Isolation and characterization of the U4 snRNP in Saccharomyces cerevisiae.

Pre-messenger RNA (pre-mRNA) splicing is a cellular phenomenon common to all eukaryotes in which non-coding sequences are removed from pre-mRNA transcripts and the coding sequences spliced together yielding mRNA. The spliceosome is the entire complement of proteins and snRNAs responsible for pre-mRNA splicing and forms around a core of five small nuclear ribonucleoprotein particles (snRNPs). There is evidence suggesting that the RNA component of the spliceosome is responsible for the two transesterification reactions that produce the spliced mRNA, highlighting the importance of understanding the behaviour of spliceosomal RNAs and the relationship between RNA and proteins in the spliceosome. With the goal of identifying the major structures of the spliceosome and their organization, there has been an effort to isolate the discrete snRNPs, identify the associated proteins and structurally characterize the particles. While the U1, U2, U4/U6, U4/U6.U5 and U2/U6 snRNPs have been isolated and studied, there are other snRNPs that have been hypothesized to exist. The U4 snRNA is believed to exist as a discrete particle prior to incorporation into the U4/U6 snRNP 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 U4 snRNP from Saccharomyces cerevisiae for the purposes of isolation and characterization. I identified the 1sub/4sub allele of prp24 ...as a mutation set that causes a shift to almost 100% of U4 RNA in the cell occurring in the free form. Analysis of lysates from this strain on cesium chloride gradients provided evidence that the accumulated U4 RNA is associated with a mass of protein and that it is indeed an RNP. In addition, I successfully modified Raghunathan & Guthrie's ...method of generating Brr2-released U4 snRNA species with the goal of creating a preparative system for generating U4 snRNA/P and confirmed that the released species is not composed of naked RNA and is als