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Project description:Non-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2′-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2′-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2′-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation.
2019-01-16 | GSE109925 | GEO
Project description:Non-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2′-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2′-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2′-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation.
2019-01-16 | GSE109926 | GEO
Project description:Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose
| PRJNA432351 | ENA
Project description:Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose [eCLIP]
| PRJNA432353 | ENA
Project description:Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose [meTH-Seq]
| PRJNA432352 | ENA
Project description:RNA secondary structure in untranslated and protein coding regions has been shown to play an important role in regulatory processes and the viral replication cycle. While structures in non-coding regions have been investigated extensively, a thorough overview of the structural repertoire of protein coding mRNAs, especially for viruses, is lacking. Secondary structure prediction of large molecules, such as long mRNAs remains a challenging task, as the contingent of structures a sequence can theoretically fold into grows exponentially with sequence length. We applied a structure prediction pipeline to Viral Orthologous Groups that first identifies the local boundaries of potentially structured regions and subsequently predicts their functional importance. Using this procedure, the orthologous groups were split into structurally homogenous subgroups, which we call subVOGs. This is the first compilation of potentially functional conserved RNA structures in viral coding regions, covering the complete RefSeq viral database. We were able to recover structural elements from previous studies and discovered a variety of novel structured regions. The subVOGs are available through our web resource RNASIV (RNA structure in viruses; http://rnasiv.bio.wzw.tum.de).
| S-EPMC6563262 | biostudies-literature
Project description:Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2? (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2? is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2? binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2? regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2? binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2? protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2?. Versions of Tra2? lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2? protein.
| S-EPMC3240583 | biostudies-literature
Project description:Cellular eukaryotic mRNAs are capped at their 5' ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2'-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2'-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.
| S-EPMC2963352 | biostudies-literature
Project description:Histone methylation modulates gene expression in response to external and internal cues. We uncovered a non-redundant role for the Arabidopsis histone methyltransferase, SDG8, which provides a unique opportunity to study the global function of a specific histone methyltransferase within in a multicellular organism. We previously used a promoter responsive to light and carbon in a positive genetic screen to identify an Arabidopsis carbon and light insensitive mutant cli186. In this study, we characterize the mutant cli186 as a complete deletion of a histone methyltransferase gene SDG8 (now renamed sdg8-5). To assess the global role of SDG8, we compared the global histone methylation patterns and the transcriptome of sdg8-5 to wild type (WT) in the context of a transient carbon and light treatment. We showed that the complete deletion of SDG8 in sdg8-5 is associated with a dramatic reduction of H3K36me3 towards the 3’ of the gene body, which correlates with significant reduction in gene expression. We uncovered 1,084 “high confidence” functional targets of SDG8 – affected in both H3K36me3 marks and gene expression – that are associated with specific biological processes including defense, photosynthesis, nutrient metabolism and energy metabolism. Importantly, 71% of these functional targets are responsive to carbon and/or light. Our model suggests that SDG8 functions to mark specific sets of genes with H3K36me3 in the gene body for active transcription, to tune genes involved in primary metabolism that are responsive to the energy level in the environment. Wild type Arabidopisis and sdg8-5 plants were grown in hydroponics system for three weeks, then starved for carbonhydrate and light for 24 h. They were then treated with carbon and/or light or remained untreated as controls.Three biological replicates were collected, resulting in 24 samples (2 genotypes X 2 carbonhydrate treatmens X 2 light treatments X3 biological replicates).
2015-03-01 | E-GEOD-62435 | biostudies-arrayexpress
Project description:The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified Rrp8 as a methyltransferase involved in m(1)A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m(1)A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase-HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-L-methionine-binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m(1)A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m(1)A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.
| S-EPMC3664796 | biostudies-literature