Structure of spliceosomal UsnRNPs

Abstract

The major snRNPs, Ul, U2, U4/U6 and U5, are essential trans-acting factors in the pre-mRNA splicing process. They assemble with a pre-mRNA and a number of other non-snRNP splicing factors prior to the splicing reaction to form an active spliceosome. We are interested in investigating the biochemical composition of UsnRNPs and their ultrastructure as well as their function in splicing. In HeLa cell nuclear extracts the spliceosomal UsnRNPs exhibit differential association behaviour depending on the salt concentration. Thus, at high salt (420 mM) the majority of the Ul, U2, U4/U6 snRNPs migrates on sucrose gradients at 10-12S, while U5 snRNP sediments at 20S. Under in vitro splicing conditions (i.e. at about 100 mM salt), U5 and U4/U6 snRNPs form a 25 S [U4/U6.U5]tri-snRNP-complex and U2 snRNPs sediment at about 17 S.We have isolated the various types of UsnRNPs under native conditions using mainly immunoaffinity chromatography procedures. Today we can distinguish more than 35 distinct snRNP proteins. They can be grouped into two classes. The first class comprises eight common snRNP proteins which are present in each of the spliceosomal UsnRNPs. In addition, the individual snRNPs contain snRNP-specific proteins. These include three (70k, A, C) for the 12 S Ul snRNP, two (A′, B″ for the 12 S U2 snRNP, an additional eight for the 17 S U2 snRNP and eight for the 20 S U5 snRNP. The 25 S [U4/U6.U5]tri-snRNP-complex contains, in addition to the common proteins and the U5-specific proteins, a third group of six proteins which are essential for the stable formation of the tri-snRNP-complex. Thus, the different S-values of a particular snRNP particle result from differences in the population of snRNP-specific proteins associated with that particle.