136 results on '"Motorin Y"'
Search Results
2. DYNAMIC RIBOSOMES IN OSTEOARTHRITIS
- Author
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Chabronova, A, van den Akker, GG, Housmans, BA, Caron, MM, Cremers, A, Surtel, DA, Peffers, MJ, van Rhijn, LW, Marchand, V, Motorin, Y, and Welting, TJ
- Subjects
Rheumatology ,Biomedical Engineering ,Orthopedics and Sports Medicine - Published
- 2022
3. Ribosomal 2’-O-methylation profiling reveals cancer-associated ribosomal signature in clinical prostate cancer
- Author
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Barros Silva, D., primary, Marchand, V., additional, Jerónimo, C., additional, Jenster, G., additional, Motorin, Y., additional, and Martens-Uzunova, E.S., additional
- Published
- 2022
- Full Text
- View/download PDF
4. Evidence of specialized ribosomes in osteoarthritic chondrocytes
- Author
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Chabronova, A., primary, Akker, G.G., additional, Ripmeester, E.G., additional, Housmans, B.A., additional, Cremers, A., additional, Surtel, D.A., additional, Wichapong, K., additional, Rhijn, L.W., additional, Marchand, V., additional, Motorin, Y., additional, and Welting, T.J., additional
- Published
- 2021
- Full Text
- View/download PDF
5. Mapping rRNA 2’-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants
- Author
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Azevedo-Favory, J., primary, Gaspin, C., additional, Ayadi, L., additional, Montacié, C., additional, Marchand, V., additional, Jobet, E., additional, Rompais, M., additional, Carapito, C., additional, Motorin, Y., additional, and Sáez-Vásquez, J., additional
- Published
- 2021
- Full Text
- View/download PDF
6. Positioning Europe for the EPITRANSCRIPTOMICS challenge
- Author
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Jantsch, M, Quattrone, A, O'Connell, M, Helm, M, Frye, M, Macias-Gonzales, M, Ohman, M, Ameres, S, Willems, L, Fuks, F, Oulas, A, Vanacova, S, Nielsen, H, Bousquet-Antonelli, C, Motorin, Y, Roignant, J, Balatsos, N, Dinnyes, A, Baranov, P, Kelly, V, Lamm, A, Rechavi, G, Pelizzola, M, Liepins, J, Kholodnyuk, I, Zammit, V, Ayers, D, Drablos, F, Dahl, J, Bujnicki, J, Jeronimo, C, Almeida, R, Neagu, M, Costache, M, Bankovic, J, Banovic, B, Kyselovic, J, Valor, L, Selbert, S, Pir, P, Demircan, T, Cowling, V, Schäfer, M, Rossmanith, W, Lafontaine, D, David, A, Carre, C, Lyko, F, Schaffrath, R, Schwartz, S, Verdel, A, Klungland, A, Purta, E, Timotijevic, G, Cardona, F, Davalos, A, Ballana, E, O Carroll, D, Ule, J, Fray, R, Jantsch MF, Quattrone A, O'Connell M, Helm M, Frye M, Macias-Gonzales M, Ohman M, Ameres S, Willems L, Fuks F, Oulas A, Vanacova S, Nielsen H, Bousquet-Antonelli C, Motorin Y, Roignant J, Balatsos N, Dinnyes A, Baranov P, Kelly V, Lamm A, Rechavi G, Pelizzola M, Liepins J, Kholodnyuk IH, Zammit V, Ayers D, Drablos F, Dahl JA, Bujnicki J, Jeronimo C, Almeida R, Neagu M, Costache M, Bankovic J, Banovic B, Kyselovic J, Valor LM, Selbert S, Pir P, Demircan T, Cowling V, Schäfer M, Rossmanith W, Lafontaine D, David A, Carre C, Lyko F, Schaffrath R, Schwartz S, Verdel A, Klungland A, Purta E, Timotijevic G, Cardona F, Davalos A, Ballana E, O Carroll D, Ule J, Fray R, Jantsch, M, Quattrone, A, O'Connell, M, Helm, M, Frye, M, Macias-Gonzales, M, Ohman, M, Ameres, S, Willems, L, Fuks, F, Oulas, A, Vanacova, S, Nielsen, H, Bousquet-Antonelli, C, Motorin, Y, Roignant, J, Balatsos, N, Dinnyes, A, Baranov, P, Kelly, V, Lamm, A, Rechavi, G, Pelizzola, M, Liepins, J, Kholodnyuk, I, Zammit, V, Ayers, D, Drablos, F, Dahl, J, Bujnicki, J, Jeronimo, C, Almeida, R, Neagu, M, Costache, M, Bankovic, J, Banovic, B, Kyselovic, J, Valor, L, Selbert, S, Pir, P, Demircan, T, Cowling, V, Schäfer, M, Rossmanith, W, Lafontaine, D, David, A, Carre, C, Lyko, F, Schaffrath, R, Schwartz, S, Verdel, A, Klungland, A, Purta, E, Timotijevic, G, Cardona, F, Davalos, A, Ballana, E, O