Yuri Motorin
University of Lorraine
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Featured researches published by Yuri Motorin.
Biochemistry | 2010
Yuri Motorin; Mark Helm
Post-transcriptional ribonucleotide modification is a phenomenon best studied in tRNA, where it occurs most frequently and in great chemical diversity. This paper reviews the intrinsic network of modifications in the structural core of the tRNA, which governs structural flexibility and rigidity to fine-tune the molecule to peak performance and to regulate its steady-state level. Structural effects of RNA modifications range from nanometer-scale rearrangements to subtle restrictions of conformational space on the angstrom scale. Structural stabilization resulting from nucleotide modification results in increased thermal stability and translates into protection against unspecific degradation by bases and nucleases. Several mechanisms of specific degradation of hypomodified tRNA, which were only recently discovered, provide a link between structural and metabolic stability.
Wiley Interdisciplinary Reviews - Rna | 2011
Yuri Motorin; Mark Helm
Methylation of RNA occurs at a variety of atoms, nucleotides, sequences and tertiary structures. Strongly related to other posttranscriptional modifications, methylation of different RNA species includes tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, and viral RNA. Different catalytic strategies are employed for RNA methylation by a variety of RNA‐methyltransferases which fall into four superfamilies. This review outlines the different functions of methyl groups in RNA, including biophysical, biochemical and metabolic stabilization of RNA, quality control, resistance to antibiotics, mRNA reading frame maintenance, deciphering of normal and altered genetic code, selenocysteine incorporation, tRNA aminoacylation, ribotoxins, splicing, intracellular trafficking, immune response, and others. Connections to other fields including gene regulation, DNA repair, stress response, and possibly histone acetylation and exocytosis are pointed out. WIREs RNA 2011 2 611–631 DOI: 10.1002/wrna.79
Nucleic Acids Research | 2010
Yuri Motorin; Frank Lyko; Mark Helm
The nucleobase modification 5-methylcytosine (m5C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m5C-methylation were extensively studied during the last decades. However, the location, the mechanism of formation and the cellular function(s) of the same modified nucleobase in RNA still remain to be elucidated. The recent development of a bisulfite sequencing approach for efficient m5C localization in various RNA molecules puts ribo-m5C in a highly privileged position as one of the few RNA modifications whose detection is amenable to PCR-based amplification and sequencing methods. Additional progress in the field also includes the characterization of several specific RNA methyltransferase enzymes in various organisms, and the discovery of a new and unexpected link between DNA and RNA m5C-methylation. Numerous putative RNA:m5C-MTases have now been identified and are awaiting characterization, including the identification of their RNA substrates and their related cellular functions. In order to bring these recent exciting developments into perspective, this review provides an ordered overview of the detection methods for RNA methylation, of the biochemistry, enzymology and molecular biology of the corresponding modification enzymes, and discusses perspectives for the emerging biological functions of these enzymes.
Molecular and Cellular Biology | 1999
Séverine Massenet; Yuri Motorin; Denis L. J. Lafontaine; Eduard C. Hurt; Henri Grosjean; Christiane Branlant
ABSTRACT Pseudouridine (Ψ) residues were localized in theSaccharomyces 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 Ψ 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 Ψ content, only the loss of the Pus1p activity was found to affect Ψ formation in spliceosomal UsnRNAs. Indeed, Ψ44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Ψ44 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 Ψ content, formation of Ψ residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.
RNA | 1999
Yuri Motorin; Henri Grosjean
Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w.
Nucleic Acids Research | 2011
Yuri Motorin; Jürgen Burhenne; Roman Teimer; Kaloian Koynov; Sophie Willnow; Elmar G. Weinhold; Mark Helm
This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNAPhe. LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments.
RNA | 1998
Yuri Motorin; G. Keith; C. Simon; D. Foiret; G. Simos; Ed Hurt; Henri Grosjean
We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.
Nature Reviews Genetics | 2017
Mark Helm; Yuri Motorin
RNA modifications are emerging players in the field of post-transcriptional regulation of gene expression, and are attracting a comparable degree of research interest to DNA and histone modifications in the field of epigenetics. We now know of more than 150 RNA modifications and the true potential of a few of these is currently emerging as the consequence of a leap in detection technology, principally associated with high-throughput sequencing. This Review outlines the major developments in this field through a structured discussion of detection principles, lays out advantages and drawbacks of new high-throughput methods and presents conventional biophysical identification of modifications as meaningful ways for validation.
Biochimie | 1997
Henri Grosjean; Z. Szweykowska-Kulinska; Yuri Motorin; F. Fasiolo; George Simos
In eukaryotic cells, especially in yeast, several genes encoding tRNAs contain introns. These are removed from pre-tRNAs during the maturation process by a tRNA-specific splicing machinery that is located within the nucleus at the nuclear envelope. Before and after the intron removal, several nucleoside modifications are added in a stepwise manner, but most of them are introduced prior to intron removal. Some of these early nucleoside modifications are catalyzed by intron-dependent enzymes while most of the others are catalyzed in an intron-independent manner. In the present paper, we review all known cases where the nucleoside modifications were shown to depend strictly on the presence of an intron. These are pseudouridines at anticodon positions 34, 35 and 36 and 5-methylcytosine at position 34 of several eukaryotic tRNAs. One common property of the corresponding intron-dependent modifying enzymes is that their activities are essentially dependent on the local specific architecture of the pre-tRNA molecule that comprises the anticodon stem and loop prolonged by the intron domain. Thus introns clearly serve as internal (cis-type) RNAs that guide nucleoside modifications by providing transient target sites in tRNA for selected nuclear modifying enzymes. This situation may be similar to the recently discovered (trans-type) snoRNA-guided process of ribose methylations of ribosomal RNAs within the nucleolus of eukaryotic cells.
Nucleic Acids Research | 2015
Ralf Hauenschild; Lyudmil Tserovski; Katharina Schmid; Kathrin Thüring; Marie-Luise Winz; Sunny Sharma; Karl-Dieter Entian; Ludvine Wacheul; Denis L. J. Lafontaine; James T. Anderson; Juan D. Alfonzo; Andreas Hildebrandt; Andres Jäschke; Yuri Motorin; Mark Helm
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.