The long-term objectives of this proposal are directed towards understanding the molecular basis of the alternative pre-mRNA processing reactions that occur during the post-meiotic stages of spermatogenesis. In spermatids, both polyadenylation and splicing at alternative sites produce a variety of unique MRNA species with structures that differ from the corresponding somatic transcripts. It is clear that the alternative processing of pre-MRNAS is an important component of the changes in gene expression that are required for the developmental pathway that generates mature spermatozoa. Thus, defects in the trans- acting factors involved in alternative pre-MRNA processing in spermatids may be one source of male infertility. In addition, these defects could produce anomalous transcripts from cellular proto-oncogenes, causing the uncontrolled proliferation of germ line cells that ultimately leads to the formation of testicular carcinomas. The research program described in this application is based on preliminary evidence that the post-meiotic expression of variant forms of several small nuclear ribonucleoprotein particles (snRNPs: the trans- acting factors required for polyadenylation and splicing) correlates with the synthesis of spermatid-specific mRNAs. Accordingly, the initial purification and characterization of the trans-acting factors and cis- acting elements that are required for alternative pre-mRNA processing in spermatids in the first priority of the proposed experiments.
In Specific Aim 1, the extent of alternative pre-mRNA processing in spermatids will be determined by measuring the frequency of inserts in a spermatid cDNA library that encode alternatively processed mRNAs. In addition, the frequency of aberrant cis-acting elements at spermatid- specific polyA addition sites will be assessed. Any regions detected by these two approaches that contain alternative polyadenylation or splice sites will be characterized and compared.
In Specific Aim 2, the structures of spermatid-specific snRNP variants will be studied by two approaches. First, the RNA components of the variants snrnps will be sequenced to define the differences between spermatid-specific and somatic snrnp RNAs. Secondly, differences in the protein composition of snrnps purified from spermatids and somatic cells will be analyzed.
In Specific Aim 3, nuclear extracts will be prepared from spermatids and somatic cells that accurately reproduce the sequence specificities of the polyadenylation and splicing reactions in these cells. By mixing the two extracts, any inhibition of spermatid-specific pre-mRNA processing by negative trans-acting factors in the somatic extracts will be detected. Finally, the ability of the variant snRNPs to alter the activity of a somatic extract so that spermatid-specific ployA addition or splice sites are correctly processed will be tested. Ultimately, these in vitro polyadenylation and splicing reactions will provide a means of determining which trans-acting factors and cis-acting elements are necessary for alternative pre-mRNA processing in spermatids.
