Regulation of Splice Site Choice in C. elegans

秀丽隐杆线虫剪接位点选择的调控

• 批准号: 7904673
• 负责人: ALAN M ZAHLER
• 金额: $ 15.18万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904673
• 关键词: 3' Splice Site; Alternative Splicing; Applications Grants; Bioinformatics; Biological Models; Caenorhabditis elegans; Complex; Defect; Eukaryota; Event; Gene Expression; Genetic; Genome; Genomics; Introns; Knowledge; Laboratories; Maps; Mutation; Nematoda; Nucleotides; Organism; Process; Proteome; RNA Splicing; Regulation; Regulatory Element; Ribonucleoproteins; Role; Signal Transduction; Site; Spliced Genes; Suppressor Mutations; System; Techniques; in vivo; mRNA Precursor; tool

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.

描述（由申请人提供）：在过去的五年中，我们的实验室开发了许多方法，以使用秀丽隐杆线虫作为模型系统来研究MRNA剪接的调节。替代剪接是控制基因表达并在蛋白质组中产生多样性的关键机制。使用秀丽隐杆线虫作为模型系统来研究此过程具有多种不同的优势，因为该生物具有与更高的真核生物相同类型的替代剪接事件和调节剂，而可用的基因组学和遗传学工具使该过程非常可拖动，以研究该过程的调节。我们开发了生物信息学技术，用于识别剪接基因和进行基因组的种间比对。我们已经将这些方法应用于内含子中的替代剪接调节元件。线虫的内含子大小相对较小，以及秀丽隐杆线虫和briggsae内含子之间的序列差异，这使我们比脊椎动物模型系统具有优势，用于识别这些CIS剪接调节剂。我们将应用这些知识来理解这些序列在调节替代剪接方面的功能。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。了解这种复合物及其在剪接站点选择中的功能将阐明MEC-8剪接调节的机制。我们已经克隆并表征了两个突变抑制因子到内含子的第一核苷酸。我们还绘制了内含子3'端的隐秘剪接位点选择的调节器。我们建议继续并扩展这些研究，以进一步了解剪接站点选择的机制。该赠款提案的具体目的是： 1-通过合并的生物信息学和体内研究来鉴定内含子MRNA剪接调节元件。这些将确定一组全面的内含子替代剪接控制信号及其相互作用的因素。 2-确定MRNA剪接因子MEC-8在调节前MRNA剪接方面的作用机理。 3-表征5'和3'剪接部位缺陷的抑制器，以阐明剪接位点选择的机制。