Significant insight has emerged from the analyses of phylogenetics, compensatory base-pair mutations, and photochemical crosslinking that indicate a specific catalytic role for U6 small nuclear RNA (snRNA) in pre-messenger RNA splicing. This notion has been strengthened by our recent discovery that the 3' intramolecular stem-loop of U6 snRNA binds a magnesium ion that is critical for the chemistry of splicing (Yean et al., Nature 408:881). It is unclear, however, whether the magnesium that we have identified is at the catalytic site and how the 3' stem-loop of U6 interacts with the rest of the spliceosome. The objectives of this pre-doctoral grant application arc two-fold: (1) test a metal-ligand interaction that would involve juxtaposing the 3' intramolecular stem loop (ISL) of U6 snRNA with the 5' splice site (SS) and (2) to investigate what protein factors stabilize, assist, or mediate RNA folding that leads to catalysis within the context of the assembled spliceosome. These interactions, once defined, would help our understanding of how the different sites of chemistry become tightly linked during spliceosome catalysis.
| Dery, Kenneth J; Yean, Shyue-Lee; Lin, Ren-Jang (2008) Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA. Methods Mol Biol 488:41-63 |
