STRUCTURAL, FUNCTIONAL, AND PROTEIN-BINDING ANALYSES OF BOVINE PAPILLOMAVIRUS TYPE-1 EXONIC SPLICING ENHANCERS

Alternative splicing plays an important role in regulation of bovine p apillomavirus type 1 (BPV-1) gene expression, We have recently identif ied in BPV-1 late pre-mRNAs two purine-rich exonic splicing enhancers (SE1 and SE2) which also stimulate splicing of a Drosophila doublesex (dsx) pre-mRNA containing a suboptimal 3' splice site. In vivo studies now demonstrate that both SE1 and SE2 are required for preferential u se of the BPV-1 nucleotide (nt) 3225 3' splice site in nonpermissive c ells, Deletion or mutation of either element in a BPV-1 late pre-mRNA switches splicing to the late-specific alternative 3' splice site at n t 3605. To investigate the sequence specificity of these exonic splici ng enhancers, various mutant SE1 or SE2 elements were connected to dsx pre-mRNAs and tested for their stimulatory effects on dsx pre-mRNA sp licing in vitro, Substitution of U residues for either A or G residues in and around potential ASF/SF2 binding sites in SE1 or SE2 resulted in a significant reduction of splicing enhancer activity, However, the G-to U substitutions in both enhancers had the largest effect, reduci ng splicing to near control levels, Further in vitro analyses showed t hat splicing enhancement by SE2 could be competed with excess unlabele d SE2 RNA, indicating that SE2 activity in HeLa nuclear extracts is me diated by trans-acting factors. UV cross-linking plus immunoprecipitat ion assays showed that both wild-type SE1 and SE2 RNAs could bind dire ctly to purified HeLa SR proteins SRp30a (ASF/SF2), SRp55, and SRp75, UV cross-linking experiments also identified a 23-kDa protein which bi nds to SE2 but not SE1. This protein is present in both HeLa nuclear e xtracts and S100 extracts but absent from SR protein preparations, sug gesting that it is not a classical SR protein. Mutant SE elements (con taining G-to U-mutations) which had minimal splicing enhancer activity also had very weak binding capacity for these proteins, strongly sugg esting that the binding of these proteins is required for splicing enh ancer function.