Spliceosome activation by EF-G like GTPase Snu114

描述（由申请人提供）： 剪接体是由5个核小RNA（snRNA）和~80种蛋白质组成的大型核糖核蛋白机器。剪接体的组装需要由RNA依赖性ATP酶的DEAD盒家族成员推动的一系列复杂的重排。剪接体还含有一个重要的GTdR，Snu 114，其功能知之甚少。完全组装的剪接体的激活需要通过U 5 snRNP相关的ATP酶Prp 28和Brr 2置换U1和U4 snRNP。一个重要的未回答的问题是如何调节这些ATP酶的活性。基于最近的遗传分析，古特里实验室提出Snu 114控制着这种调节，而且Snu 114的活性本身也受到泛素化的调节。具体来说，他们的数据表明，GTP水解Snu 114产生的剪接体通过改变Snu 114的C-末端结构域和大的U 5 snRNP蛋白Prp 8之间的相互作用的大构象重排。由于Prp 8先前已被提出负调节Prp 28和Brr 2的活性，这种GTP依赖性重排可能允许这些ATP酶的激活。该提案将测试Snu 114依赖性GTP水解激活剪接体进行催化以及泛素化对Snu 114的功能很重要的假设。

DESCRIPTION (provided by applicant): The spliceosome is a large ribonucleoprotein machine comprised of 5 small nuclear RNAs (snRNAs) and ~80 proteins. Assembly of the spliceosome requires a complex series of rearrangements fueled by members of the DEAD-box family of RNA-dependent ATPases. The spliceosome also contains a single essential GTPase, Snu114, whose function is poorly understood. Activation of the fully assembled spliceosome for catalysis requires the displacement of U1 and U4 snRNPs by the U5 snRNP-associated ATPases Prp28 and Brr2. An important unanswered question is how the activity of these ATPases is regulated. Based on recent genetic analyses, the Guthrie lab has proposed that Snu114 controls this regulation and, moreover, that the activity of Snu114 is itself regulated by ubiquitination. Specifically, their data suggest that GTP hydrolysis by Snu114 produces a large conformational rearrangement of the spliceosome via changed interactions between the C-terminal domain of Snu114 and the large U5 snRNP protein Prp8. Since Prp8 has previously been proposed to negatively regulate the activity of Prp28 and Brr2, this GTP-dependent rearrangement could likely allow activation of these ATPases. This proposal will test the hypotheses that Snu114-dependent GTP hydrolysis activates the spliceosome for catalysis and that ubiquitination is important for the function of Snu114.