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

ABSTRACT: 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 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. The positional context of m6A directs the temporal regulation of poly(A) tail lengths. CDS m6A constitutively suppresses tail length throughout MZT, while 3′-UTR m6A promotes shortening only after zygotic genome activation (ZGA). Transcriptomic analysis of ythdf2 knockout embryos revealed two unrecognized roles. Ythdf2 sets m6A stoichiometry at MZT onset, and maintains global poly(A) tail homeostasis prior to ZGA through an m6A-independent mechanism. Together, these findings identify the single-molecule logic by which m6A modifications shape transcript fate during vertebrate MZT.

ORGANISM(S): Danio rerio

PROVIDER: GSE341570 | GEO | 2026/07/27

REPOSITORIES: GEO

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