Alshawi, S. A. | Search bioRxiv

bioRxiv2026

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

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.

developmental biology↗

bioRxiv2025

Genome-wide profiling of RNA 2'-O-methylation in neurons and identification of orphan snoRNA targets

We have compared genome-wide patterns of RNA 2-O-methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2-O-methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders.

molecular biology↗