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

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.