Regulation of Splice Site Choice in C. elegans

描述(由申请人提供)：在过去的五年里，我们的实验室开发了许多方法来研究前mRNA剪接的调控，使用线虫作为模型系统。选择性剪接是控制基因表达和在蛋白质组中创造多样性的关键机制。使用线虫作为研究这一过程的模型系统有几个明显的优势，因为这种生物保持了与高等真核生物相同类型的选择性剪接事件和调控因子，而可用的基因组学和遗传学工具使其成为研究这一过程调控的一个非常容易处理的系统。我们已经开发出生物信息学技术，用于识别选择性剪接基因和进行基因组的物种间比对。我们已经应用这些方法来确定内含子中的替代剪接调控元件。线虫相对较小的内含子大小，以及线虫和桥连线虫内含子之间较大程度的序列差异，使我们在识别这些顺式剪接调控子方面比脊椎动物模型系统具有优势。我们将利用这些知识来理解这些序列在调节选择性剪接中的功能。我们正在研究剪接调控因子MEC-8在剪接位点选择中的作用，并表明它是一个大型稳定的核糖核蛋白复合体的一部分。了解这种复合体及其在剪接位点选择中的功能将有助于阐明MEC-8剪接调控的机制。我们已经克隆并鉴定了内含子第一个核苷酸的两个突变抑制子。我们还绘制了内含子3‘端隐蔽剪接位点选择的调控因子。我们建议继续和扩大这些研究，以进一步了解剪接位点选择的机制。这项拨款建议的具体目标是： 1-通过生物信息学和体内研究相结合的方法确定内含子前mRNA剪接调控元件。这些将确定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-确定前信使核糖核酸剪接因子MEC-8在调控前信使核糖核酸剪接中的作用机制。 3-鉴定5‘和3’剪接位缺陷的抑制子，以阐明剪接位选择的机制。

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.