Splice site selection in polyomavirus late pre-mRNA processing

Abstract

Polyomavirus late pre-mRNAs contain one 5' splice site and two message body 3' splice sites, which are not used at equal frequencies. As a result of alternative splicing, the total late mRNA population consists of about 5% mVP2 (no message body splice chosen), about 15% mVP3 (promoter-proximal 3' splice site chosen), and about 80% mVP1 (promoter-distal 3' splice site chosen). To determine whether it is splice site strength that determines the ratio of spliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bp surrounding the VP3 3' splice site was substituted with the corresponding region of the VP1 3' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacing between the 3' splice sites. Analysis of RNAs from transfections showed that in each construct, the 3' splice closest to the polyadenylation site was used preferentially. Analysis of a number of additional constructs indicated that there are no strong cis-acting positive or negative regulators of polyomavirus late splicing; rather, splicing choices appear to be determined largely by relative position of splice sites.