SELECTION OF THE BOVINE PAPILLOMAVIRUS TYPE-1 NUCLEOTIDE-3225 3'-SPLICE-SITE IS REGULATED THROUGH AN EXONIC SPLICING ENHANCER AND ITS JUXTAPOSED EXONIC SPLICING SUPPRESSOR

Alternative splicing is an important mechanism for the regulation of b ovine papillomavirus type 1 (BPV-1) gene expression during the virus l ife cycle. However, one 3' splice site, located at nucleotide (nt) 322 5, is used for the processing of most BPV-1 pre-mRNAs in BPV-l-transfo rmed C127 cells and at early to intermediate times in productively inf ected warts. At late stages of the viral life cycle, an alternative 3' splice site at nt 3605 is used for the processing of the late pre-mRN A. In this study, we used in vitro splicing in HeLa cell nuclear extra cts to identify cis elements which regulate BPV-1 3' splice site selec tion. Two purine-rich exonic splicing enhancers were identified downst ream of nt 3225, These sequences, designated SE1 (nt 3256 to 3305) and SE2 (nt 3477 to 3526), were shown to strongly stimulate the splicing of a chimeric Drosophila doublesex pre mRNA, which contains a weak 3' splice site. A BPV-1 late pre-mRNA containing the nt 3225 3' splice si te but lacking both SE1 and SE2 was spliced poorly, indicating that th is 3' splice site is inherently weak Analysis of the 3' splice site su ggested that this feature is due to both a nonconsensus branch point s equence and a suboptimal polypyrimidine tract. Addition of SE1 to the late pre-mRNA dramatically stimulated splicing, indicating that SE1 al so functions as an exonic splicing enhancer in its normal context. How ever, a late pre-mRNA containing both SE1 and SE2 as well as the seque nce in between was spliced inefficiently. Further mapping studies demo nstrated that a 48-nt pyrimidine-rich region immediately downstream of SE1 was responsible for this suppression of splicing. Thus, these dat a suggest that selection of the BPV-1 nt 3225 3' splice site is regula ted by both positive and negative exonic sequences.