Spermatogenesis requires extensive control of gene expression to coordinate the unique cell proliferation and differentiation events. Dysregulation of post-transcriptional processing can impair sperm function but little is known about the global germ cell-specific regulatory mechanisms. The 3'untranslated region (UTR) of male germ cell transcripts contain cis-regulatory elements, including destabilization elements and microRNA binding sites, that control subsequent mRNA metabolism including localization, stability and translational. Transcripts can be expressed as isoforms that differ only in 3'UTR length;the result of a process known as alternative polyadenylation. Short 3'UTR isoforms typically are processed at a proximal polyadenylation (poly(A)) site within the 3'UTR that will exclude cis-elements found in the long 3'UTR isoform. Alternative polyadenylation is tissue- and developmentally- regulated and differences in global poly(A) site use have been reported in different cell types. Proliferating cells, including cancer cells, display a prevalence for short 3'UTRs and, for specific transcripts, these shorter 3'UTRs have increased translation due to the loss of microRNA sites. Male germ cells also appear to have a prevalence of short 3'UTRs, compared to somatic cells, but a transcriptome- wide analysis of poly(A) site use in male germ cells has not been completed and the functional significance of 3'UTR length is not known. The overarching goal of this research program is to investigate post-transcriptional processing and subsequent mRNA metabolism in male germ cells. This proposal addresses several goals of the 2011 NICHHD Scientific Vision Workshop on Reproduction including contributing to a molecular atlas of fundamental male gametogenesis by using current high throughput sequencing and bioinformatics. A greater understanding of the molecular regulation of sperm production holds promise in identifying specific underlying causes of male infertility that can lead to more accurate tests, treatments and, in the case of germ-cell specific processes, also contraceptive targets. Our primary hypothesis is that male germ cells have a global tendency for shorter 3'UTR transcripts, compared to somatic cells, and these transcripts demonstrate increased stability and subsequent protein production.
The specific aims of this project are to: (1) compare transcriptome-wide poly(A) site use in male germ cells and somatic cells by PolyA-seq, (2) compare the stability and translational activity of the short 3'UTR and the long 3'UTR isoforms of male germ cell transcripts and (3) engage graduate and undergraduate students in current molecular biology techniques and reproductive biology to promote scientific literacy and careers. This approach has the ability to identify male germ cell specific patterns of alternative polyadenylation and candidate transcripts for further functional studies to determine the physiological significance of 3'UTR length during spermatogenesis.
A greater understanding of the molecular regulation of sperm production holds promise in identifying underlying causes of male infertility that can lead to more accurate tests and treatments in an effort to improve a patient's quality of life. With this proposl, we will identify specific patterns of male germ cell RNA regulation and determine the impact of this regulation on subsequent protein production.