The sequence and context of the 5' splice site govern the nuclear stability of polyoma virus late RNAs

Abstract

We have examined the influence of splicing signals on the stability of polyoma virus late RNAs in the nucleus. Late primary transcripts contain a single 5' splice site and three alternative 3' splice sites. In earlier work we showed that the presence of introns was not required for late RNA accumulation, however, the 5' splice site was essential, as removal of only the 5' splice site was sufficient to destabilize late RNAs up to 100-fold when compared with early RNAs. A complementary clone which retained the 5' splice site but which carried small deletions of all late region 3' splice sites produced wild-type levels of unspliced late RNA. In order to extend this work we have constructed additional types of mutants. Point mutations in the 5' splice site confirmed its importance for RNA stability. Other mutants included constructs in which the spacing between the 5' splice site and the late promoter was altered and 5' splice site insertion mutants where a 58 bp fragment containing the 5' splice site sequence was inserted separately at various restriction sites in the late region. Both types of mutants lacked all of the late 3' splice sites and had only a single 5' splice site. RNase protection analyses of late and early RNAs from these constructs revealed that moving the 5' splice site away from the late promoter (or from its normal context) destabilized late RNAs > 10-fold relative to the wild-type. We conclude that both 5' splice site integrity and its proximity to the late promoter play important roles in the nuclear stability of polyoma virus late RNAs.