Biochemical Characterization of p68 RNA Helicase

描述（由申请人提供）：从信使RNA前体（pre-mRNA）中去除内含子是真核基因表达的重要过程。为了去除内含子，必须通过有序的多步途径在前mRNA上组装一个称为剪接体的大型RNA-蛋白质复合物。尽管大量的研究已经对前体mRNA是如何拼接的有了一个大致的了解，但仍然存在许多重要的知识空白。在许多有趣的未回答的问题中，所谓的DExH/D盒解旋酶蛋白在剪接体中的作用特别有趣。许多假定的RNA解旋酶已被证明在剪接体中起作用。然而，我们的知识，连接一个假定的解旋酶一个特定的目标RNA双链体目前是有限的。该建议的目的是了解p68 RNA解旋酶的生物学功能，该酶最近被检测到在剪接体组装期间与瞬时U1：5 'ss双链体相互作用（Liu，1998）。我们的初步数据表明，p68 RNA解旋酶是必不可少的在体外前mRNA剪接。该蛋白可能在剪接体组装过程中参与介导U1 snRNA和前mRNA的相互作用。P68也可能在U 5、U4/U6三-snRNP加入前剪接体的过程中起作用。本研究将探讨p68 RNA解旋酶在前体mRNA剪接过程中的功能作用。本研究将集中在两个重要问题上：（1）p68 RNA解旋酶是否解旋U 1：5 ′ ss双链体; (2)p68是否在添加三-snRNP中起作用。我们预计，了解p68的功能作用将构成一个显着的进步知识的剪接体组装的机制。在表征p68解旋酶的酶活性的过程中，我们首先证明了dsRNA结合和dsRNA刺激的ATP酶活性。我们提出实验来阐明RNA结合机制，并研究RNA结合如何与p68的酶活性相结合。

DESCRIPTION (provided by applicant): Remove of introns from messenger RNA precursors (pre-mRNA) is an essential process in eukaryotic gene expression. To remove introns, a large RNA-protein complex, known as the spliceosome, must be assembled on pre-mRNA through an ordered multi-step pathway. Although an outline picture of how pre-mRNA is spliced has emerged from the numerous studies, many important knowledge gaps remain. Among the many interesting unanswered questions, the roles of so-called DExH/D box helicase proteins in the spliceosome are particularly intriguing. A number of putative RNA helicases have been shown to function in the spliceosome. However, our knowledge that links a putative helicase to a specific target RNA duplex is currently limited. The goal of this proposal is to understand the biological functions of the p68 RNA helicase that was recently detected interacting with the transient U1:5'ss duplex during the spliceosome assembly (Liu, 1998). Our preliminary data suggest that the p68 RNA helicase is essential for in vitro pre-mRNA splicing. The protein may be functionally involved in mediating the U1 snRNA and pre-mRNA interactions during the spliceosome assembly. P68 may also plays a role in the process of addition of U5.U4/U6 tri-snRNP to the pre-spliceosome. This proposal will investigate the functional roles of p68 RNA helicase in the pre-mRNA splicing process. The studies will focus on two important issues: (1) Does p68 RNA helicase unwind the U1:5'ss duplex. (2) Does p68 plays a role in the addition of the tri-snRNP. We anticipate that understanding the functional roles of p68 would constitute a significant advance in knowledge about the mechanism of the spliceosome assembly. In the process of characterization of enzymatic activity of p68 helicase, we first demonstrated dsRNA-binding and dsRNA stimulated ATPase activity. We propose experiments to elucidate the RNA-binding mechanism and investigate how the RNA-binding is coupled to the enzymatic activities of p68.