Carroll, D, Ule, J, Fray, R, Jantsch MF, Quattrone A, O'Connell M, Helm M, Frye M, Macias-Gonzales M, Ohman M, Ameres S, Willems L, Fuks F, Oulas A, Vanacova S, Nielsen H, Bousquet-Antonelli C, Motorin Y, Roignant J, Balatsos N, Dinnyes A, Baranov P, Kelly V, Lamm A, Rechavi G, Pelizzola M, Liepins J, Kholodnyuk IH, Zammit V, Ayers D, Drablos F, Dahl JA, Bujnicki J, Jeronimo C, Almeida R, Neagu M, Costache M, Bankovic J, Banovic B, Kyselovic J, Valor LM, Selbert S, Pir P, Demircan T, Cowling V, Schäfer M, Rossmanith W, Lafontaine D, David A, Carre C, Lyko F, Schaffrath R, Schwartz S, Verdel A, Klungland A, Purta E, Timotijevic G, Cardona F, Davalos A, Ballana E, O Carroll D, Ule J, and Fray R
- Abstract
The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.
- Published
- 2018
7. O07 - Ribosomal 2’-O-methylation profiling reveals cancer-associated ribosomal signature in clinical prostate cancer
- Author
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Barros Silva, D., Marchand, V., Jerónimo, C., Jenster, G., Motorin, Y., and Martens-Uzunova, E.S.
- Published
- 2022
- Full Text
- View/download PDF
8. PO-151 Variation of ribosome composition and translational reprogramming during human mammary epithelial-to-mesenchymal transition
- Author
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Nguyen Van Long, F., primary, Pion, N., additional, Couté, Y., additional, Morel, A.P., additional, Marchand, V., additional, Motorin, Y., additional, Namy, O., additional, Puisieux, A., additional, Diaz, J.J., additional, and Marcel, V., additional
- Published
- 2018
- Full Text
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9. The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent
- Author
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Hauenschild, R, Tserovski, L, Schmid, K, Thuring, K, Winz, M, Sharma, Sunny, Entian, Karl-Dieter, Wacheul, Ludivine, Lafontaine, Denis, Anderson, J, Alfonzo, J, Hildebrandt, A, Jaschke, A, Motorin, Y, Helm, Mark, Hauenschild, R, Tserovski, L, Schmid, K, Thuring, K, Winz, M, Sharma, Sunny, Entian, Karl-Dieter, Wacheul, Ludivine, Lafontaine, Denis, Anderson, J, Alfonzo, J, Hildebrandt, A, Jaschke, A, Motorin, Y, and Helm, Mark
- Abstract
The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m1A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3′ of m1A in the template RNA, meaning it is sequence dependent. The RT-signature of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA., SCOPUS: ar.j, info:eu-repo/semantics/published
- Published
- 2015
10. Deficiency of the tRNATyr :Psi35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery
- Author
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Muller, S., Urban, A., Hecker, A., Leclerc, F., Branlant, C., Motorin, Y., SERRE, Marie-Claude, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,[SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 2009
11. Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling
- Author
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Motorin, Y., primary, Burhenne, J., additional, Teimer, R., additional, Koynov, K., additional, Willnow, S., additional, Weinhold, E., additional, and Helm, M., additional
- Published
- 2010
- Full Text
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12. Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.
- Author
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Massenet, S, Motorin, Y, Lafontaine, Denis, Hurt, Eduard C., Grosjean, Henri, Branlant, C, Massenet, S, Motorin, Y, Lafontaine, Denis, Hurt, Eduard C., Grosjean, Henri, and Branlant, C
- Abstract
Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published
- Published
- 1999
13. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of PSI55 in both mitochondrial and cytoplasmic tRNAs.
