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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    • "m 6 A was selected for study because it exists in most of the RNAs in a variety of organisms . The abundance of m 6 A throughout the transcriptome (about 7,000 mRNAs and over 300 ncRNAs in the mammalian genome) has been demonstrated by m 6 A profiling from two independent studies (Dominissini et al., 2012; Meyer et al., 2012). Knockdown of either METTL3 or METTL14 results in a reduction in total m 6 A levels in human cells (Dominissini et al., 2012; Liu et al., 2014). "
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    ABSTRACT: NIPBL, a cohesin loader, has been implicated in transcriptional control and genome organization. Mutations in NIPBL, cohesin, and its deacetylase HDAC8 result in Cornelia de Lange syndrome. We report activation of the RNA-sensing kinase PKR in human lymphoblastoid cell lines carrying NIPBL or HDAC8 mutations, but not SMC1A or SMC3 mutations. PKR activation can be triggered by unmodified RNAs. Gene expression profiles in NIPBL-deficient lymphoblastoid cells and mouse embryonic stem cells reveal lower expression of genes involved in RNA processing and modification. NIPBL mutant lymphoblastoid cells show reduced proliferation and protein synthesis with increased apoptosis, all of which are partially reversed by a PKR inhibitor. Non-coding RNAs from an NIPBL mutant line had less m(6)A modification and activated PKR activity in vitro. This study provides insight into the molecular pathology of Cornelia de Lange syndrome by establishing a relationship between NIPBL and HDAC8 mutations and PKR activation.
    Full-text · Article · Jan 2016 · Cell Reports
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    • "Transcriptome-wide approaches to map m 6 A are based on the immunoprecipitation of fragmented, poly(A)-enriched RNA using m 6 A-specific antibodies prior to NGS (MeRIP-seq or m 6 A-seq) [20] [21] (Fig. 1B). After sequencing, the reads are mapped to the reference genome. "
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    ABSTRACT: The pervasive transcription of genomes into long noncoding RNAs has been amply demonstrated in recent years and garnered much attention. Similarly, emerging 'epitranscriptomics' research has shown that chemically modified nucleosides, thought to be largely the domain of tRNAs and other infrastructural RNAs, are far more widespread and can exert unexpected influence on RNA utilization. Both areas are characterized by the often-ephemeral nature of the subject matter in that few individual examples have been fully assessed for their molecular or cellular function, and effects might often be subtle and cumulative. Here we review available information at the intersection of these two exciting areas of biology, by focusing on four RNA modifications that have been mapped transcriptome-wide: 5-methylcytidine, N6-methyladenosine, pseudouridine as well as adenosine to inosine (A-to-I) editing, and their incidence and function in long noncoding RNAs. This article is part of a Special Issue entitled: Long Noncoding RNA. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.
    Full-text · Article · Nov 2015 · Biochimica et Biophysica Acta
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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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    ABSTRACT: Summary Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5′ cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N6-methyladenosine (m6A) in their 5′ UTR can be translated in a cap-independent manner. A single 5′ UTR m6A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5′UTR m6A. Additionally, increased m6A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m6A, resulting in increased numbers of mRNAs with 5′ UTR m6A. These data show that 5′ UTR m6A bypasses 5′ cap-binding proteins to promote translation under stresses.
    Full-text · Article · Oct 2015
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