N6-Methyladenosine (m6A) is the predominant internal RNA modification in eukaryotic messenger RNAs (mRNAs) and plays a crucial role in mRNA stability. Here, using human cells, we reveal that m6A sites in the coding sequence (CDS) trigger CDS-m6A decay (CMD), a 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. Mechanistically, CMD depends on translation, whereby m6A deposition in the CDS triggers ribosome pausing and transcript destabilization. The subsequent decay involves the translocation of the CMD target transcripts to processing bodies (P-bodies) and recruitment of the m6A reader protein YT521-B homology domain family protein 2 (YTHDF2). Our findings highlight CMD as a previously unknown pathway, which is particularly important for controlling the expression of developmental regulators and retrogenes.

Buchmann Institute for Molecular Life Sciences and Institute of Molecular Biosciences Goethe University Frankfurt 60438 Frankfurt a M Germany; Theodor Boveri Institute Biocenter University of Würzburg Am Hubland 97074 Würzburg Germany

Central European Institute of Technology Masaryk University Kamenice 5 E35 625 00 Brno Czech Republic

Institute for Organic Chemistry and Chemical Biology Goethe University Frankfurt Max von Laue Str 7 60438 Frankfurt a M Germany

Institute for Organic Chemistry and Chemical Biology Goethe University Frankfurt Max von Laue Str 7 60438 Frankfurt a M Germany; Institute for Pharmaceutical and Biomedical Sciences Johannes Gutenberg University Mainz Staudingerweg 5 55128 Mainz Germany

Institute of Molecular Biology 55128 Mainz Germany

Klaus Tschira Institute for Integrative Computational Cardiology University of Heidelberg 69120 Heidelberg Germany; German Centre for Cardiovascular Research Partner Site Heidelberg Mannheim Heidelberg Germany

Theodor Boveri Institute Biocenter University of Würzburg Am Hubland 97074 Würzburg Germany; Institute of Molecular Biology 55128 Mainz Germany

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Global analysis by LC-MS/MS of N6-methyladenosine and inosine in mRNA reveal complex incidence