- Author
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Becker, H.F., Motorin, Y., Planta, R.J., Grosjean, H., Becker, H.F., Motorin, Y., Planta, R.J., and Grosjean, H.
- Published
- 1997
- Full Text
- View/download PDF
14. Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of 2819 in 21S mitochondrial ribosomal RNA
- Author
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Ansmant, I., primary, Massenet, S., additional, Grosjean, H., additional, Motorin, Y., additional, and Branlant, C., additional
- Published
- 2000
- Full Text
- View/download PDF
15. Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNAPhe
- Author
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Khvorova, A., primary, Motorin, Y., additional, and Wolfson, A. D., additional
- Published
- 1999
- Full Text
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16. The tRNA(guanine-26,N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus: cloning, sequencing of the gene and its expression in Escherichia coli
- Author
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Constantinesco, F., primary, Benachenhou, N., additional, Motorin, Y., additional, and Grosjean, H., additional
- Published
- 1998
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17. The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity
- Author
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MOTORIN, Y., primary, KEITH, G., additional, SIMON, C., additional, FOIRET, D., additional, SIMOS, G., additional, HURT, E., additional, and GROSJEAN, H., additional
- Published
- 1998
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18. Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of Ψ2819 in 21S mitochondrial ribosomal RNA.
- Author
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Ansmant, I., Massenet, S., Grosjean, H., Motorin, Y., and Branlant, C.
- Published
- 2000
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19. Pseudouridine and ribothymidine formation in the tRNA‐like domain of turnip yellow mosaic virus RNA.
- Author
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Becker, H. F., Motorin, Y., Florentz, C., Giegé, R., and Grosjean, H.
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- 1998
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20. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of Y55 in both mitochondrial and cytoplasmic tRNAs.
- Author
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Becker, H. F., Motorin, Y., Planta, R. J., and Grosjean, H.
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- 1997
- Full Text
- View/download PDF
21. Pleiotropic effects of intron removal on base modification pattern of yeast tRNAPhe: an in vitro study.
- Author
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Jiang, H.-Q., Motorin, Y., Jin, Y.-X., and Grosjean, H.
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- 1997
- Full Text
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22. Cloning and characterization of the Schizosaccharomyces pombe tRNA:pseudouridine synthase Pus1p.
- Author
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Hellmuth, K, Grosjean, H, Motorin, Y, Deinert, K, Hurt, E, and Simos, G
- Abstract
Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S. cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.
- Published
- 2000
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- View/download PDF
23. Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA.
- Author
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Ansmant, I, Massenet, S, Grosjean, H, Motorin, Y, and Branlant, C
- Abstract
So far, four RNA:pseudouridine (Psi)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Psi-synthases responsible for several U-->Psi conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Psi-synthases, four additional open reading frames (ORFs) encoding putative RNA:Psi-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Psi residue normally present in the fully matured mitochondrial rRNAs (Psi(2819)in 21S rRNA) was missing, while Psi residues at all the tested pseudo-uridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U-->Psi conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71-->A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Psi(2819)synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Psi-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Psi(2819)in mitochondrial 21S rRNA is not essential for cell viability.
- Published
- 2000
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24. Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of Ψ<SUB>2819</SUB> in 21S mitochondrial ribosomal RNA
- Author
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Grosjean, H., Motorin, Y., Ansmant, I., Massenet, S., and Branlant, C.
- Abstract
So far, four RNA:pseudouridine (Ψ)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Ψ-synthases responsible for several U→Ψ conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Ψ-synthases, four additional open reading frames (ORFs) encoding putative RNA:Ψ-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Ψ residue normally present in the fully matured mitochondrial rRNAs (Ψ2819 in 21S rRNA) was missing, while Ψ residues at all the tested pseudouridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U→Ψ conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71→A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Ψ2819 synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5and the corresponding RNA:Ψ-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Ψ2819 in mitochondrial 21S rRNA is not essential for cell viability.
- Published
- 2000
25. Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro.
