Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance

Abstract

One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.