Although snRNPs containing the abundant snRNAs U1, U2, U4, U5, and U6 are required for removing introns from pre-mRNA, little is known about the mechanism of action of snRNPs. While we know the primary sequence of these snRNAs from divergent organisms, the higher order structures of the snRNAs, and their relationship to snRNP function during splicing remain mysterious. RNA processing is an obligate step in the pathway of gene expression for many important genes. An understanding of the mechanisms of RNA processing and how RNA processing might be regulated will be essential for a complete understanding of gene expression. The strategy proposed here for analyzing the mechanism of action of snRNPs focuses initially on the yeast U2 snRNP. Using the well established procedures for site-directed oligonucleotide mutagenesis and molecular-genetic analysis in yeast, the RNA sequences essential for U2 snRNA function during splicing in vivo will be determined. Using chemical modifying agents and single-and double-strand specific nucleases, the secondary structure of yeast U2 and the other yeast snRNAs involved in splicing will be determined and compared to available secondary structure models based on phylogenetic comparisons. By the same procedures, the secondary structures of mutant U2 RNAs will also be determined. High resolution structure probing using nuclear magnetic resonance and X-ray crystallographic techniques will be attempted using wild type and mutant U2 RNA model transcripts synthesized in vitro from T7 promoter constructs, in collaboration with Dr. Peter Moore's group at Yale university. From these data, a consistent set of structure function relationships will be extracted, and used to develop models for mechanisms of U2 snRNP action. Assays will be developed designed to identify proteins and other snRNAs that interact with U2 snRNA. The nature of the interactions will be defined by determining their effect on U2 structure. By using these assays, the proteins will be purified and their role in modulating the structure and function of U2 snRNA splicing will be determined.
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