Genomic Analysis of Alternative Splice-Site Selection

选择性剪接位点选择的基因组分析

基本信息

批准号：
7383919
负责人：
Klemens J Hertel
金额：
$ 21.56万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-12 至 2009-02-28
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): With the completion of the human genome project, it has become clear that the number of genes cannot account for the complexity of the human proteome. This conclusion has lead to a dramatic increase in our appreciation of the abundance and importance of post-transcriptional mechanisms of gene regulation. Among several proposed mechanisms, alternative pre-mRNA splicing is considered to be one of the most efficient and wide spread avenues to generate multiple protein isoforms from individual genes. Current estimates indicate that over 60% of all human genes undergo alternative splicing, thus greatly increasing the coding potential of our genome. The Hertel laboratory (Principal Investigator) has successfully used quantitative approaches to uncover important regulatory aspect of the pre-mRNA splicing reaction. The Baldi laboratory (Collaborator) has been instrumental in the development of bioinformatics approaches required for the analysis of large datasets. In this proposal, the expertise of both groups will be combined to determine the mechanisms of alternative splice-site activation, one of the most frequent forms of alternative pre-mRNA splicing. Biochemical assays will be complemented by computational analyses of large EST databases to provide a molecular understanding for alternative splice-site activation. The long- term goal of this research proposal is to understand the mechanisms by which the splicing machinery correctly identifies exons and faithfully removes intervening sequences. The experiments proposed in this application have two major goals: To characterize the activation of alternative 5' splice sites (Specific Aim 1) and to characterize the activation of alternative 3' splice sites (Specific Aim 2). Preliminary computational analysis of the human genome demonstrated an unusually high frequency of alternative 5' splice-site activation 4 nucleotides upstream or downstream of the dominant 5' splice site. However, it is not clear whether these overlapping splice sites are activated stochastically or whether they are induced by regulatory elements. In Specific Aim 1 we propose biochemical experiments and computational analyses to evaluate these possibilities. Furthermore, we will test the hypothesis that alternative splicing of overlapping 5' splice sites serves to reestablish reading the frame disrupted by additional splicing events. In Specific Aim 2, we will perform a similar set of computational and biochemical experiments to determine why 3' splice-site activation 3 nucleotides upstream or downstream of the dominant splice site is the most frequent form of alternative 3' splice-site usage within the human genome.

描述（由申请人提供）：随着人类基因组计划的完成，很明显基因的数量无法解释人类蛋白质组的复杂性。这一结论使我们对基因调控转录后机制的丰富性和重要性有了极大的认识。在几种提出的机制中，选择性前 mRNA 剪接被认为是从单个基因生成多种蛋白质亚型的最有效和最广泛的途径之一。目前的估计表明，超过 60% 的人类基因经历了选择性剪接，从而大大增加了我们基因组的编码潜力。 Hertel 实验室（首席研究员）已成功使用定量方法揭示了前 mRNA 剪接反应的重要调控方面。 Baldi 实验室（合作者）在开发分析大型数据集所需的生物信息学方法方面发挥了重要作用。在这项提案中，两个小组的专业知识将结合起来，以确定选择性剪接位点激活的机制，这是选择性前 mRNA 剪接最常见的形式之一。大型 EST 数据库的计算分析将补充生化测定，以提供对选择性剪接位点激活的分子理解。这项研究计划的长期目标是了解剪接机制正确识别外显子并忠实去除干扰序列的机制。本申请中提出的实验有两个主要目标：表征选择性 5' 剪接位点的激活（具体目标 1）和表征选择性 3' 剪接位点的激活（具体目标 2）。对人类基因组的初步计算分析表明，主要 5' 剪接位点上游或下游 4 个核苷酸的选择性 5' 剪接位点激活频率异常高。然而，尚不清楚这些重叠剪接位点是随机激活的还是由调控元件诱导的。在具体目标 1 中，我们提出生化实验和计算分析来评估这些可能性。此外，我们将测试以下假设：重叠 5' 剪接位点的选择性剪接有助于重新建立对因额外剪接事件而中断的框架的读取。在具体目标 2 中，我们将进行一组类似的计算和生化实验，以确定为什么 3' 剪接位点激活优势剪接位点上游或下游的 3 个核苷酸是替代 3' 剪接位点使用的最常见形式。人类基因组。