Investigating mechanisms of RNA regulation during spermatogenesis
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Gene regulation is a complex process that involves a variety of mechanisms throughout the lifetime of an RNA molecule, including control of transcription levels, pre-mRNA processing, and regulation of mRNA stability and translation. Dysregulation of these mechanisms can impact human health and, though many aspects of gene regulation have now been uncovered, our understanding of them and how they control biological processes is still incomplete. Spermatogenesis, as a cellular differentiation trajectory which requires particularly expensive translational and post-translational control, provides a system to understand these regulatory processes. The X and Y chromosomes are silenced during meiosis in males, and we have investigated the role of nuclear Argonaute proteins in this process. We find that AGO3 and AGO4 play partially overlapping roles in spermatogenesis, and knockout of both proteins leads to upregulation of XY genes and altered timing of spermatid gene programs. We find evidence of interaction between Argonautes and chromatin remodelers in the nucleus, which may mediate removal of these remodelers. Post-transcriptional regulation is also particularly important for spermatogenesis, and we study selection on 3′UTR regulatory regions in these genes using SNPs. Most regulatory regions show evidence of current negative selection, but we find increased signature of selection when testing for SNP depletion at these sites compared to all types. Different subtypes of regulatory regions are under different selective pressure of different strength, indicating their varying importance to post-transcriptional regulation. Nonconserved RNA binding protein motifs are also enriched for polymorphisms, in contrast to all other regulatory regions. Together, this work advances the understanding of both transcriptional and post-transcriptional regulatory mechanisms important during spermatogenesis and across biological processes. Furthermore, this work demonstrates the utility of using a complex biological system to understand regulation, and accounting for diverse methods of regulation.