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.
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