The objective of this research is to provide a molecular understanding of an unusual alternative splicing of bovine growth hormone (bGH) pre-mRNA in which the last intron is retained in a small proportion of mature message to produce an intron-encoded variant bovine growth hormone (vbGH). An efficient in vitro splicing system will be used to test the hypothesis that constitutive splicing factors mediate a collaborative interaction between short, positive-acting signals in the downstream exon and a weak, upstream 5' splice site to produce the spliced form of the message. We will define the cellular and sub-cellular localization of the 27kD vbGH protein to better understand the biological consequences of this form of alternative splicing. 1) Mutation of the 5' splice site and an adjacent """"""""pseudo 5' splice site"""""""" will be used to define the essential features of these sequences in determining the levels of alternative splicing in transfected cells and early spliceosome complex formation in vitro. A core 10-nucleotide sequence contained within a positive element in the downstream exon will be systematically mutated to test the hypothesis that this signal stimulates intron removal via an interaction with U1 snRNP. 2) UV-crosslinking and gel-retardation assays will be used to identify those splicing factor(s) which directly interact with the positive exonic sequence. Purified splicing factors and mutated forms of these factors will be used in vitro to define the observed antagonistic effects of splicing factors SF2 and hnRNP-A1 on the splicing of this bGH intron. 3) The sub-cellular localization and possible post-translational modification of the vbGH protein will be examined in transfected cells and the bovine pituitary by immunocytochemical and Western blot techniques utilizing anti-vbGH-specific monoclonal antibodies. The developmental expression of vbGH in the pituitary and recent observation of vbGH expression in ovarian tissue will be characterized at both the RNA and protein levels. Receptor binding studies and somatotroph specific transgenic expression of vbGH will ultimately be employed to probe its potential biological role. Alternative splicing is a common form of post-transcriptional regulation of gene expression which is only partially understood at the molecular level. The alternative splicing of the last intron of bGH pre-mRNA provides an opportunity to clarify the roles of multiple RNA signals and interacting splicing factors while probing the biological implications of this novel hormone isoform.
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