support
@article{,
title = {tRNA-like structure regulates translation of Brome mosaic virus RNA},
author = {S Barends and J Rudinger-Thirion and C Florentz and R Giege and C W Pleij and B Kraal},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15047816},
isbn = {15047816},
year = {2004},
date = {2004-01-01},
journal = {J Virol},
volume = {78},
number = {8},
pages = {4003-4010},
abstract = {For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.},
note = {0022-538x
Journal Article},
keywords = {ase Sequence Bromovirus/*genetics/metabolism Genetic Complementation Test Genome, FLORENTZ, FRUGIER, Genetic Triticum/virology Tyrosine/chemistry Tyrosine-tRNA Ligase/chemistry/genetics/metabolism Viral Proteins/chemistry/genetics, Non-U.S. Gov't Translation, Transfer/chemistry/genetics RNA, Unité ARN, Viral Molecular Sequence Data Nucleic Acid Conformation RNA, Viral/*chemistry/*genetics Support},
pubstate = {published},
tppubtype = {article}
}
For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.
2003
Tryoen-Toth P, Richert S, Sohm B, Mine M, Marsac C, Dorsselaer A Van, Leize E, Florentz C
Proteomic consequences of a human mitochondrial tRNA mutation beyond the frame of mitochondrial translation Journal Article
In: J Biol Chem, vol. 278, no. 27, pp. 24314-24323, 2003, ISBN: 12714596, (0021-9258 Journal Article).
Abstract | Links | BibTeX | Tags: Cultured, FLORENTZ, Genetic Tumor Cells, Human Mitochondria/genetics *Mutation Nuclear Proteins/*genetics Proteomics RNA/*genetics RNA, Non-U.S. Gov't Translation, Transfer/*genetics Structure-Activity Relationship Support, Unité ARN
@article{,
title = {Proteomic consequences of a human mitochondrial tRNA mutation beyond the frame of mitochondrial translation},
author = {P Tryoen-Toth and S Richert and B Sohm and M Mine and C Marsac and A Van Dorsselaer and E Leize and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12714596},
isbn = {12714596},
year = {2003},
date = {2003-01-01},
journal = {J Biol Chem},
volume = {278},
number = {27},
pages = {24314-24323},
abstract = {Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.},
note = {0021-9258
Journal Article},
keywords = {Cultured, FLORENTZ, Genetic Tumor Cells, Human Mitochondria/genetics *Mutation Nuclear Proteins/*genetics Proteomics RNA/*genetics RNA, Non-U.S. Gov't Translation, Transfer/*genetics Structure-Activity Relationship Support, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.
Sohm B, Frugier M, Brule H, Olszak K, Przykorska A, Florentz C
Towards understanding human mitochondrial leucine aminoacylation identity Journal Article
In: J Mol Biol, vol. 328, no. 5, pp. 995-1010, 2003, ISBN: 12729737, (0022-2836 Journal Article).
Abstract | Links | BibTeX | Tags: Base Sequence Human In Vitro Leucine/*metabolism Leucine-tRNA Ligase/*metabolism Mitochondria/*metabolism Mitochondrial Diseases/genetics/metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation RNA, ERIANI, FLORENTZ, FRUGIER, Leu/chemistry/genetics/*metabolism Recombinant Proteins/genetics/metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Variation (Genetics), Transfer, Unité ARN
@article{,
title = {Towards understanding human mitochondrial leucine aminoacylation identity},
author = {B Sohm and M Frugier and H Brule and K Olszak and A Przykorska and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12729737},
isbn = {12729737},
year = {2003},
date = {2003-01-01},
journal = {J Mol Biol},
volume = {328},
number = {5},
pages = {995-1010},
abstract = {Specific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems. Only restricted information is available for aminoacylation of human mitochondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes. A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs. Here, structural and aminoacylation properties of in vitro transcribed tRNA(Leu(UUR)) are presented. Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch. Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA(Leu(UUR)) is able to adapt to the synthetase. In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements. Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli. The impact of a subset of pathology-related mutations on aminoacylation and on tRNA structure, has been explored. These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects. Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively).},
note = {0022-2836
Journal Article},
keywords = {Base Sequence Human In Vitro Leucine/*metabolism Leucine-tRNA Ligase/*metabolism Mitochondria/*metabolism Mitochondrial Diseases/genetics/metabolism Molecular Sequence Data Mutation Nucleic Acid Conformation RNA, ERIANI, FLORENTZ, FRUGIER, Leu/chemistry/genetics/*metabolism Recombinant Proteins/genetics/metabolism Solutions Substrate Specificity Support, Non-U.S. Gov't Variation (Genetics), Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Specific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems. Only restricted information is available for aminoacylation of human mitochondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes. A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs. Here, structural and aminoacylation properties of in vitro transcribed tRNA(Leu(UUR)) are presented. Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch. Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA(Leu(UUR)) is able to adapt to the synthetase. In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements. Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli. The impact of a subset of pathology-related mutations on aminoacylation and on tRNA structure, has been explored. These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects. Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively).