- Author
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Constantinesco, F, Motorin, Y, and Grosjean, H
- Abstract
The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with S -adenosyl-l-methionine (AdoMet). The results indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro. They correspond to the formation of pseudouridines (Psi) at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and 56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49 (m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of adenosine into m1I at position 57. Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am6, Cm56and m1I57are specific modifications found exclusively in Archaea. It is also shown that the enzymatic formation of m5C49, m5U54, Psi55and m1I57does not depend on the three-dimensional architecture of the tRNA substrate, since these modi-fications also occur in fragmented tRNAs as substrate.
- Published
- 1999
- Full Text
- View/download PDF
26. Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop.
- Author
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Lecointe, F, Simos, G, Sauer, A, Hurt, E C, Motorin, Y, and Grosjean, H
- Abstract
The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).
- Published
- 1998
27. Two human valyl-tRNA synthetase-encoding cDNA sequences deposited in GenBank display extensive differences
- Author
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Motorin, Y.
- Published
- 1996
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28. Crucial role of pyrophosphate in the aminoacylation of E. coli tRNA^P^h^e by yeast phenylalanyl-tRNA synthetase
- Author
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Khvorova, A. M., Motorin, Y., and Wolfson, A. D.
- Published
- 1992
- Full Text
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29. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of {Psi}55 in both mitochondrial and cytoplasmic tRNAs
- Author
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Becker, H. F., Motorin, Y., Planta, R. J., and Grosjean, H.
- Abstract
The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w and CBF5) display a remarkable sequence homology with Escherichia coli tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene YNL292w coding for one of these proteins was cloned in an E.coli expression vector downstream of a His
6 -tag. The resulting recombinant protein (Pus4) was expressed at high level and purified to homogeneity by metal affinity chromatography on Ni2+-NTA-agarose, followed by ion-exchange chromatography on MonoQ. The purified Pus4p catalyzes the formation of pseudouridine-55 in T7 in vitro transcripts of several yeast tRNA genes. In contrast to the known yeast pseudouridine synthase (Pus1) of broad specificity, no other uridines in tRNA molecules are modified by the cloned recombinant tRNA:Ψ55 synthase. The disruption of the corresponding gene YNL292w in yeast, which has no significant effect on the growth of yeast cells, leads to the complete disappearance of the Ψ55 formation activity in a cell-free extract. These results allow the formal identification of the protein encoded by the yeast ORF YNL292w as the only enzyme responsible for the formation of Ψ55 which is almost universally conserved in tRNAs. The substrate specificity of the purified YNL292w-encoded recombinant protein was shown to be similar to that of the native protein present in yeast cell extract. Chemical mapping of pseudouridine residues in both cytoplasmic and mitochondrial tRNAs from the yeast strain carrying the disrupted gene reveals that the same gene product is responsible for Ψ55 formation in tRNAs of both cellular compartments.- Published
- 1997
- Full Text
- View/download PDF
30. DORQ-seq: high-throughput quantification of femtomol tRNA pools by combination of cDNA hybridization and Deep sequencing.
- Author
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Kristen M, Lander M, Kilz LM, Gleue L, Jörg M, Bregeon D, Hamdane D, Marchand V, Motorin Y, Friedland K, and Helm M
- Subjects
- Humans, Alzheimer Disease genetics, Sequence Analysis, RNA methods, Brain metabolism, RNA, Transfer genetics, RNA, Transfer metabolism, High-Throughput Nucleotide Sequencing methods, DNA, Complementary genetics, Nucleic Acid Hybridization methods
- Abstract
Due to its high modification content tRNAs are notoriously hard to quantify by reverse transcription and RNAseq. Bypassing numerous biases resulting from concatenation of enzymatic treatments, we here report a hybrid approach that harnesses the advantages of hybridization-based and deep sequencing-based approaches. The method renders obsolete any RNAseq related workarounds and correction factors that affect accuracy, sensitivity, and turnaround time. Rather than by reverse transcription, quantitative information on the isoacceptor composition of a tRNA pool is transferred to a cDNA mixture in a single step procedure, thereby omitting all enzymatic conversations except for the subsequent barcoding PCR. As a result, a detailed tRNA composition matrix can be obtained from femtomolar amounts of total tRNA. The method is fast, low in cost, and its bioinformatic data workup surprisingly simple. These properties make the approach amenable to high-throughput investigations including clinical samples, as we have demonstrated by application to a collection of variegated biological questions, each answered with novel findings. These include tRNA pool quantification of polysome-bound tRNA, of tRNA modification knockout strains under stress conditions, and of Alzheimer patients' brain tissues., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
