Role of SR Proteins in Trans-splicing

描述（由申请人提供）：本提案的长期目标是确定SR蛋白在反式剪接中的作用。这将在来自寄生线虫蛔虫的实验系统中完成。我们的模型系统提供了独特的能力，以测定在体外相同的提取物中的反式和顺式剪接。此外，提取物和剪接因子都可以从发育阶段的胚胎制备。拟议的实验建立在我们已经取得的进展，在建立该系统的效用，在检查反式和顺式剪接之间的相互作用，以及它的适用性，在解决许多问题有关的SR蛋白和选择性剪接的发展调节。我们还确定了反式剪接反应可以分为至少两个不同的阶段，使我们能够独立地测定SR蛋白在每个步骤中的参与。提出了三个具体目标：1。检查反式和顺式剪接的综合发育调控。2.确定在将U2 snRNP加入底物之前和之后SR蛋白参与反式剪接的要求。3.研究外显子定义的作用和U1 snRNP在反式剪接中的参与。对反式剪接反应的透彻理解可能会导致识别潜在的治疗干预点。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to determine the roles of SR proteins in trans-splicing. This will be accomplished in an experimental system derived from the parasitic nematode, Ascaris lumbricoides. Our model system offers the unique ability to assay both trans- and cis-splicing in the same extracts in vitro. Additionally, both extracts and splicing factors can be prepared from developmentally staged embryos. The proposed experiments build upon the progress that we have made in establishing the utility of this system in examining the interplay between trans- and cis-splicing as well as its applicability in addressing numerous questions related to the developmental regulation of both SR proteins and alternative splicing. We have also determined that the trans-splicing reaction can be separated into at least two distinct stages allowing us to assay the participation of SR proteins in each step independently. Three specific aims are proposed: 1. Examine the integrated developmental regulation of trans- and cis-splicing. 2. Determine the requirements for SR protein participation in trans-splicing before and after U2 snRNP addition to the substrate. 3. Investigate the role of exon definition and the involvement of U1 snRNP in trans-splicing. A thorough understanding of the trans-splicing reaction may lead to the identification of potential points of therapeutic intervention.