Spliceosome activation by EF-G like GTPase Snu114

描述(由申请人提供)： 剪接体是一个由5个小核RNA和~80个蛋白质组成的大型核糖核蛋白机器。剪接体的组装需要一系列复杂的重排，由依赖RNA的ATPase的死盒家族成员推动。剪接体还含有单一的必需GTP酶，Snu114，其功能尚不清楚。为了激活完全组装的剪接体用于催化，需要U5 SnRNP相关的ATPase Prp28和Brr2取代U1和U4 SnRNPs。一个重要的悬而未决的问题是这些ATPase的活性是如何被调控的。根据最近的基因分析，格思里实验室提出，Snu114控制着这一调控，而且Snu114的活动本身受到泛素化的调节。具体地说，他们的数据表明，Snu114的GTP水解通过改变Snu114的C-末端结构域和大的U5 SnRNP蛋白Prp8之间的相互作用，产生了剪接体的大规模构象重排。由于Prp8以前被认为是负向调节Prp28和Brr2的活性，这种依赖GTP的重排可能允许这些ATPase的激活。这一提议将检验以下假设：Snu114依赖的GTP水解会激活剪接体进行催化，泛素化对Snu114的功能很重要。

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.