Probing the 3' Loop of U6 snRNA in Spliceosome Catalysis

描述(由申请人提供)：从系统发育、碱基对补偿突变和光化学交联的分析中得出了重要的见解，这些分析表明U6小核RNA(SnRNA)在信使前RNA剪接中具有特定的催化作用。我们最近的发现加强了这一观点，即U6 SnRNA的3‘分子内茎环与一个对剪接化学至关重要的镁离子结合(Yean等人，自然408：881)。然而，目前尚不清楚我们确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。这项博士前资助申请的目标有两个：(1)测试金属-配体相互作用，涉及将U6 SnRNA的3‘分子内干环(ISL)与5’剪接位点(SS)并列；(2)研究哪些蛋白质因素稳定、辅助或介导RNA折叠，从而在组装的剪接体的背景下导致催化。一旦定义了这些相互作用，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联系在一起的。

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.