m6A sites in the coding region trigger translation-dependent mRNA decay (YTHDF2 iCLIP2)

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. To learn about m6A-dependent binding of YTHDF2 in the transcriptome, we mapped YTHDF2–RNA binding using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP2) in HEK293T cells with and without m6A depletion for 12 h. The experiment performed in 4 replicates.