Expression profiling by high throughput sequencing
Summary
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.
Overall design
RNA sequencing of mESC transfected with a plasmid encoding GFP with a 3' UTR fragment derived from SLC25A3 and subjected to m6A immunoprecipitation. RNA sequencing of HEK293T cells transfected with a massively parallel reporter assay and subjected to m6A immunoprecipitation. RNA sequencing of MCF7 cells transfected with plasmids encoding variants of the mouse Nol12, Coa3 and Calm3 genes and subjected to m6A immunoprecipitation. RNA sequencing of HEK293T cells transfected with a spliced, long massively parallel reporter assay and subjected to m6A immunoprecipitation. RNA sequencing of MCF7 cells transfected with plasmids encoding variants of the mouse Nol12, Coa3 and Calm3 genes and subjected to m6A immunoprecipitation. RNA sequencing of Mettl3 inhibitor and actD treated HEK293T cells. RNA sequencing of Y14 degron HEK293T cell time point samples with m6A immunoprecipitation.
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