m6A sites in the coding region trigger translation-dependent mRNA decay (in vitro methylation)
N6-Methyladenosine (m6A) is the predominant internal RNA modification in eukaryotic messenger RNAs (mRNAs) and plays a crucial role in mRNA stability. In this study, we reveal that m6A sites in the coding sequence (CDS) trigger CDS–m6A decay (CMD), a novel mRNA decay pathway that is distinct from previously reported m6A-dependent degradation mechanisms. Importantly, CDS m6A sites act considerably faster and more efficiently than those in the 3' untranslated region, which to date have been considered the main effectors of m6A-mediated RNA decay. Mechanistically, CMD depends on translation whereby m6A deposition in the CDS induces ribosome pausing and transcript destabilization. We found that the target transcripts of CMD are recognized by the m6A reader protein YTHDF2, selectively enriched in processing bodies (P-bodies) and degraded via the decapping factor DCP2. Our findings highlight CMD as a previously unknown pathway for m6A-mediated decay, which is particularly important for controlling the expression of developmental regulators and retrogenes. Overall design: To investigate if the sequence preference is intrinsic to the METTL3–METTL14 writer complex, we established a high-throughput in vitro methylation assay. In brief, we generated a library of 11,999 in vitro transcripts (IVTs, 183 nucleotides [nt] each), which represent 3,488 human transcript loci. The IVT sequences were chosen around DRACN motifs ("selected DRACN") that represent different DRACN 5-mers with or without m6A modification from different transcript regions. We then used a recombinantly reconstituted METTL3–METTL14 writer complex to methylate the IVT library and analyzed the resulting methylation profile using miCLIP2. Experiments were performed in duplicate.