Single-nucleotide RNA m6A mapping in bovine preimplantation development reveals site-specific regulation of RPL12 at zygotic genome activation.

RNA N6-methyladenosine (m6A) is a key regulator of gene expression during early embryogenesis. Using SAC-seq (m6A-selective allyl chemical labeling and sequencing), an antibody-independent m6A profiling method, we generated the first single-nucleotide-resolution m6A map of bovine oocytes and preimplantation embryos. We observed both coordinated and uncoupled relationships between m6A modification and expression of protein-coding and noncoding genes. Integrative analysis of the transcriptome, m6A epitranscriptome, and translatome revealed dynamic m6A remodeling, particularly in ribosomal protein genes. Functional interrogation of a specific m6A site within the RPL12 transcript demonstrated that loss of this modification reduces protein synthesis, disrupts translation-related gene expression, impairs zygotic genome activation, and compromises blastocyst formation. Notably, supplementation with wild-type RPL12 mRNA failed to rescue developmental arrest, suggesting that m6A regulates RPL12 function beyond transcript abundance. Overall, these findings provide a valuable single-nucleotide-resolution resource of m6A dynamics in mammalian embryogenesis and uncover a site-specific mechanism by which m6A regulates translation and developmental competence in early embryos.