Purification and visualization of native spliceosomes

Abstract

Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.