Regulation of Splice Site Choice in C. elegans

描述（由申请人提供）：在过去的五年中，我们的实验室开发了多种方法来研究使用秀丽隐杆线虫作为模型系统的前体mRNA剪接的调节。选择性剪接是控制基因表达和创造蛋白质组多样性的关键机制。使用线虫作为模型系统来研究这一过程具有几个明显的优势，因为该生物体保持与高等真核生物相同类型的选择性剪接事件和调节因子，而可用的基因组学和遗传学工具使其成为研究该过程调节的非常容易处理的系统。我们开发了生物信息学技术来识别可变剪接基因并进行基因组的种间比对。我们已应用这些方法来识别内含子中的选择性剪接调控元件。线虫的内含子尺寸相对较小，并且线虫和C.briggsae内含子之间的序列差异较大，使我们在识别这些顺式剪接调节因子方面比脊椎动物模型系统具有优势。我们将应用这些知识来理解这些序列在调节选择性剪接中的功能。我们正在表征剪接调节因子 MEC-8 在剪接位点选择中的作用，并表明它是大型稳定核糖核蛋白复合物的一部分。了解该复合物及其在剪接位点选择中的功能将阐明 MEC-8 剪接调节机制。我们克隆并鉴定了两个内含子第一个核苷酸的突变抑制子。我们还在内含子 3' 端绘制了神秘剪接位点选择的调节因子。我们建议继续并扩大这些研究，以进一步了解剪接位点选择的机制。该拨款提案的具体目标是： 1-通过结合生物信息学和体内研究来识别内含子前mRNA剪接调控元件。这些将识别一套全面的内含子选择性剪接控制信号以及与它们相互作用的因素。 2-确定前mRNA剪接因子MEC-8在调节前mRNA剪接中的作用机制。 3-表征5'和3'剪接位点缺陷的抑制子，以阐明剪接位点选择的机制。

DESCRIPTION (provided by applicant): Over the past five years, our laboratory has developed numerous approaches to study the regulation of pre-mRNA splicing using C. elegans as a model system. Alternative splicing is a key mechanism for controlling gene expression and creating diversity in the proteome. Using C. elegans as a model system to study this process has several distinct advantages in that this organism maintains the same types of alternative splicing events and regulators as higher eukaryotes, while the available genomics and genetics tools make this a very tractable system for studying the regulation of this process. We have developed bioinformatics techniques for identifying alternatively spliced genes and doing interspecies alignments of genomes. We have applied these approaches towards identifying alternative splicing regulatory elements in introns. The relatively small intron size of nematodes, and the large degree of sequence divergence between C. elegans and C. briggsae introns, gives us advantages over vertebrate model systems for identifying these cis splicing regulators. We will apply this knowledge to understanding the function of these sequences in regulating alternative splicing. We are characterizing the role of splicing regulatory factor MEC-8 in splice site choice and have shown that it is part of a large stable ribonucleoprotein complex. Understanding this complex and its function in splice site choice will illuminate the mechanism of MEC-8 splicing regulation. We have cloned and characterized two suppressors of mutations to the first nucleotide of an intron. We have also mapped regulators of cryptic splice site choice at the 3' end of the intron. We propose to continue and expand these studies to further understand the mechanisms of splice site selection. The specific aims of this grant proposal are: 1- To identify intronic pre-mRNA splicing regulatory elements through combined bioinformatics and in vivo studies. These will identify a comprehensive set of intronic alternative splicing control signals and the factors that interact with them. 2- To determine the mechanism of action of the pre-mRNA splicing factor MEC-8 in regulation of pre-mRNA splicing. 3- To characterize suppressors of 5' and 3' splice site defects in order to illuminate the mechanisms of splice site selection.