The spliceosome is an intricate biological nanomachine that is unique to the eukaryotic lineage of life. It performs the essential process of removing introns from the primary transcripts of eukaryotic genes, thus creating functional messenger RNAs for protein production. Failure of the spliceosome to accurately and efficiently remove introns, either due to mutations in individual introns or in spliceosome components, results in a range of human diseases including autosomal dominant retinitis pigmentosa (adRP), which causes blindness. We propose to continue our ongoing investigation into the molecular mechanisms of wild-type and mutant spliceosome function. Five small nuclear RNAs, U1, U2, U4, U5 and U6, are central to the splicing process. Each RNA associates with proteins to form a snRNP, and the five snRNPs assemble on an intron to reconstitute a spliceosome. After assembly, a complex activation process ensues that results in expulsion of the U1 and U4 snRNPs, and alignment of the intron splice sites on a U2/U6/U5 scaffold. It is thought that the U2/U6 complex then carries out the two catalytic steps of splicing in conjunction with key protein factors. Our studies will follow the progression of U6 RNA through the splicing cycle. We will determine the mechanism of assembly of the U4/U6 di-snRNP, which is thought to keep U6 in an inactive state during the spliceosome assembly process. In particular, we will test our hypothesis that U2 RNA is actively involved in this process, as well as the proteins Prp24 and Slt11. During the subsequent transition from U4/U6 complex to U2/U6 complex, U6 RNA changes structure to form a remarkably conserved internal stem-loop (ISL), which is thought to be required for catalysis of splicing. Engineered mutations that further stabilize the ISL result i a cold-sensitive block to splicing, providing a genetic and biochemical tool for studying the interconversion of U4/U6 and U2/U6. We will exploit one such mutation to probe the function of the essential U6 RNA-binding protein Cwc2 in spliceosome activation. We will determine the structures of key sub-complexes identified in our studies. The results of these studies will be a deeper understanding of an ancient molecular machine essential to all eukaryotic cells, and possible diagnostic and therapeutic interventions for devastating human diseases.
The spliceosome is an intricate cellular machine composed of 5 RNAs and more than 75 proteins. The spliceosome is so vital to the cell that even a subtle defect in just one of its components results in the disease retinitis pigmentosa, which causes blindness. Our work will result in a better understanding of the mechanism by which this remarkable molecular machine processes messenger RNA so that proteins can be made correctly by cells.
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