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.

该应用程序解决了与线虫秀丽隐杆线虫中与3'剪接站点识别有关的问题。秀丽隐杆线虫中的剪接需要识别缺乏多吡啶胺段和分支点共识序列的非常小的内含子。到目前为止，在秀丽隐杆线虫中唯一发现的3'剪接位点共识是顺式和变速器所必需的。 UAF-1和UAF-2的基因编码了必需剪接因子U2AF的大小亚基，这些剪接因子负责识别此3'剪接位点共识。我们的目的是确定使用体外结合测定，即（U）4CAG/R共识和U2AF之间相互作用的性质，以及U2AF的两个亚基的哪个结构域才能生存能，体外结合和活性。通过互补的U2AF缺乏的哺乳动物提取物，将在体外研究U2AF活性。为了识别与U2AF相互作用的剪接组件，将选择在没有蛋白质的其余部分的情况下抑制U2AF部分的主要负表型的突变体。编码U2AF65，UAF-1的GEN被剪接，包括一个额外的外显子，该外显子包含（u）CAG/r共识的十个副本。 UAF-1的替代剪接可能是一种紧密调节E2AF水平以确保准确内含子识别的方法。当U2AF过度表达时，将通过比较功能mRNA和含有rNA的功能mRNA的水平来测试这个想法。将选择改变两个UAF-1 RNA的相对水平的突变体，以确定如何调节此替代剪接事件。该外显子的存在导致含有RNA的核保留。将确定USAF与（U）4CAG/R元件的结合如何导致核保留率。为了确定防止含有这些序列的RNA所需的机械组件，将在秀丽隐杆线虫中选择抑制核保留表型的突变体。