Clinton C. MacDonald - US grants

DESCRIPTION (provided by applicant): Polyadenylation-the addition of a poly(A) tail to an mRNA 32 end-is essential for gene expression in every tissue. However, fundamental features of polyadenylation-the RNA signals used, the sites chosen, and the proteins involved-are different in male germ cells than in any other tissue. These differences are essential for the correct expression of key genes during spermatogenesis, for example altering the protein forms of transcription factors or altering translational efficiency. DCstF-64 (gene name: Cstf2t) is the testis-expressed variant of CstF-64, an RNA-binding protein that regulates polyadenylation in somatic cells. DCstF-64 is essential for normal spermatogenesis: male Cstf2t knockout mice are infertile due to severe defects in meiotic and postmeiotic sperm production, a condition that resembles oligoasthenoteratozoospermia in human patients. We have found that Cstf2t controls polyadenylation of at least two important classes of genes, long interspersed nuclear elements (LINEs) and intronless small genes (ISGs). LINEs are mobile elements that are responsible for at least 70 genetic diseases, thus being a genomic and metabolic burden. DCstF-64 reduces LINE mRNAs by promoting polyadenylation at internal sites in the gene, thus reducing their abundance and suppressing their proliferation. To determine how DCstF-64 controls LINE expression, we will test whether exogenous DCstF-64 will suppress LINE mRNA expression, whether DCstF-64 binds to LINE mRNAs at internal polyadenylation sites, and whether exogenous DCstF-64 will suppress rates of retrotransposition in a cell culture assay. ISGs are expressed retroposons that control major functions in spermatogenesis (metabolism, gene expression, chromosome structure, and more). To determine how DCstF-64 regulates polyadenylation and termination of ISG mRNAs, we will test whether DCstF-64 is associated preferentially with ISG polyadenylation sites, whether exogenous DCstF-64 is required for correct polyadenylation of ISGs, and whether DCstF-64 is required for normal transcriptional termination of ISGs. Finally, to determine germ cell-specific functions of DCstF-64, we will perform a genetic test to determine whether CstF-64 will complement the Cstf2ttm1Ccma infertility phenotype, purify DCstF-64 complexes to look for germ cell-specific components, and test functions of DCstF-64 domains using a luciferase-based cell transfection assay and transgenic mice.

Polyadenylation is an essential mRNA processing event by which eukaryotic mRNAs form their 3' ends. Nearly all mRNAs are polyadenylated, and 3' end formation controls expression of potentially hundreds of genes. In male germ cells these include germs cell-essential transcription factors, cell cycle regulatory genes, proto-oncogenes, DNA packaging genes and genes involved in fertilization. In somatic tissues the signal AAUAAA is required for mRNA polyadenylation. In striking contrast, we have found that as many as 40 percent of mRNAs expressed in male germ cells lack this essential polyadenylation signal. We propose that the 64,000 M regulatory protein of the cleavage stimulation factor (CstF-64) is specifically modified in germ cells and is responsible for polyadenylation of mRNAs that lack AAUAAA. We have shown that a testis-specific form of this protein is expressed specifically during meiosis, and the somatic form is absent in those cells. We propose experiments to determine the structure and function of the testis-specific CstF-64 protein. The gene for somatic CstF-64 is on the X chromosome, and is inactivated during meiosis. Therefore we will also test whether there is a second, autosomal gene for CstF-64 that is specifically expressed during and after meiosis. Finally, we will test whether the testis-specific CstF-64 protein can mediate the germ cell pattern of polyadenylation both in vitro and in vivo. The ability of male germ cells to process pre-mRNAs that lack AAUAAA is unique in all cells of the body, and, to our knowledge, no one is studying this phenomenon. Nevertheless, it has the potential to affect many of the genes that control male germ cell development and fertility. The experiments proposed here should allow us to begin to understand how this fundamental process operates in male germ cells.