Biochemical and genetic analyses of the U5, U65 and U4/U6 center dot U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonu cleoproteins (snRNPs) by affinity chromatography and analyzed the associate d polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of th e canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and t he 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from t he purified U4/U6.U5 snRNR A novel yeast U5 and four novel yeast U4/U6.U5 s nRNP polypeptides were characterized by genetic and biochemical means to de monstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon add ition of ATP or dATP.