Publications by authors named "Yuri Motorin"

67 Publications

Mapping and Quantification of tRNA 2'-O-Methylation by RiboMethSeq.

Methods Mol Biol 2019 ;1870:273-295

Next-Generation Sequencing Core Facility, UMS2008 IBSlor, CNRS-UL-INSERM, BioPole Lorraine University, Nancy, France.

Current development of epitranscriptomics field requires efficient experimental protocols for precise mapping and quantification of various modified nucleotides in RNA. Despite important advances in the field during the last 10 years, this task is still extremely laborious and time-consuming, even when high-throughput analytical approaches are employed. Moreover, only a very limited subset of RNA modifications can be detected and only rarely be quantified by these powerful techniques. In the past, we developed and successfully applied alkaline fragmentation-based RiboMethSeq approach for mapping and precise quantification of multiple 2'-O-methylation residues in ribosomal RNA. Here we describe a RiboMethSeq protocol adapted for the analysis of bacterial and eukaryotic tRNA species, which also contain 2'-O-methylations at functionally important RNA regions.
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http://dx.doi.org/10.1007/978-1-4939-8808-2_21DOI Listing
May 2019

AlkAniline-Seq: Profiling of m G and m C RNA Modifications at Single Nucleotide Resolution.

Angew Chem Int Ed Engl 2018 12 16;57(51):16785-16790. Epub 2018 Nov 16.

Lorraine University, UMS2008 IBSLor CNRS-UL-INSERM, Biopôle UL, 9, Avenue de la Forêt de Haye, 54505, Vandoeuvre-les-Nancy, France.

RNA modifications play essential roles in gene expression regulation. Only seven out of >150 known RNA modifications are detectable transcriptome-wide by deep sequencing. Here we describe a new principle of RNAseq library preparation, which relies on a chemistry based positive enrichment of reads in the resulting libraries, and therefore leads to unprecedented signal-to-noise ratios. The proposed approach eschews conventional RNA sequencing chemistry and rather exploits the generation of abasic sites and subsequent aniline cleavage. The newly generated 5'-phosphates are used as unique entry for ligation of an adapter in library preparation. This positive selection, embodied in the AlkAniline-Seq, enables a deep sequencing-based technology for the simultaneous detection of 7-methylguanosine (m G) and 3-methylcytidine (m C) in RNA at single nucleotide resolution. As a proof-of-concept, we used AlkAniline-Seq to comprehensively validate known m G and m C sites in bacterial, yeast, and human cytoplasmic and mitochondrial tRNAs and rRNAs, as well as for identifying previously unmapped positions.
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http://dx.doi.org/10.1002/anie.201810946DOI Listing
December 2018

Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein.

Sci Rep 2018 09 14;8(1):13815. Epub 2018 Sep 14.

Université de Lorraine, CNRS, UMR 7365 Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), F-54500, Nancy, France.

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ in Pyrococcus abyssi 23S rRNA and of Ψ in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ (Ψ in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.
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http://dx.doi.org/10.1038/s41598-018-32164-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138745PMC
September 2018

Double methylation of tRNA-U54 to 2'-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7.

Nucleic Acids Res 2018 10;46(18):9764-9775

Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University of Mainz, Staudingerweg 5, D-55128 Mainz, Germany.

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2'-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2'-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2'O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.
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http://dx.doi.org/10.1093/nar/gky644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182150PMC
October 2018

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

RNA Biol 2018 9;15(6):829-831. Epub 2018 May 9.

ac International Institute of Molecular and Cell Biology in Warsaw , Poland.

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.
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http://dx.doi.org/10.1080/15476286.2018.1460996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152430PMC
November 2018

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure.

Angew Chem Int Ed Engl 2018 06 27;57(26):7893-7897. Epub 2018 Apr 27.

Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Staudingerweg 5, 55128, Mainz, Germany.

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms i A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms i A contains a thioacetal, shown in vitro to be biosynthetically derived from ms i A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms i A from i A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.
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http://dx.doi.org/10.1002/anie.201713188DOI Listing
June 2018

Publisher Correction: A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients.

Nat Commun 2018 02 2;9(1):554. Epub 2018 Feb 2.

