Function of a bovine papillomavirus type 1 exonic splicing suppressor requires a suboptimal upstream 3' splice site

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. Previous studies in our laboratory have identified two purine-rich exonic splicing enhancers (ESEs), SE1 and SE2, located between two alternative 3' splice sites at nucleotide (nt) 3225 and nt 3605. Further analysis of BPV-1 late-pre-mRNA splicing in vitro revealed a 48-nt pyrimidine-rich region immediately downstream of SE1 that inhibits utilization of the nt 3225 3' splice site. This inhibitory element, which we named an exonic splicing suppressor (ESS), has a U-rich 5' end, a C-rich central part, and an AG-rich 3' end (Z. M. Zheng, P. He, and C. C. Baker, J. Virol. 70:4691-4699, 1996). The present study utilized in vitro splicing of both homologous and heterologous pre-mRNAs to further characterize the ESS. The BPV-1 ESS was inserted downstream of the 3' splice site in the BPV-1 late pre-mRNA, Rous sarcoma virus src pre-mRNA, human immunodeficiency virus tat-rev pre-mRNA, and Drosophila dsx pre-mRNA, all containing a suboptimal 3' splice site, and in the human beta-globin pre-mRNA, which contains a constitutive 3' splice site. These studies demonstrated that suppression of splicing by the BPV-1 ESS requires an upstream suboptimal 3' splice site but not an upstream ESE. Furthermore, the ESS functions when located either upstream or downstream of BPV-1 SE1. Mutational analyses demonstrated that the function of the ESS is sequence dependent and that only the C-rich region of the ESS is essential for suppression of splicing in all the pre-mRNAs tested.