RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

摘要

Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing. Author Summary The production of functional spermatozoa is an extraordinarily complex process that transforms a conventional round cell into the highly specialised sperm cell. These events require the coordinated activation of thousands of genes. It is likely that this complexity contributes to the large number of idiopathic infertility cases seen in humans. In an effort to improve the field's understanding of male fertility, we used a random mutagenesis screen to produce the Joey mouse line and to conclusively define RBM5 as an essential regulator of male fertility. The Joey line carries a mutation in the Rbm5 gene, which leads to a complete block of spermatid (haploid male germ cell) differentiation and ultimately a total loss of sperm production. Our results reveal a physiological role for RBM5 in the splicing of several spermatid-expressed mRNAs that are critical for the production of spermatozoa. This study is the first to show that RBM5, via its effects on mRNA splicing in the testis, is required for male fertility. These data improve our understanding of the regulatory networks of gene expression that control sperm production and as such may lead to the development of novel approaches to enhance or suppress fertility in men.