Department of Human Genetics, McGill University and Research Institute McGill University Health Centre, H4A 3J1, Montreal, Quebec, Canada.

The original version of this Article contained an error in the title, which was incorrectly given as 'APRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'. This has now been corrected in both the PDF and HTML versions of the Article to read 'A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'.
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http://dx.doi.org/10.1038/s41467-018-03054-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797229PMC
February 2018

APRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients.

Nat Commun 2018 01 4;9(1):67. Epub 2018 Jan 4.

Department of Human Genetics, McGill University and Research Institute McGill University Health Centre, Montreal, H4A 3J1, Quebec, Canada.

To date, epimutations reported in man have been somatic and erased in germlines. Here, we identify a cause of the autosomal recessive cblC class of inborn errors of vitamin B metabolism that we name "epi-cblC". The subjects are compound heterozygotes for a genetic mutation and for a promoter epimutation, detected in blood, fibroblasts, and sperm, at the MMACHC locus; 5-azacytidine restores the expression of MMACHC in fibroblasts. MMACHC is flanked by CCDC163P and PRDX1, which are in the opposite orientation. The epimutation is present in three generations and results from PRDX1 mutations that force antisense transcription of MMACHC thereby possibly generating a H3K36me3 mark. The silencing of PRDX1 transcription leads to partial hypomethylation of the epiallele and restores the expression of MMACHC. This example of epi-cblC demonstrates the need to search for compound epigenetic-genetic heterozygosity in patients with typical disease manifestation and genetic heterozygosity in disease-causing genes located in other gene trios.
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http://dx.doi.org/10.1038/s41467-017-02306-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754367PMC
January 2018

Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

Proc Natl Acad Sci U S A 2017 12 20;114(49):12934-12939. Epub 2017 Nov 20.

Centre de Recherche en Cancérologie de Lyon, UMR, INSERM 1052, CNRS 5286, Centre Léon Bérard, F-69373 Lyon, France;

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.
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http://dx.doi.org/10.1073/pnas.1707674114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724255PMC
December 2017

Quantification of 2'-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol).

Methods Mol Biol 2018 ;1649:29-48

IMoPA UMR7365 CNRS-Lorraine University, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-les-Nancy, France.

RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from bacteria, archaea, and eukarya. We and others have recently published accurate and sensitive detection of these modifications on native RNA at a single base resolution by high-throughput sequencing technologies. Relative quantification of these modifications is still under progress and would probably reduce the number of false positives due to 3D RNA structure. Therefore, here, we describe a reliable and optimized protocol for quantification of 2'-O-Methylations based on alkaline fragmentation of RNA coupled to a commonly used ligation approach followed by Illumina sequencing. For this purpose, we describe how to prepare in vitro transcribed yeast 18S and 25S rRNA used as a reference for unmodified rRNAs and to compare them to purified 18S and 25S rRNA from yeast total RNA preparation. These reconstructed rRNA mixes were combined at different ratios and processed for RiboMethseq protocol.This technique will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in normal and pathologic processes or during development.
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http://dx.doi.org/10.1007/978-1-4939-7213-5_2DOI Listing
June 2018

Engineering of a DNA Polymerase for Direct m A Sequencing.

Angew Chem Int Ed Engl 2018 01 7;57(2):417-421. Epub 2017 Dec 7.

Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N -methyladenosine (m A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m A-containing RNA prior to sequencing, since m A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N -methylation. We identified a mutant that exhibits increased misincorporation opposite m A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m A sites directly from the sequencing data of untreated RNA samples.
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http://dx.doi.org/10.1002/anie.201710209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768020PMC
January 2018

Identification of sites of 2'-O-methylation vulnerability in human ribosomal RNAs by systematic mapping.

Sci Rep 2017 09 13;7(1):11490. Epub 2017 Sep 13.

RNA Molecular Biology and Center for Microscopy and Molecular Imaging (CMMI), Fonds National de la Recherche (F.R.S./FNRS) and Université Libre de Bruxelles (ULB), BioPark campus Gosselies, Belgium.

