RNA DYNAMICS IN THE SPLICEOSOME

描述（改编自申请人摘要）：研究者将 定义了驱动RNA重排的分子相互作用， 激活剪接体以催化前体mRNA剪接。的 剪接体是负责将内含子从 前信使RNA，为它们的翻译做准备。它包括五 小的核RNA，被认为催化剪接反应， 超过60种蛋白质粗略地说，这五种RNA可以被认为是 具有以下功能：一个是组装因子（Ui），一个是负的 调节催化活性（U4），一个是特异性因子（U 5），两个是 构成活性位点（U2和U6）。这项研究将集中在积极的 特别是U6。在许多剪接蛋白中，Prp 8是最大的 (280 kDa），最保守的（酵母和人类之间> 60%相同） 并且与活性位点连接最紧密。他们发现Prp 8 控制U4和U6 RNA之间分子间螺旋的解旋， 剪接体的催化活化的必要步骤。分子 这种调节机制将在目标1中研究。U6 RNA 广泛的变化，在其纳入二级结构， 剪接体，并在随后的剪接体激活过程中， 催化作用RNA能够经历如此大的结构变化， 不寻常的，我们相信它需要专门的RNA元件，以及 蛋白质的辅助（即，RNA分子伴侣）。目标2将侧重于 剪接因子Prp 24（U6 RNA分子伴侣的主要候选者）在 在剪接体激活期间U4/U6复合物的解旋。在U4 RNA之后， 取代，U6碱基对与U2 RNA形成的催化核心， 剪接体很少有人知道这种二级结构是如何形成的， 它折叠成催化活性的三级结构。在目标3中，他们提出 鉴定U6和U2 RNA中的新突变， 对剪接循环中这一重要步骤的生物化学分析。

DESCRIPTION (Adapted from the applicant's abstract): The investigator will define the molecular interactions that drive RNA rearrangements during activation of the spliceosome for the catalysis of pre-mRNA splicing. The spliceosome is the macromolecular machine responsible for removing introns from pre-messenger RNAs, in preparation for their translation. It consists of five small nuclear RNAs, which are thought to catalyze the splicing reaction, and more than 60 proteins. Roughly speaking, the five RNAs can be thought of as having the following functions: one is an assembly factor (Ui), one negatively regulates catalytic activity (U4), one is a specificity factor (U5), and two constitute the active site (U2 and U6). This study will focus on the active site RNAs, particularly U6. Of the many splicing proteins, Prp8 is the largest (280 kDa), the most conserved (>60 percent identical between yeast and humans) and is most closely linked to the active site. They have discovered that Prp8 controls the unwinding of intermolecular helices between U4 and U6 RNAs, a necessary step in catalytic activation of the spliceosome. The molecular mechanism of this regulation will be studied in Aim 1. U6 RNA undergoes extensive changes in secondary structure during its incorporation into the spliceosome, and during the subsequent activation of the spliceosome for catalysis. The ability of an RNA to undergo such large changes in structure is unusual, and we believe it requires specialized RNA elements as well as assistance from proteins (i.e., RNA chaperones). Aim 2 will focus on the function of splicing factor Prp24, a prime candidate for a U6 RNA chaperone, in unwinding of the U4/U6 complex during spliceosome activation. After U4 RNA is displaced, U6 base-pairs with U2 RNA to form the catalytic core of the spliceosome. Little is known about how this secondary structure forms, or how it folds into a catalytically active tertiary structure. In Aim 3, they propose to identify new mutations in U6 and U2 RNAs that will enable genetic and biochemical analyses of this essential step in the splicing cycle.