m6A sites in the coding region trigger translation-dependent mRNA decay (ribosome profiling)

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 changes in translation in response to m6A depletion, we performed ribosome profiling (Ribo-seq) and matched RNA-seq in HEK293T cells subjected to m6A depletion for 6 h using METTL3 inhibitor STM2457. The Ribo-seq experiment performed in duplicate and the matched RNA-seq performed with 4 replicates.