- Full Text
- View/download PDF
31. Functional redundancy in tRNA dihydrouridylation.
- Author
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Sudol C, Kilz LM, Marchand V, Thullier Q, Guérineau V, Goyenvalle C, Faivre B, Toubdji S, Lombard M, Jean-Jean O, de Crécy-Lagard V, Helm M, Motorin Y, Brégeon D, and Hamdane D
- Subjects
- Bacterial Proteins metabolism, Bacterial Proteins genetics, RNA, Bacterial metabolism, RNA, Bacterial genetics, Gene Expression, Bacillus subtilis enzymology, Bacillus subtilis genetics, RNA, Transfer metabolism, RNA, Transfer genetics, Uridine metabolism, Uridine analogs & derivatives
- Abstract
Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
- Full Text
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32. TGF-β2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.
- Author
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van den Akker GGH, Chabronova A, Housmans BAC, van der Vloet L, Surtel DAM, Cremers A, Marchand V, Motorin Y, Caron MMJ, Peffers MJ, and Welting TJM
- Subjects
- Humans, Internal Ribosome Entry Sites, Cell Line, Chondrocytes metabolism, Chondrocytes drug effects, Ribosomes metabolism, Protein Biosynthesis, RNA, Ribosomal metabolism, RNA, Ribosomal genetics, Transforming Growth Factor beta2 metabolism, Transforming Growth Factor beta2 pharmacology
- Abstract
Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-β2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-β2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.
- Published
- 2024
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33. Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.
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Sklias A, Cruciani S, Marchand V, Spagnuolo M, Lavergne G, Bourguignon V, Brambilla A, Dreos R, Marygold SJ, Novoa EM, Motorin Y, and Roignant JY
- Subjects
- Animals, Ribosomes genetics, Ribosomes metabolism, Base Sequence, RNA, Ribosomal metabolism, Methylation, RNA, Small Nucleolar metabolism, Drosophila melanogaster genetics, Drosophila melanogaster metabolism
- Abstract
During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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34. Mapping the tRNA modification landscape of Bartonella henselae Houston I and Bartonella quintana Toulouse.
- Author
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Quaiyum S, Sun J, Marchand V, Sun G, Reed CJ, Motorin Y, Dedon PC, Minnick MF, and de Crécy-Lagard V
- Abstract
Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens- Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana . Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. Bartonella quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Quaiyum, Sun, Marchand, Sun, Reed, Motorin, Dedon, Minnick and de Crécy-Lagard.)
- Published
- 2024
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35. Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.
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Lucas MC, Pryszcz LP, Medina R, Milenkovic I, Camacho N, Marchand V, Motorin Y, Ribas de Pouplana L, and Novoa EM
- Subjects
- RNA, RNA, Transfer chemistry, Sequence Analysis, RNA methods, Nanopore Sequencing, Nanopores
- Abstract
Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress., (© 2023. The Author(s).)
- Published
- 2024
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36. NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.
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Montacié C, Riondet C, Wei L, Darrière T, Weiss A, Pontvianne F, Escande ML, de Bures A, Jobet E, Barbarossa A, Carpentier MC, Aarts MGM, Attina A, Hirtz C, David A, Marchand V, Motorin Y, Curie C, Mari S, Reichheld JP, and Sáez-Vásquez J
- Subjects
- DNA, Ribosomal metabolism, Methylation, Iron metabolism, RNA, Ribosomal genetics, RNA, Ribosomal metabolism, Arabidopsis genetics, Arabidopsis metabolism
- Abstract
In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2023
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37. Depletion of SNORA33 Abolishes ψ of 28S-U4966 and Affects the Ribosome Translational Apparatus.
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Chabronova A, van den Akker G, Housmans BAC, Caron MMJ, Cremers A, Surtel DAM, Peffers MJ, van Rhijn LW, Marchand V, Motorin Y, and Welting TJM
- Subjects
- Humans, Chromatography, Liquid, Ribosomes genetics, RNA Processing, Post-Transcriptional, RNA, Ribosomal genetics, Proteome genetics, Tandem Mass Spectrometry
- Abstract
Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by
35 S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33 , but not SNORA22 , reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.- Published
- 2023
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38. METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer.
