THE SEQUENCE AND CONTEXT OF THE 5'-SPLICE-SITE GOVERN THE NUCLEAR-STABILITY OF POLYOMA-VIRUS LATE RNAS

We have examined the influence of splicing signals on the stability of polyoma virus late RNAs in the nucleus. Late primary transcripts cont ain a single 5' splice site and three alternative 3' splice sites. In earlier work we showed that the presence of introns was not required f or late RNA accumulation, however, the 5' splice site was essential, a s removal of only the 5' splice site was sufficient to destabilize lat e RNAs up to 100-fold when compared with early RNAs. A complementary c lone which retained the 5' splice site but which carried small deletio ns of all late region 3' splice sites produced wild-type levels of uns pliced late RNA. In order to extend this work we have constructed addi tional types of mutants. Point mutations in the 5' splice site confirm ed 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 fragme nt containing the 5' splice site sequence was inserted separately at v arious restriction sites in the late region. Both types of mutants lac ked all of the late 3' splice sites and had only a single 5' splice si te. RNase protection analyses of late and early RNAs from these constr ucts revealed that moving the 5' splice site away from the late promot er (or from its normal context) destabilized late RNAs >10-fold relati ve to the wild-type. We conclude that both 5' splice site integrity an d its proximity to the late promoter play important roles in the nucle ar stability of polyoma virus late RNAs.