The role of m6A RNA modification in RNA regulation

Abstract

N6-methyladenosine (m6A) is the most abundant internal modification found in eukaryotic messenger RNA (mRNA) and serves as a dynamic regulatory signal that controls multiple aspects of mRNA metabolism, including splicing, nuclear export, translation, and degradation. Among these, m6A’s most prominent function is the regulation of mRNA stability. Typically, m6A sites are recognized by members of the YTH domain family of reader proteins, particularly YTHDF1–3, which facilitate transcript degradation by recruiting effector complexes. Despite extensive research into m6A-mediated decay, key questions remain unresolved: Which specific m6A sites trigger degradation? Do all m6A-modified regions contribute equally to mRNA destabilization? And how is the choice of decay pathway determined? In this thesis, I describe the discovery and characterization of a novel, translation-dependent mRNA decay pathway driven by m6A modifications located specifically within the coding sequence (CDS) of transcripts. We term this mechanism CDS–m6A decay (CMD). In contrast to canonical pathways that require reader protein binding to initiate decay, CMD is triggered by impaired decoding of methylated codons by the ribosome. This establishes a direct mechanistic link between m6A and translation, where ribosomal slowdown or stalling at modified codons initiates rapid degradation of the transcript. This pathway operates independently of initial reader recognition, although we find that YTHDF2 acts downstream to facilitate transcript clearance, suggesting a multilayered control system. Our data also show that CMD targets are preferentially enriched in processing bodies (P-bodies), which serve as key cytoplasmic hubs for mRNA decay and storage. This spatial segregation may represent a protective or regulatory layer in the execution of CMD. Intriguingly, the deposition of m6A in the CDS is not random; recent findings reveal that the exon junction complex (EJC), deposited near exon–exon boundaries during splicing, restricts m6A methylation in its vicinity. This implies that the decision to methylate specific codons—and thus commit the transcript to CMD—can be made co-transcriptionally in the nucleus, long before translation occurs in the cytoplasm. The work presented here provides new insights into how site-specific m6A deposition and ribosome dynamics intersect to regulate mRNA fate. It reveals that CDS m6A modifications couple translation fidelity with transcript turnover. This pathway is especially relevant for transcripts involved in cellular stress responses, development, and

cancer, pointing to a broader physiological significance of CMD in controlling gene expression programs that require precise temporal regulation.