Biochemical Characterization of p68 RNA Helicase

描述(申请人提供)：从信使RNA前体中去除内含子(前-mRNA)是真核基因表达中的一个必不可少的过程。为了去除内含子，一个大的RNA-蛋白质复合体，称为剪接体，必须通过有序的多步骤途径组装在前-mRNA上。尽管大量研究已经勾勒出了Pre-mRNA是如何剪接的轮廓，但许多重要的知识空白仍然存在。在许多有趣的悬而未决的问题中，所谓的DExH/D盒解旋酶蛋白在剪接体中的作用尤其耐人寻味。许多假定的RNA解旋酶已被证明在剪接体中起作用。然而，我们目前对将假定的解旋酶与特定的靶RNA双链联系在一起的知识是有限的。这项建议的目的是了解最近被检测到的p68RNA解旋酶在剪接体组装过程中与瞬时U1：5‘s双链相互作用的生物学功能(Liu，1998)。我们的初步数据表明，p68RNA解旋酶对于体外Pre-mRNA剪接是必不可少的。该蛋白可能在功能上参与了剪接体组装过程中U1SnRNA和Pre-mRNA的相互作用。P68也可能在将U5.U4/U6 tri-SnRNP添加到预剪接体的过程中发挥作用。这项建议将研究p68 RNA解旋酶在前mRNA剪接过程中的功能作用。这些研究将集中在两个重要的问题上：(1)p68 RNA解旋酶是否解开U1：5‘s双链。(2)p68在Tri-SnRNP的增加中起作用。我们预计，了解p68的功能作用将构成对剪接体组装机制的认识的重大进步。在p68解旋酶的活性鉴定过程中，我们首先证明了dsRNA结合和dsRNA刺激的ATPase活性。我们建议通过实验来阐明RNA结合的机制，并研究RNA结合是如何与p68的酶活性偶联的。

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.