RNA DYNAMICS IN THE SPLICEOSOME

描述(改编自申请人的摘要)：调查员将 定义驱动RNA重排的分子相互作用 剪接体的激活对前信使核糖核酸剪接的催化。这个 剪接体是负责去除内含子的大分子机器 前信使RNA，为它们的翻译做准备。它由五个部分组成 被认为催化剪接反应的小核RNA，以及 超过60种蛋白质。粗略地说，这五个RNA可以被认为是 具有以下功能：一个是装配因子(Ui)，一个是负值 调节催化活性(U4)，一个是特异性因子(U5)，两个 构成活性部位(U2和U6)。这项研究将重点放在积极的 站点RNA，特别是U6。在众多剪接蛋白中，Prp8是最大的 (280 KDa)，最保守的(&gt；60%在酵母和人类之间相同) 并且与活动站点链接最紧密。他们发现Prp8 控制U4和U6 RNA之间分子间螺旋的解离，a 剪接体催化激活的必要步骤。分子 这种调节的机制将在目标1中进行研究。U6 RNA经历了 在其并入的过程中，二级结构的广泛变化 剪接体，并在随后的剪接体激活过程中 催化作用。RNA经历如此大的结构变化的能力是 不同寻常，我们认为它需要专门的RNA元素以及 来自蛋白质的协助(即，RNA伴侣)。目标2将专注于 剪接因子Prp24的作用，Prp24是U6 RNA伴侣的主要候选者，在 剪接体激活过程中U4/U6复合体的解离。在U4 RNA被 取代的U6碱基对与U2 RNA形成催化核心 剪接体。关于这种二级结构是如何形成的，或者是如何形成的，我们知之甚少 它折叠成具有催化活性的三级结构。在目标3中，他们提出 识别U6和U2 RNA中的新突变，使基因和 对剪接循环中这一关键步骤的生化分析。

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.