YTHDF2 Recognition of N¹‑Methyladenosine (m¹A)-Modified RNA Is Associated with Transcript Destabilization

Epitranscriptomic modifications play an important role in RNA function and can impact gene expression. Here, we apply a chemical proteomics approach to investigate readers of N¹-methyladenosine (m¹A), a poorly characterized modification on mammalian mRNA. We find that YTHDF proteins, known m⁶A readers, recognize m¹A-modified sequences in a methylation-specific manner. We characterize binding of recombinant YTHDF1/2 proteins to m¹A-modified oligonucleotides to demonstrate that these interactions can exhibit comparable affinity to m⁶A-recognition events and occur in diverse sequence contexts. Further, we demonstrate YTHDF2 interacts specifically with endogenously modified m¹A transcripts. Finally, we deplete cellular YTHDF2 to show that the abundance of m¹A-modified transcripts is increased in its absence. Similarly, increasing m¹A levels through depletion of ALKBH3, an m¹A eraser protein, destabilizes known m¹A-containing RNAs. Our results shed light on the function of m¹A on mRNA and provide a mechanistic framework to further evaluate the role of m¹A in biological processes.