Levinger L, Giege R, Florentz C
Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro Journal Article
In: Nucleic Acids Res, vol. 31, no. 7, pp. 1904-1912, 2003, ISBN: 12655007, (1362-4962 Journal Article).
Abstract | Links | BibTeX | Tags: Base Sequence DNA, FLORENTZ, Ile/*genetics/metabolism Support, Mitochondrial/*genetics Endoribonucleases/metabolism Hela Cells Human Kinetics Molecular Sequence Data Mutation RNA Precursors/genetics/metabolism *RNA Processing, Non-U.S. Gov't Support, P.H.S., Post-Transcriptional RNA, Transfer, U.S. Gov't, Unité ARN
@article{,
title = {Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro},
author = {L Levinger and R Giege and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12655007},
isbn = {12655007},
year = {2003},
date = {2003-01-01},
journal = {Nucleic Acids Res},
volume = {31},
number = {7},
pages = {1904-1912},
abstract = {The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA(Ile) precursor 3' end maturation in vitro by 3'-tRNase. Strikingly, four pathogenic tRNA(Ile) mutations reduce 3'-tRNase processing efficiency (V(max) / K(M)) to approximately 10-fold below that of wild-type, principally due to lower V(max). The structural impact of mutations was sought by secondary structure probing and wild-type tRNA(Ile) precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA(Ile) precursors with the greatest 3' end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA(Ile) precursor 3' end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.},
note = {1362-4962
Journal Article},
keywords = {Base Sequence DNA, FLORENTZ, Ile/*genetics/metabolism Support, Mitochondrial/*genetics Endoribonucleases/metabolism Hela Cells Human Kinetics Molecular Sequence Data Mutation RNA Precursors/genetics/metabolism *RNA Processing, Non-U.S. Gov't Support, P.H.S., Post-Transcriptional RNA, Transfer, U.S. Gov't, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA(Ile) precursor 3' end maturation in vitro by 3'-tRNase. Strikingly, four pathogenic tRNA(Ile) mutations reduce 3'-tRNase processing efficiency (V(max) / K(M)) to approximately 10-fold below that of wild-type, principally due to lower V(max). The structural impact of mutations was sought by secondary structure probing and wild-type tRNA(Ile) precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA(Ile) precursors with the greatest 3' end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA(Ile) precursor 3' end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.
Florentz C, Sohm B, Tryoen-Toth P, Putz J, Sissler M
Human mitochondrial tRNAs in health and disease Journal Article
In: Cell Mol Life Sci, vol. 60, no. 7, pp. 1356-1375, 2003, ISBN: 12943225, (1420-682x Journal Article Review Review, Academic).
