U2AF AND SPLICE SITE RECOGNITION IN C ELEGANS

该应用程序解决了线虫3'剪接位点识别的相关问题。秀丽隐杆线虫的剪接需要识别非常小的内含子，这些内含子缺乏聚嘧啶束和分枝点一致序列。3'剪接位点共识，(U)4CAG/R，迄今为止只在秀丽隐杆线虫中发现，对顺式和反式剪接都是必需的。uaf-1和uaf-2基因编码基本剪接因子U2AF的大小亚基，负责识别这种3'剪接位点共识。我们的目的是利用体外结合试验确定(U)4CAG/R共识和U2AF之间相互作用的性质，以及U2AF的两个亚基的哪些结构域是生存能力、体外结合和活性所必需的。U2AF的活性将通过补充U2AF耗尽的哺乳动物提取物在体外进行研究。为了鉴定与U2AF相互作用的剪接成分，将选择在缺乏其余蛋白的情况下抑制U2AF部分显性负表型的突变体。编码U2AF65的基因uaf-1被选择性地拼接到包括一个额外的外显子，该外显子包含10个(U)CAG/R共识的拷贝。uaf-1 pre-mRNA的选择性剪接可能是严格调节E2AF水平以确保准确识别内含子的一种方法。这一想法将通过比较U2AF过表达时功能性mRNA和外显子RNA的水平来验证。将选择改变两种uaf-1 rna相对水平的突变体来确定这种可选剪接事件是如何调节的。这个外显子的存在导致含有它的RNA的核保留。将确定USAF与(U)4CAG/R元素的结合如何导致核保留。为了确定防止含有这些序列的RNA输出所需的机制的其他组成部分，将在秀丽隐杆线虫中选择抑制核保留表型的突变体。

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.