U2AF AND SPLICE SITE RECOGNITION IN C ELEGANS

本申请解决与线虫线虫3‘剪接位点识别相关的问题。线虫的剪接需要识别非常小的内含子，这些内含子缺乏多嘧啶序列和分支点共有序列。3‘剪接位点共识，(U)4CAG/R，到目前为止在线虫中是唯一发现的，顺式和反式剪接都需要。UAF-1和UAF-2基因编码基本剪接因子U2AF的大小亚基，负责识别这个3‘剪接位点的共识。我们的目的是利用体外结合试验，确定(U)4CAG/R共识与U2AF之间相互作用的性质，以及U2AF的两个亚单位的哪些结构域是活性、体外结合和活性所必需的。通过补充缺乏U2AF的哺乳动物提取物，将在体外研究U2AF的活性。为了确定与U2AF相互作用的剪接成分，将选择在没有蛋白质剩余部分的情况下抑制U2AF部分表达的显性负表型的突变体。编码U2AF65的Gen，UAF-1被选择性地剪接，以包括一个额外的外显子，该外显子包含一个10个拷贝的(U)CAG/R共识的阵列。UAF-1、Pre-mRNA的选择性剪接可能是一种严格调控E2AF水平的方法，以确保准确的内含子识别。这一想法将通过比较U2AF过度表达时功能mRNA和包含外显子的RNA的水平来检验。将选择改变两个UAF-1 RNA相对水平的突变体来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该外显子的RNA的核保留。将确定USAF与(U)4CAG/R元素的结合如何导致核滞留。为了确定阻止含有这些序列的RNA出口所需的机制的其他组件，将在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.