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

The maternal-to-zygotic transition (MZT) requires coordinated clearance and deadenylation of maternally deposited mRNAs, yet the underlying molecular mechanisms remain poorly understood. N6-methyladenosine (m⁶A) has emerged as a key regulator of maternal mRNA fate, but prior studies have 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 on zebrafish embryos across MZT to resolve the interplay between m⁶A deposition, mRNA clearance, and poly(A) tail length 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 to maternal mRNA clearance than 3'-UTR m⁶A. The positional context of m⁶A alone is sufficient to determine the temporal regulation of poly(A) tail lengths. CDS m⁶A constitutively suppresses tail length throughout MZT, while 3'-UTR m⁶A acquires shortening activity only after zygotic genome activation (ZGA). Transcriptomic analysis of ythdf2 knockout embryos reveals two unrecognized roles. Ythdf2 stabilizes m⁶A-marked maternal transcripts to set stoichiometry at MZT onset, and is also responsible for maintaining global poly(A) tail homeostasis prior to ZGA through an m⁶A-independent mechanism. Together, these findings define the single-molecule logic by which m⁶A modifications shape transcript fate during vertebrate MZT.