Isolation and Characterization of the U4 SnRNP in Saccharomyces Cerevisiae

The U4 snRNA may exist as a discrete particle prior to U4/U6 snRNP formation; however, the free U4 snRNP has not been isolated, as it does not accumulate in cellular lysates. This investigation utilized both genetic and biochemical means to generate and characterize U4 snRNP from Saccharomyces cerevisiae. I identified Prp24 alleles that cause a shift to free U4 RNA. Analysis of such lysates on cesium chloride gradients provided evidence that the accumulated U4 RNA occurs as an RNP. In addition, I modified Raghunathan & Guthrie's (1998b) method of generating U4 snRNA species for the preparative generation of U4 snRNA/P and confirmed that it is an RNP. I compared the sedimentation of the two U4 RNA-containing species by rate-zonal and isopycnic sedimentation and found them to differ. In addition, I successfully captured free U4 and U4/U6 RNA-containing species by targeting U4 RNA residues with a complementary 2'-O-methyl RNA oligonucleotide.