U2AF AND SPLICE SITE RECOGNITION IN C ELEGANS

线虫中的 U2AF 和剪接位点识别

• 批准号: 6363304
• 负责人: Thomas Blumenthal
• 金额: $ 19.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363304
• 关键词: Caenorhabditis elegans; RNA binding protein; RNA splicing; gene expression; gene mutation; life cycle; messenger RNA; nucleic acid sequence; protein binding

This application addresses issues related to 3' splice site recognition in the nematode C. elegans. Splicing in C. elegans requires recognition of very small introns which lack polypyrimidine tract and branch-point consensus sequences. The 3' splice site consensus, (U)4CAG/R, so far found uniquely in C. elegans, is required for both cis- and trans-splicing. The genes, uaf-1 and uaf-2, encode large and small subunits of the essential splicing factor U2AF, which are responsible for recognition of this 3' splice site consensus. Our aims are to determine using in vitro binding assays, the nature of the interaction between the (U)4CAG/R consensus and U2AF, and which domains of both subunits of U2AF are required for viability, in vitro binding, and activity. U2AF activity will be studied in vitro by complementation of a U2AF-depleted mammalian extract. In order to identify splicing components that interact with U2AF, mutants that suppress the dominant negative phenotype of part of U2AF expressed in the absence of the remainder of the protein will be selected. The gen encoding U2AF65, uaf-1, is alternatively spliced to include an extra exon which contains an array of ten copies of the (U)CAG/R consensus. The alternative splicing of uaf-1, pre-mRNA may be a way to tightly regulate levels of E2AF to insure accurate intron recognition. This idea will be tested by comparing levels of functional mRNA and extra-exon- containing RNA when U2AF is over-expressed. Mutants that change the relative levels of the two uaf-1 RNAs will be selected to determine how this alternative splicing event is regulated. The presence of this exon results in nuclear retention of RNA containing it. It will be determined how binding of USAF to the (U)4CAG/R elements causes nuclear retention. In order to identify additional components of the machinery needed to prevent export of RNA containing these sequences, mutants that suppress the nuclear retention phenotype will be selected in C. elegans.

该应用解决了与线虫秀丽隐杆线虫中 3' 剪接位点识别相关的问题。秀丽隐杆线虫中的剪接需要识别非常小的内含子，这些内含子缺乏多嘧啶束和分支点共有序列。迄今为止，仅在秀丽隐杆线虫中发现的 3' 剪接位点共有 (U)4CAG/R 是顺式和反式剪接所必需的。基因 uaf-1 和 uaf-2 编码必需剪接因子 U2AF 的大小亚基，负责识别该 3' 剪接位点共有序列。我们的目标是使用体外结合测定法确定 (U)4CAG/R 共有序列和 U2AF 之间相互作用的性质，以及 U2AF 两个亚基的哪些结构域是活力、体外结合和活性所必需的。 U2AF 活性将通过补充 U2AF 耗尽的哺乳动物提取物进行体外研究。为了鉴定与U2AF相互作用的剪接组分，将选择抑制在蛋白质剩余部分不存在的情况下表达的U2AF部分的显性失活表型的突变体。编码 U2AF65 的基因 uaf-1 被选择性剪接以包含一个额外的外显子，该外显子包含 (U)CAG/R 共有序列的十个拷贝的阵列。 uaf-1、pre-mRNA 的选择性剪接可能是严格调节 E2AF 水平以确保准确的内含子识别的一种方法。当 U2AF 过度表达时，将通过比较功能性 mRNA 和含有外显子的 RNA 的水平来检验这一想法。将选择改变两个 uaf-1 RNA 相对水平的突变体来确定如何调节这种选择性剪接事件。该外显子的存在导致含有它的 RNA 被保留在核中。将确定 USAF 与 (U)4CAG/R 元件的结合如何导致核滞留。为了鉴定防止含有这些序列的RNA输出所需的额外组件，将在秀丽隐杆线虫中选择抑制核滞留表型的突变体。