RNA Immunoprecipitation(RIP)-seq of BUD31 in the mouse reproductive system

The testis is the organ with the most transcriptome diversity and alternative splicing (AS) is considered to be the most important source. However, the physiological role of AS in spermatogenesis is poorly understood. Here, we report that spliceosome component BUD31 is a prominent AS regulator in early stage of spermatogenesis in mice. Bud31 expression in the testis is initiated soon after birth. Conditional knock out of Bud31 in germ cells in Vasa-Cre mice led to loss of spermatogonial stem cells (SSCs) and to infertility. We further demonstrate that BUD31was required for both SSC pool maintenance and initiation of spermatogenesis. Mechanistically, BUD31 regulates the alternative splicing of multiple genes involved in cell cycle checkpoint and cell differentiation. In particular, we identified CDK2 as a direct splicing target of BUD31 through integrative analysis of SMART-seq and RIP-seq data. Knockout of BUD31 results in the intron retention as well as exon skipping of CDK2, which led to decrease in CDK2 expression. Our findings highlight the significance of BUD31 and its regulated AS in SSC self-renewal and differentiation. The assay was performed in the mouse Germ cells with BUD31anti-BUD31 antibody.10% of total RNA was used as input.