Exon junction complex shapes m6A epitranscriptome during splicing
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PROVIDER: PRJNA856642 | ENA |
REPOSITORIES: ENA
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Project description:N6-methyladenosine (m6A) is the most abundant post-transcriptional methylation of mature mRNA and plays a crucial regulatory role in various biological functions, involving development and cancer progression. m6A is deposited by methyltransferase complex composed of METTL3 and METTL14 cotranscriptionally, and removed by demethylase FTO or ALKBH5. m6A is highly enriched within the 3’ UTRs and in the vicinity of the stop codon of mature mRNA. However, the mechanism that causes this distribution pattern is still enigmatic. Here, we show that EJC packages mRNAs to shape mRNA m6A landscape. We first tested the possibility that demethylase removes m6A during spicing and found that ALKBH5 depletion had a minor effect on m6A levels. Thus, we ruled out the demethylation model. We then hypothesized that splicing factors inhibit the methylation process of internal exons, but not the 3’ UTR. Knockdown of EIF4A3, a core component of the exon junction complex, significantly increased m6A levels in mature mRNAs. The hypermethylated sites are enriched in short internal exons. Furthermore, EIF4A3 depletion upregulates METTL3 binding affinity to mRNA to deposit m6As. In conclusion, Our results demonstrate that EIF4A3 blocks METTL3 binding and methylating internal short exons of mRNA. These findings shed light on the fact that RNA packaging formed during splicing acts as a regulator that shapes mRNA modifications.
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Project description:N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we uncover that m6A deposition is not selective. Instead, m6A distribution is exclusion-based: m6A-consensus harboring sites are methylated by default, unless they are within a window of up to ~200 nt from an exon-intron junction. A simple model which we extensively validate, relying exclusively on presence of m6A motifs and exon-intron architecture allows high accuracy recapitulation of experimentally-measured m6A profiles and of the vast majority of m6A hallmarks. Finally, we provide strong evidence that this exclusion is mediated by the exon-junction complex. Our findings establish a mechanism by which the memory of nuclear RNA splicing is covalently etched on an mRNA in the form of m6A, and determines its cytoplasmic stability, with broad implications on the regulation, function, and evolution of the exon-junction complex and m6A.
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Project description:N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (âm6A levelsâ), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3â untranslated regions (3â-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. m6A-LAIC-seq of H1-ESC and GM12878 cell lines, each cell line has two replicates
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