Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal

Abstract

An integral component of the splicing machinery, the U1 snRNP, is here implicated in the efficient polyadenylation of SV40 late mRNAs. This occurs as a result of an interaction between U1 snRNP-A protein and the upstream efficiency element (USE) of the polyadenylation signal. UV cross-linking and immunoprecipitation demonstrate that this interaction can occur while U1 snRNP-A protein is simultaneously bound to U1 RNA as part of the snRNP. The target RNA of the first RRM (RRM1) has been shown previously to be the second stem-loop of U1 RNA. We have found that a target for the second RRM (RRM2) is within the AUUUGURA motifs of the USE of the SV40 late polyadenylation signal. RNA substrates containing the wild-type USE efficiently bind to U1 snRNP-A protein, whereas substrates fail to bind when motifs of the USE were replaced by linker sequences. The addition of an oligoribonucleotide containing a USE motif to an in vitro polyadenylation reaction inhibits polyadenylation of a substrate representing the SV40 late polyadenylation signal, whereas a mutant oligoribonucleotide, a nonspecific oligoribonucleotide, and an oligoribonucleotide containing the U1 RNA-binding site had much reduced or no inhibitory effects. In addition, antibodies to bacterially produced, purified U1 snRNP-A protein specifically inhibit in vitro polyadenylation of the SV40 late substrate. These data suggest that the U1 snRNP-A protein performs an important role in polyadenylation through interaction with the USE. Because this interaction can occur when U1 snRNP-A protein is part of the U1 snRNP, our data provide evidence to support a link between the processes of splicing and polyadenylation, as suggested by the exon definition model.