Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3 ' UTRs and near Stop Codons

Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA.
Cell (Impact Factor: 32.24). 05/2012; 149(7):1635-46. DOI: 10.1016/j.cell.2012.05.003
Source: PubMed


Methylation of the N(6) position of adenosine (m(6)A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity risk gene, encodes an m(6)A demethylase implicates m(6)A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m(6)A localization, which combines m(6)A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m(6)A, indicating that m(6)A is a common base modification of mRNA. The m(6)A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m(6)A sites are enriched near stop codons and in 3' UTRs, and we uncover an association between m(6)A residues and microRNA-binding sites within 3' UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome.

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    • "The m 6 A miCLIP mapping read clusters are shown in pink. To show that the m 6 A or eIF3 peaks are not an artifact of uneven RNA recovery, the RNA-Seq reads (Meyer et al., 2012) are also displayed (purple). Lastly, to determine if eIF3 binding is occurring at the related nucleotide m 6 Am, the CAGE tags (Lykke-Andersen et al., 2014) (black) are shown. "

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    • "Analysis of published m 6 A peak-calling data Data from the previous m 6 A publications were downloaded directly from the NCBI Gene Expression Omnibus (GEO) and aligned to the same reference genomes by TopHat/Bowtie according to Meyer et al. (2012) and Batista et al. (2014) for mm9 and Schwartz et al. (2014) for hg19. We determined m 6 A peak regions using either the methods described in these studies or a strategy similar to that described above except implementing a 100-nt sliding window (as their RNA fragments were all longer); the same findings were obtained for the figure citing these data (Supplemental Fig. 3). "
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    ABSTRACT: We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice.
    Genes & development 09/2015; 29(19). DOI:10.1101/gad.269415.115 · 10.80 Impact Factor
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    • "Of these, N 6 -methyladenosine (m 6 A) is the most abundant, with more than 12,000 m 6 A sites in over 7000 genes in the human transcriptome [2–4,7]. A reversible modification, m 6 A occurs within RRm 6 ACH motifs (R = A/G, H = A/C/U), with a high density of m 6 A sites near stop codons and in long internal exons [3] [4]. The m 6 A methyltransferase complex is IMF YJMBI-64840; No. of pages: 12; 4C: 2, 3, 5, 6, 7 0022-2836/© 2015 Elsevier Ltd. "
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    ABSTRACT: N6-methyladenosine (m(6)A) is a reversible and abundant internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) with roles in RNA processing, transport, and stability. Although m(6)A does not preclude Watson-Crick base pairing, the N6-methyl group alters the stability of RNA secondary structure. Since changes in RNA structure can affect diverse cellular processes, the influence of m(6)A on mRNA and lncRNA structure has the potential to be an important mechanism for m(6)A function in the cell. Indeed, an m(6)A site in the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently shown to induce a local change in structure that increases the accessibility of a U5-tract for recognition and binding by heterogeneous nuclear ribonucleoprotein C (HNRNPC). This m(6)A-dependent regulation of protein binding through a change in RNA structure, termed 'm(6)A-switch,' affects transcriptome-wide mRNA abundance and alternative splicing. To further characterize this first example of an m(6)A-switch in a cellular RNA, we used nuclear magnetic resonance (NMR) and Förster resonance energy transfer (FRET) to demonstrate the effect of m(6)A on a 32-nucleotide RNA hairpin derived from the m(6)A-switch in MALAT1. The observed imino proton NMR resonances and FRET efficiencies suggest that m(6)A selectively destabilizes the portion of the hairpin-stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure. The m(6)A-modified hairpin has a predisposed conformation that resembles the hairpin conformation in the RNA-HNRNPC complex more closely than the unmodified hairpin. The m(6)A-induced structural changes in the MALAT1 hairpin can serve as a model for a large family of m(6)A-switches that mediate the influence of m(6)A on cellular processes.
    Journal of Molecular Biology 09/2015; DOI:10.1016/j.jmb.2015.08.021 · 4.33 Impact Factor
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