Although snRNPs containing the abundant snRNAs are required for removing introns from pre-mRNA, little is known about the mechanism of action of snRNPs. While the primary sequences of snRNAs have been determined, and secondary structure models have been developed, the higher order structures of snRNAs and their relationship to snRNP function remain mysterious. We propose to analyze the mechanism of action of snRNPs focusing initially on the U2 snRNP. Based on comparison of U2 sequences from different organisms, and initial chemical modification experiments, we suspect that U2 snRNA is folded into an intriguing RNA structure called a pseudoknot. Pseudoknots are found in the reaction center os group I self-splicing introns and 16S rRNA. Our hypothesis is that this structure plays an important role at the reaction center of the spliceosome. We will use oligonucleotide directed mutagenesis to alter a clones copy of the yeast U2 gene, and introduce the mutant U2 gene into yeast cells lacking any other US gene. US mutations that allow normal splicing and growth will be considered not to affect U2 function, and those mutations that do not allow growth or allow only abnormal or conditional growth with defects in splicing will be considered to affect U2 function in vivo. We will use chemical structure probes to determine the effect of U2 mutations on U2 structure. We will also pursue approaches designed to identify proteins that interact with U2. A search for extragenic suppressors of a cold sensitive U2 mutation may identify genes encoding such proteins.
