Reconstitution of exon-bridging activity with purified U2AF and U1 snRNP components

Abstract

For pre-mRNAs containing multiple introns, the exon definition hypothesis has been proposed to account for the interactions that specify relatively short exons and prevent inappropriate exon-skipping [1]. Support for this hypothesis includes the finding that naturally occurring, or engineered mutations in 5' splice sites that weaken base complementary to U1 snRNA result in exon skipping due to a decrease in upstream 3' splice site activity. The reciprocal effect is also observed. For example, we found previously that the selection of the alternatively spliced rat preprotachykinin exon 4 is improved under conditions in which the adjacent 5' splice site is converted to a site with strengthened base pairing to U1 snRNA [2]. In the latter study, 3' splice site activity is improved in parallel with strengthened U1 snRNP binding to the downstream 5' splice site. Subsequent RNA-protein crosslinking experiments have provided evidence for exon bridging interactions between U2AF bound to the 3' splice site and U1 snRNP bound to the downstream 5' splice site in the preprotachykinin substrates [3]; see Figure 1. U2AF, a polypyrimidine tract binding protein composed of 65 and 35 kD subunits, is required for U2 snRNP binding to the adjacent branch site [4], [5]. In this work we have reconstituted exon bridging activity with purified components. These results show that U1 snRNP in addition to U2AF are the two components required to reconstitute full activity in vitro. The purified system has been used to test variants of U2AF and U1 snRNP. Our results show that the U1-A and U1-C proteins are dispensable for exon bridging activity. In addition, the 35 kD subunit of U2AF appears to be dispensable, at least under certain conditions.