INTRODUCTION

During preimplantation development, the mammalian embryo undergoes a dramatic maternal-to-zygotic transition (MZT) after fertilization, followed by precise cell fate specification at the blastocyst stage. This process is tightly regulated to ensure the widespread degradation of maternally stored RNAs and proteins, as well as the gradual activation of the embryonic genome.1,2 Among key regulatory mechanisms, RNA _N_6-methyladenosine (m6A) has emerged as a critical modulator of gene expression. As the most abundant internal mRNA modification in eukaryotic mRNA, m6A plays essential roles in regulating RNA metabolism, including mRNA stability, splicing, nuclear export, and translation.3-5 These effects are mediated by a coordinated network of m6A “writers” (METTL3/METTL14), “erasers” (FTO and ALKBH5), and “reader” proteins (YTH domain family proteins), which selectively recognize methylated transcripts and regulate their fate.5,6 For example, m6A can promote mRNA decay via YTHDF2-mediated recruitment of degradation machinery,6 while enhancing translation efficiency through YTHDF1-dependent mechanisms.7 In addition, m6A influences RNA structure and ribosome dynamics,8 thereby regulating gene expression programs in a context-dependent manner. These regulatory functions are particularly important during early embryogenesis, where transcription is limited and post-transcriptional control is essential for developmental progression. m6A also plays a major role in early embryonic development by influencing RNA stability and degradation, particularly during the MZT.4,5,9-11

To date, transcriptome-wide m6A dynamics during mammalian preimplantation development have been characterized in mouse10,12-14 and humans,15 but not in other mammalian species, including bovine, which has substantial agricultural value and serves as a highly informative large animal model for human early embryonic development.16-18 However, profiling m6A in early embryos remains technically challenging due to limited material, low sensitivity, and low coverage.

Current embryo m6A profiling studies10,12-15 have primarily relied on the antibody-based methyl RNA immunoprecipitation sequencing (MeRIP-seq). However, these approaches are limited to identifying m6A-enriched regions and are biased toward canonical (DRACH) sequences. Recently, single-nucleotide-resolution m6A mapping methods have been developed, including GLORI (glyoxal- and nitrite-mediated deamination of unmethylated adenosines),19,20 SAC-seq (m6A-selective allyl chemical labeling and sequencing),21 eTAM-seq (evolved TadA-assisted N6-methyladenosine sequencing),22 and CAM-seq (chemical cooperative catalysis-assisted m6A sequencing),23 providing a quantitative view of m6A in biological regulation. Despite these advances, such high-resolution approaches have not yet been applied to mammalian oocytes and embryos. Thus, the functional roles of site-specific m6A modifications in regulating key developmental genes during early embryogenesis remain largely unknown.

To overcome this limitation, we applied SAC-seq21 to low-input samples and mapped single-nucleotide-resolution m6A landscapes across bovine oocytes and preimplantation embryos. Unlike antibody-based approaches such as MeRIP-seq, SAC-seq enables unbiased and motif-independent detection of m6A sites, resolving m6A dynamics with higher specificity, including transcripts with non-DRACH motifs. Moreover, unlike other single-nucleotide-resolution approaches such as eTAM-seq, GLORI and CAM-seq, which convert all A to I and reduce the 4-letter genome to 3, SAC-seq detects m6A as a mutation signature while preserving sequence complexity, which is important for mapping repetitive RNAs.21 Our results reveal dynamic, stage-specific patterns of m6A deposition across coding and non-coding transcripts, which coincide with major developmental events, including maternal RNA decay and zygotic genome activation (ZGA), as well as changes in translational capacity.

m6A is known to regulate mRNA stability, translation, and decay.3,7,24 Site- and region-specific m6A modifications have been shown to regulate translation efficiency and mRNA stability,25 and m6A can also influence ribosome dynamics to promote selective transcript decay.26 Together, these findings highlight the context-dependent role of m6A in gene expression regulation. However, whether m6A selectively governs the translation of specific gene classes, particularly ribosomal protein genes (RPGs), during early embryogenesis remains unknown. RPGs, traditionally viewed as structural components of the translational machinery, can also regulate selective translation and developmental competence.27,28 Notably, during oocyte maturation and early embryogenesis, when transcription is largely silent, cells rely heavily on translational control to regulate gene expression.29,30

To investigate this regulatory layer, we identified a previously uncharacterized m6A site in the ribosomal protein L12 (RPL12) transcript that is essential for embryonic development. This finding links m6A regulation to ribosome function and global protein synthesis during early development. Together, our results uncover a critical mechanism by which site-specific m6A fine-tunes gene expression and translation to support developmental competence, expanding our understanding of RNA modifications in mammalian embryogenesis.