Probing the 3' Loop of U6 snRNA in Spliceosome Catalysis

描述（由申请人提供）： 重要的洞察力已经出现了从分析的遗传学，补偿碱基对突变，和光化学交联，表明一个特定的催化作用，U6小核RNA（snRNA）在前信使RNA剪接。我们最近发现U6 snRNA的3'分子内茎-环结合对剪接化学至关重要的镁离子，这一发现加强了这一观点（Yean et al.，Nature 408：881）。然而，目前尚不清楚我们所鉴定的镁是否位于催化位点，以及U6的3'茎环如何与剪接体的其余部分相互作用。这个博士前资助申请的目的是双重的：（1）测试金属-配体相互作用，这将涉及将U6 snRNA的3'分子内茎环（ISL）与5'剪接位点（SS）并置，以及（2）研究什么蛋白质因子稳定，协助或介导RNA折叠，导致组装剪接体中的催化作用。这些相互作用一旦被定义，将有助于我们理解不同的化学位点在剪接体催化过程中是如何紧密相连的。

DESCRIPTION (provided by applicant): 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.