Alternative splicing regulation in perspective: A global analysis of exon skippin

描述（由申请人提供）：选择性剪接是高等真核生物采用的细胞过程，不仅调节基因表达，而且产生蛋白质变异性。在过去的十年中，来自基因组计划的数据以及从EST文库中提取的测序信息有助于定义受此过程影响的转录组的延伸，突出了其重要性。就在最近，这些信息被编译并用于开发专门的微阵列平台;使我们能够以以前不可能的方式进行高通量分析和研究选择性剪接。我们建议使用一种全球性的方法来解决与选择性剪接调控相关的重要问题。我们的目标是：具体目标1。我们将把我们的研究重点放在一种特殊类型的选择性剪接事件，命名为外显子跳跃。在此过程中，“选择性外显子”被排除或包括在成熟mRNA转录物中。HeLa细胞将用作模型系统，并将选择6种核RNA结合蛋白（RBP）进行分析。将采用定制的“外显子跳跃微阵列”来鉴定由每个选择的RBP调节的外显子跳跃事件的子集。具体目标2。将采用CLIP方法（交联和免疫沉淀）鉴定与选择用于分析的各RBP结合的RNA序列。来自微阵列分析和CLIP实验的数据将用计算生物学的不同工具进行探索。对于每一个选择的RBP，我们将确定和表征它在其靶基因/RNA中识别的顺式调控元件。最后，将开发一个开放获取的网页，作为我们数据集的储存库。具体目标3。将通过体内和体外方法验证特异性目标2中鉴定的推定顺式调节元件。对于每个待分析的RBP，我们将选择一组靶基因/RNA通过mini-gene系统进行分析。在每种情况下，将含有鉴定的外显子跳跃事件的基因组区域克隆到表达载体中。将完成先前鉴定的顺式调控元件的缺失和突变。然后分析剪接产物以确认这些元件直接参与外显子跳跃调控。此外，为了进一步证明所研究的RBP直接参与剪接调节，将进行体外结合测定以确定它们与所选靶RNA和所鉴定的顺式调节元件的直接相互作用。公共卫生相关性：选择性剪接是基因调控中非常重要的分子过程。由于选择性剪接，单个基因可以产生不同的蛋白质产物;在某些情况下，这些产物可以具有不同的生物学功能。负责调节选择性剪接的蛋白质和元件的改变可能导致癌症和疾病。我们建议使用新的高通量技术来提高我们对选择性剪接是如何调节的知识，并使用这些信息来更好地了解它与癌症和疾病的联系。

DESCRIPTION (provided by applicant): Alternative splicing is a cellular process that is employed by higher eukaryotes to not only regulate gene expression but to also generate protein variability. During the last decade, data derived from the Genome Project as well as sequencing information extracted from EST libraries helped to define the extension of the transcriptome that is affected by this process, highlighting its importance. Just recently, this information was compiled and used in the development of specialized microarray platforms; allowing us to perform high throughput analyses and investigate alternative splicing in ways that were not possible before. We propose to use a global approach to address important questions relevant to alternative splicing regulation. Our Aims are: Specific Aim 1. We will focus our research on a particular type of alternative splicing event, named exon skipping. In this process, an "alternative exon" is either excluded or included in the mature mRNA transcript. HeLa cells will be used as a model system and 6 nuclear RNA binding proteins (RBPs) will be selected for analysis. A custom made "exon skipping microarray" will be employed to identify the subset of exon skipping events that are regulated by each selected RBP. Specific Aim 2. The CLIP method (Crosslinking and Immunoprecipitation) will be employed to identify the RNA sequences that are bound by each RBP selected for analysis. The data derived from the both microarray analysis and CLIP experiments will be explored with different tools of Computational Biology. For each selected RBP, we will identify and characterize the cis-regulatory elements it recognizes in its target genes/RNAs. Finally, a webpage with open access will be developed to serve as repository of our data set. Specific Aim 3. Putative cis-regulatory elements identified in Specific Aim 2 will be validated by in vivo and in vitro methods. For each RBP under analysis, we will select a group of target genes/RNAs to be analyzed via mini-gene system. In each case, the genomic region containing the identified exon skipping event will be cloned in an expressing vector. Deletions and mutations of the previously identified cis-regulatory elements will be done. The splicing products will be then analyzed to confirm the direct participation of these elements in exon skipping regulation. Moreover, to establish further proof of the direct involvement of the studied RBPs in splicing regulation, in vitro binding assays will be performed to determine their direct interaction with selected target RNAs and identified cis- regulatory elements. PUBLIC HEALTH RELEVANCE: Alternative splicing is a very important molecular process implicated in gene regulation. Thanks to alternative splicing a single gene can produce different protein products; in some cases these products can have different biological functions. Alterations in the proteins and elements that are responsible for the regulation of alternative splicing can lead to cancer and diseases. We propose to use novel high throughput technology to improve our knowledge in how alternative splicing is regulated and use this information to better understand its connection to cancer and diseases.