We are trying to understand how mRNA polyadenylation occurs in male germ cells in the absence of the canonical AAUAAA polyadenylation signal. During our previous grant period we discovered and characterized a variant form of the CstF-64 polyadenylation protein that is expressed at high levels in meiotic and postmeiotic germ cells, and in no other tissue except the brain. We cloned and sequenced this new protein, called """"""""tauCstF-64,"""""""" and it is the leading candidate to account for AAUAAA-independent polyadenylation in germ cells. Our overall laboratory hypothesis is that (1) the somatic CstF-64 gene (Cstf2) is on the X chromosome in mice and humans, (2) the Xchromosomal CstF-64 is inactivated during male meiosis, (3) since CstF-64 function is essential, an auxiliary CstF-64 gene must be expressed from an autosome, and (4) the auxiliary CstF-64 protein is responsible for AAUAAA-independent polyadenylation of mRNAs in male germ cells and is essential for spermatogenesis. In the previous grant period we addressed items 1-3 of our laboratory hypothesis. In this application we propose to address item 4 or our laboratory hypothesis: we will determine whether specific domains of tauCstF-64 contribute to germ cell polyadenylation. We will also determine whether tauCstF-64 is sufficient for AAUAAA-independent polyadenylation by expressing it ectopically in cells in culture, and whether it is necessary for spermatogenesis by creating a transgenic strain of mice that lack the Cstf2t gene for tauCstF-64. Finally, we will begin to examine tauCstF-64 function in human germ cells by examining its distribution in human testis and other tissues.
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