Regulation of Splice Site Choice in C. elegans

描述（申请人提供）：在过去的五年中，我们的实验室已经开发了许多方法来研究前mrna剪接的调控，以秀丽隐杆线虫为模型系统。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。使用秀丽隐杆线虫作为模型系统来研究这一过程有几个明显的优势，因为这种生物保持着与高等真核生物相同类型的可选剪接事件和调节因子，而现有的基因组学和遗传学工具使其成为研究这一过程调控的非常容易的系统。我们已经开发了生物信息学技术，用于鉴定选择性剪接基因和进行种间基因组比对。我们已经将这些方法应用于识别内含子中的可变剪接调控元件。线虫相对较小的内含子大小，以及秀丽隐杆线虫和秀丽隐杆线虫内含子之间较大程度的序列差异，使我们在鉴定这些顺式剪接调节因子方面优于脊椎动物模型系统。我们将运用这些知识来理解这些序列在调节选择性剪接中的功能。我们正在描述剪接调节因子MEC-8在剪接位点选择中的作用，并表明它是一个大型稳定核糖核蛋白复合物的一部分。了解该复合体及其在剪接位点选择中的作用将有助于阐明MEC-8剪接调控的机制。我们克隆并鉴定了内含子第一个核苷酸突变的两个抑制子。我们还绘制了在内含子3'端选择隐剪接位点的调控因子。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：

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.