THE SACCHAROMYCES-CEREVISIAE PRP21 GENE-PRODUCT IS AN INTEGRAL COMPONENT OF THE PRESPLICEOSOME

In Saccharomyces cerevisiae, the prp21 mutation causes accumulation of unspliced pre-mRNA at the nonpermissive temperature. We have cloned the PRP21 gene by complementation of its temperature-sensitive phenotype and found it to be the same as SPP91, an extragenic suppressor of the prp9 mutation previously studied in vivo by Chapon and Legrain [Chapon, C. & Legrain, P. (1992) EMBO J. 11, 3279-3288]. We have analyzed the effects of the prp21 mutation on splicing in vitro and have found that PRP21 is a splicing factor required for prespliceosome assembly. We also have analyzed the interaction of PRP21 with splicing complexes using anti-PRP21 antibodies and found that the RNA components of the prespliceosome-U1 and U2 small nuclear RNA (snRNA) particles and pre-mRNA-are specifically coimmunoprecipitated under splicing conditions in the presence of 0.2 M KCl. At higher KCl concentrations, U1 snRNP dissociates from splicing complexes; nevertheless, U2 snRNA and pre-mRNA are still efficiently immunoprecipitated. Immunoprecipitation of both U1 and U2 snRNA as well as pre-mRNA is ATP-dependent and requires a pre-mRNA capable of supporting prespliceosome assembly. Analysis of the unbound complexes in native gels confirmed that prespliceosomes are specifically immunoprecipitated by anti-PRP21 antibodies. These results demonstrate that PRP21 is an integral component of the prespliceosome and establishes a stable interaction with U2 snRNP and/or pre-mRNA in that complex.