60 The RNA m6A landscape in bovine oocytes and pre-implantation embryo development

Mammalian preimplantation embryonic development is initiated by massive degradation of oocyte-stored maternal RNA/proteins and gradual activation of the embryonic genome. However, the precise mechanisms to regulate this process is not well understood. N6-methyladenosine (m6A) found in eukaryotic RNA plays key regulatory roles in gene regulation. However, mapping m6A in oocytes and early embryos using m6A antibody-based methyl RNA immunoprecipitation and sequencing has been challenging owing to the requirement for large amounts of materials and the inability for genome-wide single-base resolution detection. In this study, by using a newly developed m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq) approach, we defined the landscape of m6A in bovine oocytes (GV and MII stages), and preimplantation embryos (4-cell, 8-cell, 16-cell, and blastocyst stages) at genome-wide single-base resolution. We further compared the m6A epitranscriptome to the transcriptome and translatome data sets generated from matched bovine oocytes and a different stage of preimplantation embryos to gain deeper insights of gene regulation. Approximately 200 oocytes/embryos per replicate (n = 2) were used for RNA extraction and subsequent m6A-SAC-seq library preparation. We sequenced ~100 million pair-end reads per sample (60 million per SAC sample and 40 million per Input control sample) to ensure the m6A coverage. We identified by far the most m6A sites from mammalian oocytes and embryos: 34 746 (GV), 22 327 (MII), 16 414 (4-cell), 4534 (8-cell), 4084 (16-cell), and 9935 (blastocyst). Specifically, the m6A abundance peaked in GV oocytes, showed a marked drop during oocyte maturation and across cleavage stage embryos, and gradually increased in blastocysts. Given the relative even number of genes expressed in bovine oocytes and different stage of preimplantation embryos, the dynamics of m6A identified here suggest that m6A marks maternal transcripts largely reflecting the suppression of transcription and translation in oocytes. This modification is then increased in embryos at 4-, 8-, and 16-cell stages to boost the activation of the embryonic genome, and finally to selectively mark genes for earlier lineage differentiation. Importantly, we identified genes with abundant m6A modification that were specifically associated with each developmental stage, indicating their key regulatory roles. By integrating the m6A epitranscriptome to our published transcriptome and translatome data sets, we found that m6A modification precisely regulates the translational selectivity associated with oocyte maturation and embryo development. In conclusion, we present the first landscape of m6A modification in bovine oocytes and preimplantation embryos. Our results provide a foundation and insights for m6A-mediated complex epigenetic reprogramming in mammalian early embryonic development