LACE-seq and full-length sequencing reveals hnRNPM regulate alternative splicing during oocyte development and is essential for female fertility.

Growing oocytes accumulate maternal mRNA to sustain subsequent meiotic maturation and maternal-zygotic transition. However, the regulatory mechanisms governing maternal mRNA fate determination remain poorly understood. Here, we identify hnRNPM as a critical regulator of alternative splicing (AS) during oocyte growth, whose ablation leads to widespread AS defects and consequent female infertility. Phenotypic analysis reveals that Hnrnpm-null oocytes manifest cytoskeletal defects, abnormal distribution of mitochondria, accumulation of lipid droplets and failure of meiotic maturation and maternal-zygotic transition. Employing SCAN-seq, a novel high-sensitivity, high-accuracy technique based on third-generation sequencing (TGS) platforms for single-cell analysis-we identified novel oocyte transcripts and characterized hnRNPM-regulated AS events. Furthermore, using LACE-seq, a high-resolution method compatible with low-input samples, we mapped hnRNPM binding sites at single-nucleotide resolution in oocytes. In addition, our results reveal that hnRNPM interacts with BCAS2 and recruits it to pre-mRNA loci to regulate alternative splicing of transcripts(Cenpf and Prc1) associated with meiosis. Overall, our study not only elucidates a critical regulatory network governing alternative splicing during oocyte development but also illustrates the power of SCAN-seq and LACE-seq for investigations of RNA-binding protein in oocytes.