Polyadenylation in mammalian male germ cells: The role of CstF-64 protein
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Polyadenylation is the process of 3' end formation of eukaryotic mRNAs. It has been noted that 90% of mRNAs in somatic cells contain AAUAAA, which is required as a signal for polyadenylation in vitro and in vivo. However, we have found that 60% of mRNAs in mammalian male germ cells lack this canonical sequence, and yet seem to be polyadenylated normally. This suggests that the polyadenylation machinery is different in germ cells as compared to somatic cells. To account for this difference we have shown that there are two immunologically distinct forms of the 64 KD subunit of CstF (Cleavage Stimulation Factor) in mouse germ cells. These two forms are encoded by two different genes. The gene for the somatic CstF-64 is present on the X chromosome in both mouse and human and is subject to inactivation due to XY body formation during meiosis in the testis. Since CstF-64 is a housekeeping gene it is required to be functional at all stages of spermatogenesis for the cells to be viable. Loss of the somatic protein due to inactivation of the X-linked gene creates a need for an alternative source of the CstF-64 protein. We propose that TCstF-64 is the alternate protein that fulfills this requirement. TCstF-64 is encoded by an autosomal gene (mouse chromosome 19, human chromosome 10), has a slower mobility of -70,000 Mr on SDS-PAGE gels, is reactive to only the 6A9 antibody and shares a similar peptide fragment pattern with the resident testis 6A9-reactive protein. These data support our hypothesis that male germ cells utilize an alternative form of CstF-64 to recognize non-canonical polyadenylation signals. We propose that the somatic form of CstF-64 recognizes AAUAAA and its gene is inactivated during male meiosis creating the need for an alternative autosomal source of the protein which is capable of polyadenylating non-canonical messages. Experiments are underway to determine the role of TCstF-64 in male germ cell mRNA processing.