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

选择性剪接调控的视角：外显子skippin的全球分析

• 批准号: 7589632
• 负责人: Luiz Otavio Penalva
• 金额: $ 25.9万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-14 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589632
• 关键词: Address; Affect; Ally; Alternative Splicing; Area; Binding; Binding Sites; Biological; Biological Assay; Biological Models; Biological Process; Cell physiology; Characteristics; Communities; Computational Biology; Computing Methodologies; Consensus; Custom; Data; Data Analyses; Data Set; Deletion Mutation; Development; Disease; EST Library; Elements; Eukaryota; Event; Exons; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genome; Genomics; Goals; Hela Cells; Human; Immunoprecipitation; In Vitro; Indium; Knowledge; Lead; Malignant Neoplasms; Maps; Messenger RNA; Methods; Microarray Analysis; Molecular; Mus; Names; Nuclear RNA; Phylogenetic Analysis; Procedures; Process; Protein Binding; Proteins; Qualifying; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Regulatory Element; Research; System; Techniques; Transcript; Validation; crosslink; design; high throughput technology; improved; in vitro testing; in vivo; mRNA Precursor; novel; public health relevance; repository; research study; statistics; tool; vector; web page

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.

描述(申请人提供)：选择性剪接是高等真核生物不仅用来调节基因表达，而且还用来产生蛋白质变异性的一种细胞过程。在过去十年中，来自基因组项目的数据以及从EST文库提取的测序信息帮助确定了受这一过程影响的转录组的延伸，突出了其重要性。就在最近，这些信息被汇编并用于专门的微阵列平台的开发；使我们能够以以前不可能实现的方式进行高通量分析和研究替代剪接。我们建议使用全球方法来解决与替代剪接监管相关的重要问题。我们的目标是：具体目标1.我们将重点研究一种特殊类型的选择性剪接事件，称为外显子跳跃。在这个过程中，成熟的信使核糖核酸转录本中会排除或包含一个“替代外显子”。将使用HeLa细胞作为模型系统，并选择6个核RNA结合蛋白(RBPs)进行分析。一个定制的“外显子跳过微阵列”将被用来鉴定由每个选择的RBP调控的外显子跳过事件的子集。具体目的2.将使用夹法(交联法和免疫沉淀法)来鉴定被选定用于分析的每个RBP结合的RNA序列。来自微阵列分析和CLIP实验的数据将使用不同的计算生物学工具进行探索。对于每个选定的RBP，我们将识别和表征它在其目标基因/RNA中识别的顺式调节元件。最后，将开发一个开放访问的网页作为我们的数据集的存储库。特异靶3.特异靶2中确定的顺式调控元件将通过体内和体外方法进行验证。对于每个待分析的RBP，我们将选择一组目标基因/RNA通过微基因系统进行分析。在每种情况下，包含已识别的外显子跳过事件的基因组区域将被克隆到表达载体中。将对之前发现的顺式调控元件进行删除和突变。然后对剪接产物进行分析，以确定这些元件是否直接参与了外显子跳跃调控。此外，为了进一步证明所研究的限制性商业惯例直接参与剪接调控，将进行体外结合分析，以确定它们与选定的靶RNA和识别的顺式调控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的一个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，这些产物可能具有不同的生物功能。负责调节选择性剪接的蛋白质和元件的变化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在如何调控替代剪接方面的知识，并利用这些信息更好地了解它与癌症和疾病的联系。