BCAS2 and hnRNPH1 orchestrate alternative splicing for DNA double-strand break repair and synapsis in meiotic prophase I [RNAs-seq]

Understanding the intricacies of homologous recombination during meiosis is crucial for reproductive biology. However, the role of alternative splicing (AS) in DNA double-strand breaks (DSBs) repair and synapsis remains elusive. In this study, we investigated the impact of conditional knockout (cKO) of the splicing factor gene Bcas2 in mouse germ cells, revealing impaired DSBs repair and synapsis, resulting in non-obstructive azoospermia (NOA). Employing crosslinking immunoprecipitation and sequencing (CLIP-seq), we globally mapped BCAS2 binding sites in the testis, uncovering its predominant association with 5' splice sites (5'SS) of introns and a preference for GA-rich regions. Integrating CLIP-seq with RNA-seq, we identified BCAS2 as a key player in the AS of pre-mRNAs, including Spo11, Six6os1, Sycp1, Msh5, Cdk2, Sun2, Stag3, and Spdya, crucial for DSBs repair and synapsis. Notably, BCAS2 exhibited direct binding and regulatory influence on Trp53bp1 and Six6os1 through AS, unveiling novel insights into DSBs repair and synapsis during meiotic prophase I. Furthermore, the interaction between BCAS2, hnRNPH1, and SRSF3 was discovered to orchestrate Trp53bp1 expression via AS, underscoring its role in meiotic prophase I DSBs repair. In summary, our findings delineate the indispensable role of BCAS2-mediated post-transcriptional regulation in DSBs repair and synapsis during male meiosis. This study provides a comprehensive framework for unraveling the molecular mechanisms governing the post-transcriptional network in male meiosis, contributing to the broader understanding of reproductive biology. STRA8+ cell suspensions were isolated and prepared from mouse testes. Total RNA was extracted from STRA8+ cells.解析减数分裂过程中同源重组的复杂机制，对于生殖生物学研究至关重要。然而，可变剪接（alternative splicing, AS）在DNA双链断裂（DNA double-strand breaks, DSBs）修复与联会中的作用仍不明确。本研究针对小鼠生殖细胞中剪接因子基因Bcas2的条件性敲除（conditional knockout, cKO）展开探究，发现其会导致DSBs修复与联会受损，进而引发非阻塞性无精子症（non-obstructive azoospermia, NOA）。通过交联免疫沉淀测序（crosslinking immunoprecipitation and sequencing, CLIP-seq）技术，我们在全基因组范围内定位了BCAS2在睾丸中的结合位点，揭示其主要与内含子5'剪接位点（5' splice sites, 5'SS）结合，并偏好GA富集区域。将CLIP-seq数据与RNA-seq数据整合分析后，我们鉴定出BCAS2是前体mRNA（pre-mRNA）可变剪接的关键调控因子，其靶基因包括Spo11、Six6os1、Sycp1、Msh5、Cdk2、Sun2、Stag3及Spdya，这些基因均对DSBs修复与联会具有重要作用。值得注意的是，BCAS2可直接结合并通过可变剪接调控Trp53bp1与Six6os1的表达，为减数分裂I前期的DSBs修复与联会机制提供了全新的研究视角。此外，本研究发现BCAS2与hnRNPH1、SRSF3的相互作用可通过可变剪接协同调控Trp53bp1的表达，进一步阐明了其在减数分裂I前期DSBs修复中的调控功能。综上，本研究明确了BCAS2介导的转录后调控在雄性减数分裂DSBs修复与联会过程中不可或缺的核心作用。本研究为解析雄性减数分裂中转录后调控网络的分子机制提供了全面的研究框架，有助于深化对生殖生物学的整体认知。本研究从小鼠睾丸中分离并制备了STRA8+细胞悬液，并从该类细胞中提取了总RNA。