Abstract | Links | BibTeX | Tags: Base Sequence Genetic Diseases, FLORENTZ, Genetic, Inborn/*genetics Genome Human Mitochondria/*genetics Molecular Sequence Data Nucleic Acid Conformation RNA/chemistry/*genetics RNA, Messenger/genetics/metabolism RNA, Non-U.S. Gov't Translation, SISSLER, Transfer/chemistry/*genetics Reference Values Support, Unité ARN
@article{,
title = {Human mitochondrial tRNAs in health and disease},
author = {C Florentz and B Sohm and P Tryoen-Toth and J Putz and M Sissler},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12943225},
isbn = {12943225},
year = {2003},
date = {2003-01-01},
journal = {Cell Mol Life Sci},
volume = {60},
number = {7},
pages = {1356-1375},
abstract = {The human mitochondrial genome encodes 13 proteins, all subunits of the respiratory chain complexes and thus involved in energy metabolism. These genes are translated by 22 transfer RNAs (tRNAs), also encoded by the mitochondrial genome, which form the minimal set required for reading all codons. Human mitochondrial tRNAs gained interest with the rapid discovery of correlations between point mutations in their genes and various neuromuscular and neurodegenerative disorders. In this review, emerging fundamental knowledge on the structure/function relationships of these particular tRNAs and an overview of the large variety of mechanisms within translation, affected by mutations, are summarized. Also, initial results on wide-ranging molecular consequences of mutations outside the frame of mitochondrial translation are highlighted. While knowledge of mitochondrial tRNAs in both health and disease increases, deciphering the intricate network of events leading different genotypes to the variety of phenotypes requires further investigation using adapted model systems.},
note = {1420-682x
Journal Article
Review
Review, Academic},
keywords = {Base Sequence Genetic Diseases, FLORENTZ, Genetic, Inborn/*genetics Genome Human Mitochondria/*genetics Molecular Sequence Data Nucleic Acid Conformation RNA/chemistry/*genetics RNA, Messenger/genetics/metabolism RNA, Non-U.S. Gov't Translation, SISSLER, Transfer/chemistry/*genetics Reference Values Support, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The human mitochondrial genome encodes 13 proteins, all subunits of the respiratory chain complexes and thus involved in energy metabolism. These genes are translated by 22 transfer RNAs (tRNAs), also encoded by the mitochondrial genome, which form the minimal set required for reading all codons. Human mitochondrial tRNAs gained interest with the rapid discovery of correlations between point mutations in their genes and various neuromuscular and neurodegenerative disorders. In this review, emerging fundamental knowledge on the structure/function relationships of these particular tRNAs and an overview of the large variety of mechanisms within translation, affected by mutations, are summarized. Also, initial results on wide-ranging molecular consequences of mutations outside the frame of mitochondrial translation are highlighted. While knowledge of mitochondrial tRNAs in both health and disease increases, deciphering the intricate network of events leading different genotypes to the variety of phenotypes requires further investigation using adapted model systems.
Florentz C, Sissler M
Mitochondrial tRNA aminoacylation and human diseases. Book Chapter
In: Lapointe, J; Brakier-Gringas, L (Ed.): Translation Mechanisms, pp. 129-143, Landes Bioscience, 2003.
