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）：在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，大于93％的人类基因经历替代剪接。这些剪接形式被认为有助于人类蛋白质组的复杂性。此外，已知替代剪接可以允许调节基因表达，例如在发育和对环境刺激的响应中。另外，至少有15％的人类疾病是由于剪接中的错误而导致的。因此，要解释人类基因组的功能并调查人类疾病，我们必须在定性和定量上描述人类转录组在多种条件下剪接的方式，这是该项目的长期目标。尽管开发了全基因组的方法来分析替代剪接，但在一个方面或另一个方面仍缺乏当前的方法。例如，尽管剪接敏感的微阵列提供了剪接的第一个全基因组视图，但此类微阵列需要先验了解剪接连接，并遭受使用未填充的MRNA的剪接连接探针的跨杂交。深度测序避免了这些局限性并提供了巨大的希望，但深度测序对剪接的当前应用无法利用深度测序的全部功能。此外，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，无法将替代剪接与转录调节区分开。我们建议通过开发和验证一种新的，互补和变革性的方法来克服现有方法的局限性，以测定剪接全基因组的剪接。当前方法的局限性可以归因于其几乎独家关注剪接的mRNA产物。我们建议确定询问剪接的其他产物的可行性 - 切除的内含子。尽管这种方法的一定风险部分是由于切除的内含子产品的一般功能无关，但切除的内含子提供了利用深层测序的全部功能来定量和定性地以成本效益的方式分析剪接的潜力。为了开发和测试这种方法，我们建议实现以下三个特定目标。首先，我们旨在净化切除的内含子，以进行图书馆的构建和深度测序。其次，我们旨在开发构建具有内含子剪接位点的转录组的亚纤维的方法。第三，我们旨在深入序列内含子剪接位点文库，并将对该数据的分析与微阵列和深度测序分析进行比较。我们建议最初在便利模型的生物体萌芽的酵母中测试这种方法，因为其基因组大小，较小的内含子数量和剪接调节的简单模式。此外，萌芽的酵母提供现有的微阵列和深度测序数据集，这些数据集将立即对这种新方法进行比较评估。通过将测序的全部功能集中在切除的内含子中反映的剪接事件上，我们希望能够对当前无法访问的新发现和剪接分析进行新的发现和分析。 公共卫生相关性：将我们DNA中编码的信息转换为细胞的分子工作主场需要一个中间步骤，称为RNA剪接，其中删除了信息中断。 RNA剪接中的错误占所有人类疾病的至少15％。在这个项目中，我们旨在开发一种新方法来分析剪接基因组的全基因组，以揭示前所未有的见解。