Site-Specific Chemical Modification and Crosslinking Studies of U6 snRNA in the Yeast spliceosome

Citation

Kim, Chang Hee (1999) Site-Specific Chemical Modification and Crosslinking Studies of U6 snRNA in the Yeast spliceosome. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/cfw0-z473. https://resolver.caltech.edu/CaltechTHESIS:01092024-222740445

Abstract

Pre-mRNA splicing is an essential step in eukaryotic gene expression. Most eukaryotic genes are interrupted by sequences, called introns, that do not code for sequences in the final protein gene product. Pre-mRNA splicing is the process by which introns are removed and the coding sequences, called exons, are joined together following transcription of a gene into RNA. The spliced mRNA subsequently acts as the coding template for protein synthesis. Pre-mRNA splicing occurs in a large ribonucleoprotein particle called the spliceosome which includes five essential small nuclear RNAs (snRNAs) and more than 50 different proteins. It is thought that the active site of the spliceosome consists of U2, U6 and U5 snRNAs. In order to probe the structure of the catalytic site, we have done an extensive site-specific chemical modification and crosslinking studies of U6 RNA in the yeast spliceosome. From an extensive screen of site-specific 2'-deoxy modifications, we have found four that block the first step of splicing, yet are able to assemble spliceosome complexes which precede the reactive spliceosome, suggesting that the 2'-hydroxyls at these nucleotides may be required for catalysis. In the crosslinking experiments, we found crosslinks between 4-thioU placed in the central conserved sequences in U6 with the 5' splice site of the pre-mRNA, which participates in the first chemical step of splicing. We were able to determine the order of this crosslink by doing the experiment in yeast mutant spliceosomes which were blocked in well-defined stages of splicing. We also found that a conserved nucleotide in the 3' stem-loop of U6 crosslinks to a nucleotide in a region of the actin pre-mRNA intron which is required for its splicing in vitro. We also found crosslinks to snRNAs -- U2 and U4. Certain positions in U6 can crosslink first to U4 snRNA, then subsequently to the pre-mRNA, consistent with the notion that the spliceosome is a dynamic machine.

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