Ribosomal RNA modifications are important in optimizing ribosome function. Sugar 2'-O-methylation performed by fibrillarin-associated box C/D antisense guide snoRNAs impacts all steps of translation, playing a role in disease etiology (cancer). As it renders adjacent phosphodiester bonds resistant to alkaline treatment, 2'-O-methylation can be monitored qualitatively and quantitatively by applying next-generation sequencing to fragments of randomly cleaved RNA. We remapped all sites of 2'-O-methylation in human rRNAs in two isogenic diploid cell lines, one producing and one not producing the antitumor protein p53. We identified sites naturally modified only partially (confirming the existence in cells of compositionally distinct ribosomes with potentially specialized functions) and sites whose 2'-O-methylation is sensitive to p53. We mapped sites particularly vulnerable to a reduced level of the methyltransferase fibrillarin. The remarkable fact that these are largely sites of natural hypomodification provides initial insights into the mechanism of partial RNA modification. Sites where methylation appeared vulnerable lie peripherally on the 3-D structure of the ribosomal subunits, whereas the numerous modifications present at the core of the subunits, where the functional centers lie, appeared robustly made. We suggest that vulnerable sites of 2'-O-methylation are highly likely to undergo specific regulation during normal and pathological processes.
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http://dx.doi.org/10.1038/s41598-017-09734-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597630PMC
September 2017

High-Throughput Mapping of 2'-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol).

Methods Mol Biol 2017 ;1562:171-187

IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 Avenue de la Foret de Haye, 54505, Vandoeuvre-Les-Nancy, France.

Detection of RNA modifications in native RNAs is a tedious and laborious task, since the global level of these residues is low and most of the suitable physico-chemical methods require purification of the RNA of interest almost to homogeneity. To overcome these limitations, methods based on RT-driven primer extension have been developed and successfully used, sometimes in combination with a specific chemical treatment. Nowadays, some of these approaches have been coupled to high-throughput sequencing technologies, allowing the access to transcriptome-wide data. RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from bacteria, archaea, and eukarya. Here, we describe a reliable and optimized protocol based on alkaline fragmentation of total RNA coupled to a commonly used ligation approach followed by Illumina sequencing. We describe the methodology for detection and relative quantification of 2'-O-methylations with a high sensitivity and reproducibility even with a limited amount of starting material (1 ng of total RNA). Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in pathologies.
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http://dx.doi.org/10.1007/978-1-4939-6807-7_12DOI Listing
February 2018

Detecting RNA modifications in the epitranscriptome: predict and validate.

Nat Rev Genet 2017 05 20;18(5):275-291. Epub 2017 Feb 20.

IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France.

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.
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http://dx.doi.org/10.1038/nrg.2016.169DOI Listing
May 2017

Next-Generation Sequencing-Based RiboMethSeq  Protocol for Analysis of tRNA 2'-O-Methylation.

Biomolecules 2017 02 9;7(1). Epub 2017 Feb 9.

IMoPA UMR7365 CNRS-UL, Biopole Lorraine University, 54505 Vandoeuvre-les-Nancy, France.

Analysis of RNA modifications by traditional physico-chemical approaches is labor  intensive,  requires  substantial  amounts  of  input  material  and  only  allows  site-by-site  measurements.  The  recent  development  of  qualitative  and  quantitative  approaches  based  on   next-generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA  species.  The  Illumina  sequencing-based  RiboMethSeq  protocol  was  initially  developed  and  successfully applied for mapping of ribosomal RNA (rRNA) 2'-O-methylations. This method also  gives excellent results in the quantitative analysis of rRNA modifications in different species and  under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA,  and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved  RNA species. A deep understanding of RNA modification functions requires large and global  analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are  well known to contain a great variety of functionally-important modified residues. Here, we  evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2'-O-methylation in  Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic  pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for  known modified positions in different tRNA species.
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http://dx.doi.org/10.3390/biom7010013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372725PMC
February 2017

CoverageAnalyzer (CAn): A Tool for Inspection of Modification Signatures in RNA Sequencing Profiles.

Biomolecules 2016 11 10;6(4). Epub 2016 Nov 10.

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.

