Genomic Analysis of Alternative Splice-Site Selection

描述(申请人提供)：随着人类基因组计划的完成，基因的数量显然不能解释人类蛋白质组的复杂性。这一结论使我们对转录后基因调控机制的丰富性和重要性有了极大的了解。在已提出的几种机制中，选择性前mRNA剪接被认为是从单个基因产生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，从而极大地提高了我们基因组的编码潜力。Hertel实验室(首席调查员)已经成功地使用定量方法来揭示前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，将结合两个小组的专业知识来确定选择性剪接位点激活的机制，选择性剪接位点激活是选择性前mRNA剪接最常见的形式之一。生化分析将与大型EST数据库的计算分析相辅相成，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地移除插入序列的机制。本申请中提出的实验有两个主要目标：表征替代5‘剪接位点的激活(特异性目标1)和表征替代3’剪接位点的激活(特异性目标2)。对人类基因组的初步计算分析表明，优势5‘剪接位点上游或下游的4个核苷酸异常频繁地被替代的5’剪接位点激活。然而，目前还不清楚这些重叠的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，我们建议进行生化实验和计算分析来评估这些可能性。此外，我们将测试这一假设，即重叠的5‘剪接位点的替代剪接有助于重建被额外剪接事件破坏的帧的阅读。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3个核苷酸是人类基因组中最常见的替代3’剪接位点使用形式。

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.