Single-molecule m6A profiling reveals position-dependent mRNA regulation and non-canonical roles for Ythdf2 in early embryogenesis

N6-methyladenosine (m6A) has emerged as a key regulator of maternal mRNA fate, but prior studies have mainly relied on population-averaged short-read methods that cannot resolve modification state, poly(A) tail length, or isoform identity on the same molecule.

Here, we employ nanopore direct RNA sequencing across the zebrafish MZT to resolve the interplay between m6A deposition, mRNA clearance, and poly(A) tail dynamics at single-molecule resolution. We find that 78% of expressed maternal genes harbor m6A-modified isoforms, significantly exceeding prior bulk estimates. Within-isoform comparisons demonstrate that m6A promotes mRNA decay, with CDS m6A contributing more than 3’-UTR m6A.