Functional Spliceosome and Extrinsic Splicing Factors

描述（由申请人提供）：真核生物中的大多数信使RNA序列都在片段中，需要正确剪接才能翻译成功能蛋白。前mrna剪接缺陷与包括神经功能障碍和癌症在内的许多人类疾病有关。这项资助的广泛、长期目标是了解大型核糖核蛋白复合物（称为剪接体）的结构和功能，剪接体执行mrna前剪接反应。剪接体领域自近20年前提出以来已经取得了巨大的进展；然而，剪接体的核心中存在什么因子以及核心如何被激活或重塑以进行催化尚不清楚。本课题将研究U6小核RNA在剪接体催化中的作用以及剪接体核心外源剪接蛋白因子的功能。具体目的1是研究酵母U6 snRNA的3'分子内茎环（ISL），它与第一次酯交换反应所需的镁离子结合。我们将通过双硫取代和亲硫金属离子修复来检测U6/3‘ISL上的镁是否在pre-mRNA的5’剪接位点上。我们将使用交联策略来测试U6/3'ISL是由RNA还是由蛋白质保持在剪接体核心。具体目标2是研究一种名为Prp2的RNA解旋酶如何重塑剪接体的核心。我们将研究Prp2抑制蛋白或分子间U2/U6螺旋RNA如何促进Prp2的剪接体识别。我们还将使用交联和酵母遗传学方法来测试Prp2和剪接体rna之间的相互作用。具体目的3是研究Prp2对剪接体的激活在人体内是否保守。我们将通过在人细胞中表达针对DBP2的小干扰RNA （siRNA）或显性阴性突变体来抑制DBP2。我们将分析DBP2功能在体内或体外受损时的剪接机制。本研究将揭示酵母和人类剪接体核心的动态特征和重塑。

DESCRIPTION (provided by applicant): The majority of messenger RNA sequences in eukaryotes are in fragments that need to be correctly spliced in order to be translated into functional proteins. Defects in pre-mRNA splicing have been linked to many human diseases including neurological dysfunction and cancer. The broad, long-term objectives of this grant are to understand the structure and function of the large ribonucleoprotein complex, called the spliceosome, that carries out the pre-mRNA splicing reaction. The spliceosome field has made tremendous progress since its conception almost 20 years ago; however, it is still not clear what factors exist in the core of the spliceosome and how the core is activated or remodeled for catalysis. This proposal will investigate the role of the U6 small nuclear RNA in spliceosome catalysis as well as the function of extrinsic splicing protein factors at the spliceosome core. Specific aim 1 is to study the 3' intramolecular stem-loop (ISL) of yeast U6 snRNA, which binds a magnesium ion required for the first transesterification reaction. We will test whether the magnesium at the U6/3'ISL is at the 5' splice site of the pre-mRNA by using double sulfur substitutions and rescuing with a thiophilic metal ion. We will use crosslinking strategies to test whether the U6/3'ISL is held in the spliceosome core by RNA or by protein. Specific aim 2 is to study how an RNA helicase called Prp2 remodels the core of the spliceosome. We will investigate how a prp2-suppressor protein or an intermolecular U2/U6 helix RNA contributes to the spliceosome recognition by Prp2. We will also use crosslinking and yeast genetics approaches to test the interaction between Prp2 and the spliceosomal RNAs. The specific aim 3 is to investigate whether the activation of the spliceosome by Prp2 is conserved in human. We will inhibit DBP2 by expressing a small interference RNA (siRNA) or a dominant-negative mutant targeting DBP2 in human cells. We will analyze the splicing machinery when DBP2 function is impaired in vivo or in vitro. The proposed study shall reveal the dynamic features and the remodeling of the spliceosome core in yeast and human.