U2AF AND SPLICE SITE RECOGNITION IN C ELEGANS

本申请解决了线虫C中3'剪接位点识别相关的问题。优雅C.剪接秀丽线虫需要识别非常小的内含子，其缺乏多聚嘧啶段和分支点共有序列。（U）4CAG/R是迄今为止在C. elegans，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U2 AF的大小亚基，其负责识别该3'剪接位点共有序列。我们的目的是使用体外结合试验，确定（U）4CAG/R共识和U2 AF之间的相互作用的性质，以及U2 AF的两个亚基的哪些结构域是活力，体外结合和活性所需的。将通过补充U2 AF缺失的哺乳动物提取物在体外研究U2 AF活性。为了鉴定与U2 AF相互作用的剪接组分，将选择抑制在蛋白质的剩余部分不存在的情况下表达的U2 AF的部分的显性阴性表型的突变体。编码U2 AF 65的基因uaf-1被选择性剪接以包括含有（U）CAG/R共有序列的十个拷贝的阵列的额外外显子。uaf-1前体mRNA的选择性剪接可能是一种严格调节E2 AF水平以确保准确识别内含子的方法。当U2 AF过度表达时，将通过比较功能性mRNA和含有外显子外的RNA的水平来测试这一想法。将选择改变两种uaf-1 RNA的相对水平的突变体，以确定这种选择性剪接事件是如何调节的。该外显子的存在导致含有它的RNA的核滞留，这将确定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输出所需的机制的其他组分，将在C.优雅

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.