Combination of reverse transcription (RT) and deep sequencing has emerged as a powerful instrument for the detection of RNA modifications, a field that has seen a recent surge in activity because of its importance in gene regulation. Recent studies yielded high-resolution RT signatures of modified ribonucleotides relying on both sequence-dependent mismatch patterns and reverse transcription arrests. Common alignment viewers lack specialized functionality, such as filtering, tailored visualization, image export and differential analysis. Consequently, the community will profit from a platform seamlessly connecting detailed visual inspection of RT signatures and automated screening for modification candidates. CoverageAnalyzer (CAn) was developed in response to the demand for a powerful inspection tool. It is freely available for all three main operating systems. With SAM file format as standard input, CAn is an intuitive and user-friendly tool that is generally applicable to the large community of biomedical users, starting from simple visualization of RNA sequencing (RNA-Seq) data, up to sophisticated modification analysis with significance-based modification candidate calling.
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http://dx.doi.org/10.3390/biom6040042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5197952PMC
November 2016

DNA and RNA Pyrimidine Nucleobase Alkylation at the Carbon-5 Position.

Adv Exp Med Biol 2016 ;945:19-33

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128, Mainz, Germany.

The carbon 5 of pyrimidine nucleobases is a privileged position in terms of nucleoside modification in both DNA and RNA. The simplest modification of uridine at this position is methylation leading to thymine. Thymine is an integral part of the standard nucleobase repertoire of DNA that is synthesized at the nucleotide level. However, it also occurs in RNA, where it is synthesized posttranscriptionally at the polynucleotide level. The cytidine analogue 5-methylcytidine also occurs in both DNA and RNA, but is introduced at the polynucleotide level in both cases. The same applies to a plethora of additional derivatives found in nature, resulting either from a direct modification of the 5-position by electrophiles or by further derivatization of the 5-methylpyrimidines. Here, we review the structural diversity of these modified bases, the variety of cofactors that serve as carbon donors, and the common principles shared by enzymatic mechanisms generating them.
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http://dx.doi.org/10.1007/978-3-319-43624-1_2DOI Listing
June 2017

Next-generation sequencing technologies for detection of modified nucleotides in RNAs.

RNA Biol 2017 09 28;14(9):1124-1137. Epub 2016 Oct 28.

b Laboratoire IMoPA, UMR7365 CNRS-UL, Biopole Lorraine University , Vandoeuvre-les-Nancy , France.

Our ability to map and quantify RNA modifications at a genome-wide scale have revolutionized our understanding of the pervasiveness and dynamic regulation of diverse RNA modifications. Recent efforts in the field have demonstrated the presence of modified residues in almost any type of cellular RNA. Next-generation sequencing (NGS) technologies are the primary choice for transcriptome-wide RNA modification mapping. Here we provide an overview of approaches for RNA modification detection based on their RT-signature, specific chemicals, antibody-dependent (Ab) enrichment, or combinations thereof. We further discuss sources of artifacts in genome-wide modification maps, and experimental and computational considerations to overcome them. The future in this field is tightly linked to the development of new specific chemical reagents, highly specific Ab against RNA modifications and use of single-molecule RNA sequencing techniques.
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http://dx.doi.org/10.1080/15476286.2016.1251543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5699547PMC
September 2017

Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA.

Nucleic Acids Res 2016 09 14;44(16):e135. Epub 2016 Jun 14.

IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France

RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly used ligation approach, and Illumina sequencing. We describe a methodology to detect 2'-O-methylations with high sensitivity and reproducibility even with limited amount of starting material (1 ng of total RNA). The method provides a quantification of the 2'-O-methylation occupancy of a given site, allowing to detect relatively small changes (>10%) in 2'-O-methylation profiles. Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in pathologies.
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http://dx.doi.org/10.1093/nar/gkw547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027498PMC
September 2016

High-throughput sequencing for 1-methyladenosine (m(1)A) mapping in RNA.

Methods 2016 09 24;107:110-21. Epub 2016 Feb 24.

IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France; Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France. Electronic address:

Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m(1)A) residues using high-throughput next generation sequencing (NGS). Since m(1)A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types of cDNA products essential for further bioinformatic analysis. We demonstrate that m(1)A residues produce characteristic arrest and mismatch rates and combination of both can be used for their detection as well as for discrimination of m(1)A from other modified A residues present in RNAs.
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http://dx.doi.org/10.1016/j.ymeth.2016.02.012DOI Listing
September 2016

The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent.

Nucleic Acids Res 2015 Nov 13;43(20):9950-64. Epub 2015 Sep 13.

