Role of RNA modifications in spermatogenesis.

National Institute Of Environmental Health Sciences

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Abstract

Infertility affects approximately 10% of couples due to a combination of both male and female reproductive disorders. The proportion of infertile men and women is similar, but the etiology of the condition is sex-specific. The project aims to characterize molecular mechanisms required for sperm production. Genetic studies in rodents showed that several RNA binding proteins (RBPs) are necessary at different stages of gametogenesis; however, little is known about their functions mechanistically. We aim to gain insights into the role of RNA metabolism in sperm production by focusing on the role of RNA modifications. RBPs can promote transcript translation, storage, or degradation and is the balance between different protein complexes that decides the fate of the mRNA. In the process, the molecular machinery leaves various marks/modifications on the RNA that determine the molecular pathways the transcripts will follow. In particular, the 3 ends of mRNAs are tagged by specialized complexes that can sort the transcript into the different RNA processing compartments. For example, the Terminal Uridylyl-Transferases, TUT4 and TUT7, tag the mRNAs tails with non-templated Us, a process known as uridylation, that leads to transcript decay. We are now depleting TUT4 and TUT7 in different types of male germ cell, where they are highly expressed, to assess the relevance of Us additions to mRNAs in gametogenesis. The most common type of mRNA 3 end modification is the addition of a poly(A) tail. Although polyadenylation is believed to be critical in the germline, little is known about cytoplasmic poly(A) tail dynamics and the molecular machinery responsible for the modification in germ cells. Several Terminal Nucleotidyl Transferases (TENTs) with polyadenylation potential are expressed during spermatogenesis. To evaluate the physiological relevance of the TENTs, we are generating animal models to deplete the proteins at various stages of male gametogenesis. This year, we generated several floxed and null alleles that showed germline transmission, and we established reporter lines to identify and isolate particular populations of germ cells. Additionally, we set up a crucial protocol to capture RNA modifications, including the development of a bioinformatics pipeline to analyze the data generated. By applying this and other RNA quantification techniques to samples derived from genetically modified germ cells, we will better understand how RNA processing regulates sperm production.