Abstract | Links | BibTeX | Tags: FLORENTZ, SISSLER, Unité ARN
@inbook{,
title = {Mitochondrial tRNA aminoacylation and human diseases.},
author = {C Florentz and M Sissler},
editor = {J Lapointe and L Brakier-Gringas},
url = {http://www.landesbioscience.com/curie/chapter/911},
year = {2003},
date = {2003-01-01},
booktitle = {Translation Mechanisms},
pages = {129-143},
publisher = {Landes Bioscience},
abstract = {The human mitochondrial (mt) genome encodes for only 13 proteins which are all subunits of transmembranar respiratory chain complexes. These complexes contribute to a major mt functions namely the synthesis of energy in the way of ATP. Translation of the mRNAs is performed by a set of 22 tRNAs, also encoded by the mt genome, and aminoacylated by nuclear encoded aminoacyl-tRNA synthetases imported into the mitochondria. More and more point mutations affecting mt tRNA genes are reported as correlated to severe neurodegenerative disorders. Since these mutations generally lead to decreased mt protein synthesis, understanding the genotype/phenotype relationships is primarily based on investigation of the possible impacts of individual mutations on various aspects of tRNA structural and functional properties. Here, the present knowledge on human mt aminoacylation systems, as well as the strategies developed to investigate mt aminoacylation, and the effects of point mutations in tRNAs on this process are reviewed. The diversity in the effects observed so far, for a same mutation as well as for various mutations, highlights the ongoing technical limitations in studying human mt aminoacylation. They also suggest that aminoacylation may be a focus for therapeutic strategies for some mutations, while the impact of other mutations needs to be searched as well at other levels of the tRNA structure/function relationship as at unforeseen levels in mitochondria.},
keywords = {FLORENTZ, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
The human mitochondrial (mt) genome encodes for only 13 proteins which are all subunits of transmembranar respiratory chain complexes. These complexes contribute to a major mt functions namely the synthesis of energy in the way of ATP. Translation of the mRNAs is performed by a set of 22 tRNAs, also encoded by the mt genome, and aminoacylated by nuclear encoded aminoacyl-tRNA synthetases imported into the mitochondria. More and more point mutations affecting mt tRNA genes are reported as correlated to severe neurodegenerative disorders. Since these mutations generally lead to decreased mt protein synthesis, understanding the genotype/phenotype relationships is primarily based on investigation of the possible impacts of individual mutations on various aspects of tRNA structural and functional properties. Here, the present knowledge on human mt aminoacylation systems, as well as the strategies developed to investigate mt aminoacylation, and the effects of point mutations in tRNAs on this process are reviewed. The diversity in the effects observed so far, for a same mutation as well as for various mutations, highlights the ongoing technical limitations in studying human mt aminoacylation. They also suggest that aminoacylation may be a focus for therapeutic strategies for some mutations, while the impact of other mutations needs to be searched as well at other levels of the tRNA structure/function relationship as at unforeseen levels in mitochondria.
Du W, Marsac C, Kruschina M, Ortigao F, Florentz C
Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations Journal Article
In: Anal Biochem, vol. 322, no. 1, pp. 14-25, 2003, ISBN: 14705775, (0003-2697 Journal Article).
Abstract | Links | BibTeX | Tags: Alleles *Gold Human MELAS Syndrome/genetics MERRF Syndrome/genetics Nucleic Acid Hybridization/genetics Oligonucleotide Array Sequence Analysis Point Mutation/*genetics Polymerase Chain Reaction Polymorphism, FLORENTZ, Non-U.S. Gov't, Single Nucleotide/*genetics RNA/*genetics RNA, Transfer/*genetics Support, Unité ARN
@article{,
title = {Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations},
author = {W Du and C Marsac and M Kruschina and F Ortigao and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14705775},
isbn = {14705775},
year = {2003},
date = {2003-01-01},
journal = {Anal Biochem},
volume = {322},
number = {1},
pages = {14-25},
abstract = {We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA(Leu(UUR)) and tRNA(Lys) genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.},
note = {0003-2697
Journal Article},
keywords = {Alleles *Gold Human MELAS Syndrome/genetics MERRF Syndrome/genetics Nucleic Acid Hybridization/genetics Oligonucleotide Array Sequence Analysis Point Mutation/*genetics Polymerase Chain Reaction Polymorphism, FLORENTZ, Non-U.S. Gov't, Single Nucleotide/*genetics RNA/*genetics RNA, Transfer/*genetics Support, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA(Leu(UUR)) and tRNA(Lys) genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.
2002
Florentz C
Molecular investigations on tRNAs involved in human mitochondrial disorders Journal Article
In: Biosci Rep, vol. 22, no. 1, pp. 81-98, 2002, ISBN: 12418552, (0144-8463 Journal Article Review Review Literature).