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany

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 (m(1)A) 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, m(1)A 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 m(1)A, 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 m(1)A in the template RNA, meaning it is sequence dependent. The RT-signature of m(1)A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m(1)A residues in trypanosomal tRNA.
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http://dx.doi.org/10.1093/nar/gkv895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787781PMC
November 2015

Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120.

PLoS One 2015 21;10(7):e0133321. Epub 2015 Jul 21.

Laboratoire IMoPA, UMR 7365 UL-CNRS, BioPole de UL, Vandoeuvre-les-Nancy, France.

Modified nucleotide 5-methylcytosine (m5C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA:m5C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m5C- MTase activity, which restores m5C formation at position 2870 in domain V of 25S rRNA in a nop2Δ yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m5C in domains V and IV, respectively. In addition, we do not find any evidence of m5C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m5C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m5C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m5C:RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133321PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510066PMC
May 2016

Pseudouridine: still mysterious, but never a fake (uridine)!

RNA Biol 2014 ;11(12):1540-54

a Institute of Pharmacy and Biochemistry ; Johannes Gutenberg-University of Mainz ; Mainz , Germany.

Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA. Although Ψ was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of Ψ synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.
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http://dx.doi.org/10.4161/15476286.2014.992278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615568PMC
October 2015

RNA nucleotide methylation.

Wiley Interdiscip Rev RNA 2011 Sep-Oct;2(5):611-31. Epub 2011 Mar 23.

Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), Université Henri Poincaré, Vandoeuvre-les-Nancy, France.

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 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wrna.79DOI Listing
March 2012

Use of specific chemical reagents for detection of modified nucleotides in RNA.

J Nucleic Acids 2011 13;2011:408053. Epub 2011 Apr 13.

Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), UMR 7214 CNRS-UHP, Nancy Université, boulevard des Aiguillettes, BP 70239, 54506 Vandoeuvre-les-Nancy, France.

Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups frequently does not change base-pairing properties. In this paper, we give a summary of various chemical approaches exploiting the specific reactivity of modified nucleotides in RNA for their detection.
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http://dx.doi.org/10.4061/2011/408053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3118635PMC
July 2011

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA.

Nucleic Acids Res 2011 Sep 6;39(16):7348-60. Epub 2011 Jun 6.

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudinger Weg 5, D-55128 Mainz, Germany.

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.
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http://dx.doi.org/10.1093/nar/gkr449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167637PMC
September 2011

Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.

RNA Biol 2011 Mar-Apr;8(2):325-42. Epub 2011 Mar 1.

Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), UMR7214 CNRS-UHP, Faculté des Sciences et Technologies, Nancy Université, Bld des Aiguillettes, Vandoeuvre-les-Nancy, France.

HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.
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http://dx.doi.org/10.4161/rna.8.2.13984DOI Listing
November 2011

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling.

Nucleic Acids Res 2011 Mar 30;39(5):1943-52. Epub 2010 Oct 30.

Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.

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 tRNA(Phe). 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.
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http://dx.doi.org/10.1093/nar/gkq825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061074PMC
March 2011

tRNA stabilization by modified nucleotides.

Biochemistry 2010 Jun;49(24):4934-44

Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), UMR 7214 CNRS-UHP Faculté des Sciences et Techniques, Université Henri Poincaré, Nancy 1, Bld des Aiguillettes, BP 70239, 54506 Vandoeuvre-les-Nancy, France.

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.
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http://dx.doi.org/10.1021/bi100408zDOI Listing
June 2010

5-methylcytosine in RNA: detection, enzymatic formation and biological functions.

Nucleic Acids Res 2010 Mar 8;38(5):1415-30. Epub 2009 Dec 8.

Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), UMR 7214 CNRS-UHP Faculté des Sciences et Techniques, Université Henri Poincaré, Nancy 1, Bld des Aiguillettes, BP 70239, 54506 Vandoeuvre-les-Nancy, France.

The nucleobase modification 5-methylcytosine (m(5)C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m(5)C-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 m(5)C localization in various RNA molecules puts ribo-m(5)C 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 m(5)C-methylation. Numerous putative RNA:m(5)C-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.
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http://dx.doi.org/10.1093/nar/gkp1117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836557PMC
March 2010