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García-Vílchez R, Añazco-Guenkova AM, Dietmann S, López J, Morón-Calvente V, D'Ambrosi S, Nombela P, Zamacola K, Mendizabal I, García-Longarte S, Zabala-Letona A, Astobiza I, Fernández S, Paniagua A, Miguel-López B, Marchand V, Alonso-López D, Merkel A, García-Tuñón I, Ugalde-Olano A, Loizaga-Iriarte A, Lacasa-Viscasillas I, Unda M, Azkargorta M, Elortza F, Bárcena L, Gonzalez-Lopez M, Aransay AM, Di Domenico T, Sánchez-Martín MA, De Las Rivas J, Guil S, Motorin Y, Helm M, Pandolfi PP, Carracedo A, and Blanco S
- Subjects
- Male, Humans, Cell Transformation, Neoplastic, Transcription, Genetic, RNA Processing, Post-Transcriptional, Methyltransferases genetics, Carcinogenesis genetics, Prostatic Neoplasms genetics
- Abstract
Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N
7 -methylguanosine (m7 G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7 G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7 G tRNA methylation in cancer cell translation control and tumour biology., (© 2023. The Author(s).)- Published
- 2023
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39. A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine.
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Huber LB, Kaur N, Henkel M, Marchand V, Motorin Y, Ehrenhofer-Murray AE, and Marx A
- Subjects
- RNA, Messenger metabolism, RNA, RNA, Untranslated, RNA Processing, Post-Transcriptional, Pseudouridine metabolism, Nucleoside Q
- Abstract
More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis. To overcome the drawbacks associated with indirect detection strategies, we have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for Ψ or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of Ψ and Q sites of untreated RNA samples using a single enzymatic tool., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2023
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40. FUS regulates a subset of snoRNA expression and modulates the level of rRNA modifications.
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Gawade K, Plewka P, Häfner SJ, Lund AH, Marchand V, Motorin Y, Szczesniak MW, and Raczynska KD
- Subjects
- Humans, RNA, Small Nucleolar genetics, HEK293 Cells, RNA, Ribosomal genetics, RNA-Binding Protein FUS genetics, Amyotrophic Lateral Sclerosis, Neuroblastoma
- Abstract
FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2'-O methylation (2'-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2'-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2'-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression., (© 2023. The Author(s).)
- Published
- 2023
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41. The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance.
- Author
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Brazane M, Dimitrova DG, Pigeon J, Paolantoni C, Ye T, Marchand V, Da Silva B, Schaefer E, Angelova MT, Stark Z, Delatycki M, Dudding-Byth T, Gecz J, Plaçais PY, Teysset L, Préat T, Piton A, Hassan BA, Roignant JY, Motorin Y, and Carré C
- Subjects
- Humans, Methylation, Methyltransferases genetics, RNA, Transfer genetics, RNA, Transfer metabolism, Neurons metabolism, Nuclear Proteins genetics, Ribose, Intellectual Disability genetics
- Abstract
FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology., (© 2023 Brazane et al.)
- Published
- 2023
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42. Quantification of substoichiometric modification reveals global tsRNA hypomodification, preferences for angiogenin-mediated tRNA cleavage, and idiosyncratic epitranscriptomes of human neuronal cell-lines.
- Author
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Pichot F, Hogg MC, Marchand V, Bourguignon V, Jirström E, Farrell C, Gibriel HA, Prehn JHM, Motorin Y, and Helm M
- Abstract
Modification of tRNA is an integral part of the epitranscriptome with a particularly pronounced potential to generate diversity in RNA expression. Eukaryotic tRNA contains modifications in up to 20% of their nucleotides, but not all sites are always fully modified. Combinations and permutations of partially modified sites in tRNAs can generate a plethora of tRNA isoforms, termed modivariants. Here, we investigate the stoichiometry of incompletely modified sites in tRNAs from human cell lines for their information content. Using a panel of RNA modification mapping methods, we assess the stoichiometry of sites that contain the modifications 5-methylcytidine (m
5 C), 2'-O-ribose methylation (Nm), 3-methylcytidine (m3 C), 7-methylguanosine (m7 G), and Dihydrouridine (D). We discovered that up to 75% of sites can be incompletely modified and that the differential modification status of a cellular tRNA population holds information that allows to discriminate e.g. different cell lines. As a further aspect, we investigated potential causal connectivity between tRNA modification and its processing into tRNA fragments (tiRNAs and tRFs). Upon exposure of cultured living cells to cell-penetrating angiogenin, the modification patterns of the corresponding RNA populations was changed. Importantly, we also found that tsRNAs were significantly less modified than their parent tRNAs at numerous sites, suggesting that tsRNAs might derive chiefly from hypomodified tRNAs., Competing Interests: Mark Helm is a consultant for Moderna Inc., (© 2022 The Author(s).)- Published
- 2022
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43. Synthesis of point-modified mRNA.