Abstract | Links | BibTeX | Tags: FLORENTZ, Human Mitochondrial Diseases/*genetics/*physiopathology Mutation Nucleic Acid Conformation RNA, Non-U.S. Gov't, Transfer/*chemistry/*genetics Support, Unité ARN
@article{,
title = {Molecular investigations on tRNAs involved in human mitochondrial disorders},
author = {C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12418552},
isbn = {12418552},
year = {2002},
date = {2002-01-01},
journal = {Biosci Rep},
volume = {22},
number = {1},
pages = {81-98},
abstract = {Over the last decade, human neurodegenerative disorders which correlate with point mutations in mitochondrial tRNA genes became more and more numerous. Both the number of mutations (more than 70) and the variety of phenotypes (cardiopathies, myopathies, encephalopathies as well as diabetes, deafness or others) render the understanding of the genotype/phenotype relationships very complex. Here we first summarize the efforts undertaken to decipher the initial impact of various mutations on the structure/function relationships of tRNAs. This includes several lines of research, namely (i) investigation of human mitochrondrial tRNA structures, (ii) comparison of disease-related and polymorphic mutations at a theoretical level, and (iii) experimental investigations of affected tRNAs in the frame of mitochondrial protein synthesis. A new approach aimed at searching for long-range effects of mitochondrial tRNA mutations on a broader global mitochondrial level will also be presented. Initial results obtained by comparative mitochondrial proteomics turn out to be very promising for deciphering unexpected molecular partners involved in the pathological status of the mitochondria.},
note = {0144-8463
Journal Article
Review
Review Literature},
keywords = {FLORENTZ, Human Mitochondrial Diseases/*genetics/*physiopathology Mutation Nucleic Acid Conformation RNA, Non-U.S. Gov't, Transfer/*chemistry/*genetics Support, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Over the last decade, human neurodegenerative disorders which correlate with point mutations in mitochondrial tRNA genes became more and more numerous. Both the number of mutations (more than 70) and the variety of phenotypes (cardiopathies, myopathies, encephalopathies as well as diabetes, deafness or others) render the understanding of the genotype/phenotype relationships very complex. Here we first summarize the efforts undertaken to decipher the initial impact of various mutations on the structure/function relationships of tRNAs. This includes several lines of research, namely (i) investigation of human mitochrondrial tRNA structures, (ii) comparison of disease-related and polymorphic mutations at a theoretical level, and (iii) experimental investigations of affected tRNAs in the frame of mitochondrial protein synthesis. A new approach aimed at searching for long-range effects of mitochondrial tRNA mutations on a broader global mitochondrial level will also be presented. Initial results obtained by comparative mitochondrial proteomics turn out to be very promising for deciphering unexpected molecular partners involved in the pathological status of the mitochondria.
Rabilloud T, Strub J M, Carte N, Luche S, Dorsselaer A Van, Lunardi J, Giege R, Florentz C
Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders Journal Article
In: Biochemistry, vol. 41, no. 1, pp. 144-150, 2002, ISBN: 11772011, (0006-2960 Journal Article).
Abstract | Links | BibTeX | Tags: Amino Acid Sequence Cell Line Cell Nucleus/physiology Comparative Study DNA, FLORENTZ, Gel, Inborn/*metabolism Human Mitochondria/*metabolism Mitochondrial Proteins/*metabolism Molecular Sequence Data *Point Mutation Proteome RNA/*genetics RNA, Mitochondrial/physiology Electrophoresis, Non-U.S. Gov't, Transfer/*genetics Support, Two-Dimensional/methods Genetic Diseases, Unité ARN
@article{,
title = {Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders},
author = {T Rabilloud and J M Strub and N Carte and S Luche and A Van Dorsselaer and J Lunardi and R Giege and C Florentz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11772011},
isbn = {11772011},
year = {2002},
date = {2002-01-01},
journal = {Biochemistry},
volume = {41},
number = {1},
pages = {144-150},
abstract = {More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.},
note = {0006-2960
Journal Article},
keywords = {Amino Acid Sequence Cell Line Cell Nucleus/physiology Comparative Study DNA, FLORENTZ, Gel, Inborn/*metabolism Human Mitochondria/*metabolism Mitochondrial Proteins/*metabolism Molecular Sequence Data *Point Mutation Proteome RNA/*genetics RNA, Mitochondrial/physiology Electrophoresis, Non-U.S. Gov't, Transfer/*genetics Support, Two-Dimensional/methods Genetic Diseases, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.
