GENETICS OF YEAST PREMRNA SPLICING

描述：将采取遗传和生物化学相结合的方法， 研究了两种酵母剪接因子Spp2p和Prp31p的功能。 Spp2p Prp2是5种死亡（H）盒蛋白之一， 作为RNA依赖性ATP酶或RNA解旋酶在不同的步骤起作用， 剪接体组装或剪接。 目前尚不清楚这些死亡（H）中的每一个是如何死亡的 盒蛋白是专门针对剪接复合物，也不是什么 蛋白质靶或RNA配体被它们识别。 spp2p是第一个 一种被鉴定为可能与DEAD（H）盒剪接因子相互作用的蛋白质。 Spp2p的研究旨在帮助理解Prp2p和Spp2p如何在 组装成剪接体并在剪接中起作用。 Prp2p被认为是 在大多数剪接体组装之后，当它可能促进 启动第一个催化反应所必需的剪接体重排 剪接反应。 遗传和生化实验表明， Spp2p与Prp2p相互作用，是Prp2与 剪接体 使用生物化学测定，将测试Spp2p是否 直接与Prp2p相互作用，如果是这样，这两种蛋白质的哪些区域 是这种互动的必要条件。 为了确定 对于Spp2p和Prp2发挥功能的剪接体，将使用遗传筛选来 寻找与Spp2p相互作用的蛋白质或RNA分子。 UV交联试验也将用于确定Spp2p是否特异性地 与Prp2p的疑似配体前mRNA或U2 snRNA结合。 Prp31p是一种U4/U6.U5三-snRNP蛋白，是三-snRNP组装所必需的 与剪接复合体结合。 目前还不清楚U4/U6和U5 snRNP如何组装。 U4/U6.U5 tri-snRNP，或tri-snRNP如何与前剪接体结合 形成剪接体。 Prp31与U4/U6.U5 tri-snRNP相关， 与preplicosome。 在prp31突变体中，tri-snRNP的水平部分地降低。 减少，从前剪接体形成剪接体完全 被挡出. 这些结果表明，Prp31 p可能是组装 来自U4/U6和U5 snRNP的三-snRNP，并且可以将三-snRNP拴系到U4/U6和U5 snRNP。 剪接复合体 紫外线交联分析和遗传筛选将是 用于研究Prp31p是否与 前剪接体通过与前mRNA或snRNA接触或通过蛋白质-蛋白质 交互. 申请人将检查Prp31p与哪些snRNP结合。 与snRNP分子Prp31p相互作用， 免疫共沉淀和紫外交联试验以及遗传学 卡位 额外的prp31突变对snRNP生物发生和 还将测定剪接体组装。

DESCRIPTION: A combined genetic and biochemical approach will be taken to study the functions of two yeast splicing factors, Spp2p and Prp31p. Spp2p functions together with Prp2, one of 5 DEAD(H) box proteins thought to function as RNA-dependent ATPases or RNA helicases at different steps of spliceosome assembly or splicing. It is not known how each of these DEAD(H) box proteins is specifically targeted to the splicing complex, nor what protein targets or RNA ligands are recognized by them. Spp2p is the first protein identified that may interact with a DEAD(H) box splicing factor. Studies of Spp2p are designed to help understand how Prp2p and Spp2p assemble into the spliceosome and function in splicing. Prp2p is thought to function after most of the spliceosome is assembled, when it might promote rearrangements of the spliceosome necessary to initiate the first catalytic reaction of splicing. Genetic and biochemical experiments suggest that Spp2p interacts with Prp2p and is required for association of Prp2 with the spliceosome. Using biochemical assays, it will be tested whether Spp2p directly interacts with Prp2p, and if so, which regions of the two proteins are necessary for this interaction. To identify the niche within the spliceosome where Spp2p and Prp2 function, genetic screens will be used to search for protein or RNA molecules that interact with Spp2p. UV-cross-linking assays also will be used to determine if Spp2p specifically binds to pre-mRNA or U2 snRNA, suspected ligands of Prp2p. Prp31p is a U4/U6.U5 tri-snRNP protein necessary for assembly of tri-snRNPs with the splicing complex. It is not known how U4/U6 and U5 snRNPs assemble into U4/U6.U5 tri-snRNPs, or how tri-snRNPs associate with prespliceosomes to form spliceosomes. Prp31 is associated with the U4/U6.U5 tri-snRNP and with the prespliceosome. In prp31 mutants, levels of tri-snRNPs are partly diminished and formation of spliceosomes from pre-spliceosomes is completely blocked. These results suggest that Prp31p may be necessary for assembly of tri-snRNPs from U4/U6 and U5 snRNPs and may tether the tri-snRNP to the splicing complex. UV-cross-linking assays and genetic screens will be employed to investigate whether Prp31p is associated with the pre-spliceosome via contacts with pre-mRNA or snRNAs or by protein-protein interactions. The applicant will examine with which snRNPs Prp31p is associated and with which snRNP molecules Prp31p interacts by co-immunoprecipitation and UV-cross-linking assays as well as genetic screens. Effects of additional prp31 mutations on snRNP biogenesis and spliceosome assembly will also be assayed.