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Hertler J, Slama K, Schober B, Özrendeci Z, Marchand V, Motorin Y, and Helm M
- Subjects
- Methylation, RNA, Messenger chemical synthesis, RNA, Messenger genetics, Sepharose, Nucleotides metabolism, Genetic Engineering methods
- Abstract
Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed 'point modification' in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatable and internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
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44. Pseudouridylation of Epstein-Barr virus noncoding RNA EBER2 facilitates lytic replication.
- Author
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Henry BA, Marchand V, Schlegel BT, Helm M, Motorin Y, and Lee N
- Subjects
- Humans, RNA, Viral genetics, RNA, Untranslated genetics, RNA Stability, Virus Replication genetics, Herpesvirus 4, Human genetics, Epstein-Barr Virus Infections
- Abstract
Epstein-Barr virus (EBV) expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2, which are preserved in all clinical isolates of EBV, thus underscoring their essential function in the viral life cycle. Recent epitranscriptomics studies have uncovered a vast array of distinct RNA modifications within cellular as well as viral noncoding RNAs that are instrumental in executing their function. Here we show that EBER2 is marked by pseudouridylation, and by using HydraPsiSeq the modification site was mapped to a single nucleotide within the 3' region of EBER2. The writer enzyme was identified to be the snoRNA-dependent pseudouridine synthase Dyskerin, which is the catalytic subunit of H/ACA small nucleolar ribonucleoprotein complexes, and is guided to EBER2 by SNORA22. Similar to other noncoding RNAs for which pseudouridylation has a positive effect on RNA stability, loss of EBER2 pseudouridylation results in a decrease in RNA levels. Furthermore, pseudouridylation of EBER2 is required for the prolific accumulation of progeny viral genomes, suggesting that this single modification in EBER2 is important for efficient viral lytic replication. Taken together, our findings add to the list of RNA modifications that are essential for noncoding RNAs to implement their physiological roles., (© 2022 Henry et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2022
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45. SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA.
- Author
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Maharana S, Kretschmer S, Hunger S, Yan X, Kuster D, Traikov S, Zillinger T, Gentzel M, Elangovan S, Dasgupta P, Chappidi N, Lucas N, Maser KI, Maatz H, Rapp A, Marchand V, Chang YT, Motorin Y, Hubner N, Hartmann G, Hyman AA, Alberti S, and Lee-Kirsch MA
- Subjects
- Antiviral Agents, Autoimmune Diseases of the Nervous System, Exonucleases genetics, Humans, Immunity, Innate genetics, Nervous System Malformations, SAM Domain and HD Domain-Containing Protein 1 genetics, Interferon Type I genetics, RNA, Double-Stranded genetics
- Abstract
Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses., Competing Interests: Declaration of interests A.A.H. is a founder of Dewpoint Therapeutics and Caraway Therapeutics and is an advisor to Dewpoint therapeutics. S.A. is an advisor to Dewpoint Therapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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46. Studies of mutations of assembly factor Hit1 in budding yeast suggest translation defects as the molecular basis for PEHO syndrome.