2000
Wientges J, Putz J, Giege R, Florentz C, Schwienhorst A
Selection of viral RNA-derived tRNA-like structures with improved valylation activities Journal Article
In: Biochemistry, vol. 39, no. 20, pp. 6207-6218, 2000, ISBN: 10821696, (0006-2960 Journal Article).
Abstract | Links | BibTeX | Tags: 3' Untranslated Regions Acylation Anticodon/chemistry Base Sequence Cloning, FLORENTZ, Molecular Gene Library Kinetics Molecular Sequence Data Nucleic Acid Conformation Oligonucleotides/chemistry RNA, Non-U.S. Gov't Tymovirus/enzymology/genetics Valine-tRNA Ligase/chemistry Variation (Genetics), RNA Support, Transfer, Unité ARN, Val/*chemistry RNA, Viral/*chemistry Sequence Analysis
@article{,
title = {Selection of viral RNA-derived tRNA-like structures with improved valylation activities},
author = {J Wientges and J Putz and R Giege and C Florentz and A Schwienhorst},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10821696},
isbn = {10821696},
year = {2000},
date = {2000-01-01},
journal = {Biochemistry},
volume = {39},
number = {20},
pages = {6207-6218},
abstract = {The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS). This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain. Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains. Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1. After nine rounds of selection by aminoacylation, 42 have been isolated. Among them, 17 RNAs could be efficiently charged by yeast ValRS. Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53). A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered. The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination. All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant. This is in line with the role of the TLS in viral replication.},
note = {0006-2960
Journal Article},
keywords = {3' Untranslated Regions Acylation Anticodon/chemistry Base Sequence Cloning, FLORENTZ, Molecular Gene Library Kinetics Molecular Sequence Data Nucleic Acid Conformation Oligonucleotides/chemistry RNA, Non-U.S. Gov't Tymovirus/enzymology/genetics Valine-tRNA Ligase/chemistry Variation (Genetics), RNA Support, Transfer, Unité ARN, Val/*chemistry RNA, Viral/*chemistry Sequence Analysis},
pubstate = {published},
tppubtype = {article}
}
The tRNA-like structure (TLS) of turnip yellow mosaic virus (TYMV) RNA was previously shown to be efficiently charged by yeast valyl-tRNA synthetase (ValRS). This RNA has a noncanonical structure at its 3'-terminus but mimics a tRNA L-shaped fold, including an anticodon loop containing the major identity nucleotides for valylation, and a pseudoknotted amino acid accepting domain. Here we describe an in vitro selection experiment aimed (i) to verify the completeness of the valine identity set, (ii) to elucidate the impact of the pseudoknot on valylation, and (iii) to investigate whether functional communication exists between the two distal anticodon and amino acid accepting domains. Valylatable variants were selected from a pool of 2 x 10(13) RNA molecules derived from the TYMV TLS randomized in the anticodon loop nucleotides and in the length (1-6 nucleotides) and sequence of the pseudoknot loop L1. After nine rounds of selection by aminoacylation, 42 have been isolated. Among them, 17 RNAs could be efficiently charged by yeast ValRS. Their sequence revealed strong conservation of the second and the third anticodon triplet positions (A(56), C(55)) and the very 3'-end loop nucleotide C(53). A large variability of the other nucleotides of the loop was observed and no wild-type sequence was recovered. The selected molecules presented pseudoknot domains with loop L1 varying in size from 3-6 nucleotides and some sequence conservation, but did neither reveal the wild-type combination. All selected variants are 5-50 times more efficiently valylated than the wild-type TLS, suggesting that the natural viral sequence has emerged from a combination of evolutionary pressures among which aminoacylation was not predominant. This is in line with the role of the TLS in viral replication.