Analyzing pre-mRNA splicing by deep sequencing of splice sites

通过剪接位点深度测序分析前 mRNA 剪接

• 批准号: 7991208
• 负责人: JONATHAN P STALEY
• 金额: $ 22.75万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-31 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991208
• 关键词: 3' Splice Site; Accounting; Alternative Splicing; Amino Acids; Animal Model; Antibodies; Biological Assay; Cells; Chicago; Complex; DNA; Data Analyses; Data Set; Development; Disease; Evaluation; Event; Gene Expression Profile; Gene Expression Regulation; Genes; Genome; Goals; Human; Human Genome; Immunoglobulin Fragments; In Vitro; Interruption; Introns; Kinetics; Knowledge; Lead; Libraries; Messenger RNA; Methodology; Methods; Microarray Analysis; Molecular; Positioning Attribute; Process; Proteome; RNA; RNA Splicing; Reading; Regulation; Reporting; Risk; Saccharomycetales; Sequence Analysis; Signal Transduction; Site; Specificity; Spliceosomes; Starvation; Stimulus; Structure; Testing; Transcriptional Regulation; Translations; comparative; cost; genome-wide; human disease; insight; mRNA Precursor; public health relevance; response

DESCRIPTION (provided by applicant): The requirement for splicing in humans is nearly ubiquitous. Human genes contain eight introns on average and greater than 93% of human genes undergo alternative splicing. These alternatively spliced forms are thought to contribute to the complexity of the human proteome. Further, alternative splicing is known to permit regulation of gene expression, such as during development and in response to environmental stimuli. Additionally, at least 15% of human diseases result from errors in splicing. Thus, to interpret the function of the human genome and to investigate human disease, we must describe qualitatively and quantitatively how the human transcriptome is spliced under a variety of conditions, the long-term goal of this project. Despite the development of genome-wide methodologies to assay alternative splicing, current methods are lacking in one respect or another. For example, while splicing-sensitive microarrays provided the first genome-wide view of splicing, such microarrays require prior knowledge of splice junctions and suffer from cross hybridization of splice junction probes with unspliced pre-mRNAs. While deep sequencing circumvents these limitations and offers tremendous promise, current applications of deep sequencing to splicing fail to exploit the full power of deep sequencing. Further, both approaches fail to reveal critical features of the splicing mechanism, often fail to report changes in splicing promptly, and in many cases fail to distinguish alternative splicing from transcriptional regulation. We propose to overcome the limitations of existing approaches by developing and validating a new, complementary and transformative method to assay splicing genome-wide. The limitations of current methods can be attributed to their nearly exclusive focus on the mRNA product of splicing. We propose to determine the feasibility of interrogating the other product of splicing - the excised intron. While this approach carries some risk in part due to the general functional irrelevance of the excised intron product, the excised intron offers the potential to utilize the full power of deep sequencing to analyze splicing quantitatively and qualitatively in a cost-effective manner. Toward developing and testing such a method, we propose to accomplish the following three specific aims. First, we aim to purify excised introns for library construction and deep sequencing. Second, we aim to develop methods for constructing sub-libraries of the transcriptome that are rich in intronic splice sites. Third, we aim to deep sequence intronic splice site libraries and to compare an analysis of this data with microarray and deep sequencing analysis of mRNA. We propose to test this methodology initially in the facile model organism budding yeast, because of its small genome size, small intron number and simple mode of splicing regulation. Additionally, budding yeast offers existing microarray and deep sequencing datasets that will permit an immediate comparative evaluation of this new method. By focusing the entire power of sequencing on splicing events reflected in excised introns, we expect to enable a new level of discovery and analysis of splicing that is currently inaccessible. PUBLIC HEALTH RELEVANCE: Translation of the information encoded in our DNA into the molecular workhorses of the cell requires an intermediate step, termed RNA splicing, in which interruptions of the information are deleted. Errors in RNA splicing account for at least 15% of all human diseases. In this project, we aim to develop a new method to analyze splicing genome-wide that will reveal unprecedented insights.

描述（由申请人提供）：人类中剪接的要求几乎无处不在。人类基因平均包含八个内含子，超过 93% 的人类基因经历选择性剪接。这些选择性剪接形式被认为有助于人类蛋白质组的复杂性。此外，已知选择性剪接可以调节基因表达，例如在发育过程中和响应环境刺激时。此外，至少 15% 的人类疾病是由剪接错误引起的。因此，为了解释人类基因组的功能并研究人类疾病，我们必须定性和定量地描述人类转录组在各种条件下如何剪接，这是该项目的长期目标。尽管开发了全基因组方法来测定选择性剪接，但目前的方法在某一方面还是存在缺陷。例如，虽然剪接敏感微阵列提供了第一个全基因组范围的剪接视图，但此类微阵列需要剪接连接的先验知识，并且会遭受剪接连接探针与未剪接的前mRNA的交叉杂交。虽然深度测序规避了这些限制并提供了巨大的前景，但目前深度测序在剪接中的应用未能充分利用深度测序的全部功能。此外，这两种方法都无法揭示剪接机制的关键特征，通常无法及时报告剪接的变化，并且在许多情况下无法区分选择性剪接和转录调控。我们建议通过开发和验证一种新的、互补的和变革性的方法来检测全基因组剪接，从而克服现有方法的局限性。当前方法的局限性可归因于它们几乎只关注剪接的 mRNA 产物。我们建议确定询问另一种剪接产物——切除的内含子的可行性。虽然这种方法存在一定风险，部分原因是切除的内含子产物通常与功能无关，但切除的内含子提供了利用深度测序的全部功能以经济有效的方式定量和定性分析剪接的潜力。为了开发和测试这种方法，我们建议实现以下三个具体目标。首先，我们的目标是纯化切除的内含子以用于文库构建和深度测序。其次，我们的目标是开发构建富含内含子剪接位点的转录组子文库的方法。第三，我们的目标是对内含子剪接位点文库进行深度测序，并将该数据的分析与 mRNA 的微阵列和深度测序分析进行比较。我们建议首先在简单的模型生物芽殖酵母中测试这种方法，因为它的基因组大小小、内含子数量少且剪接调节模式简单。此外，芽殖酵母提供了现有的微阵列和深度测序数据集，可以立即对这种新方法进行比较评估。通过将测序的全部力量集中在切除的内含子中反映的剪接事件上，我们期望能够将目前无法实现的剪接发现和分析提高到一个新的水平。 公共健康相关性：将DNA中编码的信息翻译成细胞的分子主力需要一个中间步骤，称为RNA剪接，其中信息的中断被删除。 RNA剪接错误至少占所有人类疾病的15%。在这个项目中，我们的目标是开发一种新方法来分析全基因组剪接，这将揭示前所未有的见解。