- Author
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Dreggors-Walker RE, Cohen LN, Khoshnevis S, Marchand V, Motorin Y, and Ghalei H
- Subjects
- Humans, Infant, Newborn, Mutation, RNA, Small Nucleolar metabolism, Brain Edema genetics, Neurodegenerative Diseases genetics, Nuclear Proteins genetics, Optic Atrophy genetics, Ribonucleoproteins, Small Nucleolar genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Spasms, Infantile genetics, Transcription Factors genetics
- Abstract
Regulation of protein synthesis is critical for control of gene expression in all cells. Ribosomes are ribonucleoprotein machines responsible for translating cellular proteins. Defects in ribosome production, function, or regulation are detrimental to the cell and cause human diseases, such as progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome. PEHO syndrome is a devastating neurodevelopmental disorder caused by mutations in the ZNHIT3 gene, which encodes an evolutionarily conserved nuclear protein. The precise mechanisms by which ZNHIT3 mutations lead to PEHO syndrome are currently unclear. Studies of the human zinc finger HIT-type containing protein 3 homolog in budding yeast (Hit1) revealed that this protein is critical for formation of small nucleolar ribonucleoprotein complexes that are required for rRNA processing and 2'-O-methylation. Here, we use budding yeast as a model system to reveal the basis for the molecular pathogenesis of PEHO syndrome. We show that missense mutations modeling those found in PEHO syndrome patients cause a decrease in steady-state Hit1 protein levels, a significant reduction of box C/D snoRNA levels, and subsequent defects in rRNA processing and altered cellular translation. Using RiboMethSeq analysis of rRNAs isolated from actively translating ribosomes, we reveal site-specific changes in the rRNA modification pattern of PEHO syndrome mutant yeast cells. Our data suggest that PEHO syndrome is a ribosomopathy and reveal potential new aspects of the molecular basis of this disease in translation dysregulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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47. Phosphorylation found inside RNA.
- Author
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Helm M and Motorin Y
- Subjects
- Phosphorylation, RNA genetics, RNA, Long Noncoding genetics
- Published
- 2022
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48. Ribosomal RNA 2'- O -methylations regulate translation by impacting ribosome dynamics.
- Author
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Khoshnevis S, Dreggors-Walker RE, Marchand V, Motorin Y, and Ghalei H
- Subjects
- Methylation, RNA metabolism, RNA, Ribosomal metabolism, Ribosomes metabolism
- Abstract
SignificanceThe presence of RNA chemical modifications has long been known, but their precise molecular consequences remain unknown. 2'- O -methylation is an abundant modification that exists in RNA in all domains of life. Ribosomal RNA (rRNA) represents a functionally important RNA that is heavily modified by 2'- O -methylations. Although abundant at functionally important regions of the rRNA, the contribution of 2'- O -methylations to ribosome activities is unknown. By establishing a method to disturb rRNA 2'- O -methylation patterns, we show that rRNA 2'- O -methylations affect the function and fidelity of the ribosome and change the balance between different ribosome conformational states. Our work links 2'- O -methylation to ribosome dynamics and defines a set of critical rRNA 2'- O -methylations required for ribosome biogenesis and others that are dispensable.
- Published
- 2022
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49. Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis.
- Author
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Delhermite J, Tafforeau L, Sharma S, Marchand V, Wacheul L, Lattuca R, Desiderio S, Motorin Y, Bellefroid E, and Lafontaine DLJ
- Subjects
- Animals, Eye growth & development, Eye metabolism, Gene Knockdown Techniques, Methylation, Neural Crest growth & development, Neural Crest metabolism, Neural Plate growth & development, Neural Plate metabolism, RNA Processing, Post-Transcriptional, Xenopus Proteins genetics, Xenopus Proteins metabolism, Xenopus laevis genetics, Chromosomal Proteins, Non-Histone genetics, Chromosomal Proteins, Non-Histone metabolism, RNA Precursors metabolism, RNA, Ribosomal, 18S metabolism, RNA, Ribosomal, 28S metabolism, Xenopus laevis growth & development
- Abstract
Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
- Full Text
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50. Balancing of mitochondrial translation through METTL8-mediated m 3 C modification of mitochondrial tRNAs.
- Author
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Schöller E, Marks J, Marchand V, Bruckmann A, Powell CA, Reichold M, Mutti CD, Dettmer K, Feederle R, Hüttelmaier S, Helm M, Oefner P, Minczuk M, Motorin Y, Hafner M, and Meister G
- Subjects
- Animals, Anticodon, Cell Proliferation, Codon, Cytoplasm, DNA, Mitochondrial metabolism, Electron Transport, Green Fluorescent Proteins metabolism, HEK293 Cells, Humans, Mice, Mitochondria metabolism, Mitochondrial Membranes, Mitochondrial Proteins chemistry, Oxygen Consumption, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms mortality, Ribosomes metabolism, Up-Regulation, Methyltransferases metabolism, RNA, Mitochondrial chemistry, RNA, Transfer chemistry
- Abstract
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m
3 C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr . METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN) - and mt-tRNAThr -dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN) - and mt-tRNAThr -dependent codons through METTL8-mediated m3 C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
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