Global measurement of splicing kinetics

剪接动力学的全局测量

• 批准号: 9206210
• 负责人: Lee Stirling Churchman
• 金额: $ 21.53万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-28 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9206210
• 关键词: Affect; Alternative Splicing; Base Sequence; Bioinformatics; Biological; Cells; Characteristics; Chromatin; Classification; Complex; DNA Maintenance; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Data Set; Development; Disease; Evaluation; Event; Exons; Genes; Genetic Transcription; Genetic study; Genome; Gold; Health; High-Throughput Nucleotide Sequencing; Human; Human Genome; Individual; Introns; Kinetics; Label; Lead; Life; Light; Link; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Metabolic; Mutation; Mutation Analysis; Nonsense-Mediated Decay; Nuclear Export; Phenotype; Positioning Attribute; Process; Protein Isoforms; Proteins; Protocols documentation; Public Health; RNA; RNA Splicing; Rare Diseases; Recurrence; Regulation; Research; Resolution; Time; Transcript; Work; cancer genome; computerized tools; disease phenotype; functional gain; genome sequencing; genome-wide; insight; millisecond; mutant; nervous system disorder; novel strategies; novel therapeutic intervention; spatiotemporal; temporal measurement; tool

ABSTRACT: Alternative splicing (AS) of human genes is pervasive and greatly expands the repertoire of protein and RNA products arising from the human genome. AS is fundamental to gene and genome function, and its dysregulation has been causally linked with a broad and expanding array of both common and rare diseases and cancers. Despite its central importance, we currently have limited insight into the occurrence and regulation of AS at both the local (gene) and global (genome-wide) levels, due chiefly to a lack of tools that provide direct, high-resolution, and quantitative views into the splicing process. This deficit has in turn roadblocked progress in characterizing the wealth of mutations affecting various components of the splicing machinery now emerging from genetic studies of disease. To date, global studies of splicing have relied chiefly on analysis of mature cytosolic RNA isoforms, which represent the combined result of transcription, splicing, nuclear export and degradation and thus a very incomplete view. For example, at least 1/3rd of splicing events are estimated to go undetected since the resulting RNA product is rapidly targeted for nonsense-mediated decay, and many others go undetected due to limited sensitivity of current sequencing-based approaches. Thus, it is critical to map splicing events rapidly when and where they occur. Here we propose to develop a novel approach that will provide a direct, dynamic, and global window into the alternative splicing of human exons. Our approach enables simultaneous determination of RNA polymerase position and the splicing state of individual RNA molecules. This will in turn open a new kinetic window on splicing that will have uniquely powerful utility for basic mapping and mechanistic studies, and for characterization and functional analysis of mutations affecting the splicing machinery. In Aim 1, we develop an approach that interrogates entire nascent transcripts, to simultaneously map 3' ends as well as the splicing state of the nascent RNA, in a strand-specific manner. We will then develop computational tools to quantify the average distance transcribed by Pol II when splicing occurs, and to integrate these data with average transcription rates and high resolution Pol II mapping to reveal mean splicing times – thus opening a new and global window into splicing kinetics. This approach has a temporal resolution of tens of milliseconds that greatly eclipses other strategies to measure splicing kinetics, such as metabolic labeling of nascent RNA. In Aim 2, we apply this approach to characterize the global effects of recurrent mutations in 2 splicing regulators found in malignancies. An unexpected discovery from cancer genome sequencing studies has been the identification of frequent mutations in genes encoding splicing machinery components. As proof-of-principle for complex biological applications, we propose to apply our approach to characterize and gain functional insights into mutations in splicing regulators characteristic of several major malignancies. Our comprehensive approach for measuring global splicing kinetics has the positive impact of enabling rapid discovery and characterization of splicing regulation in health and disease.

摘要：人类基因的选择性剪接（AS）非常普遍，并且极大地扩展了基因库。 来自人类基因组的蛋白质和RNA产物是基因和基因组功能的基础， 它的失调与广泛且不断扩大的常见和罕见疾病有因果关系 尽管其至关重要，但我们目前对其发生和发展的了解有限。 AS 在局部（基因）和全局（全基因组）水平上的调控，主要是由于缺乏工具 为拼接过程提供直接、高分辨率和定量的视图。 在表征影响剪接各个组成部分的大量突变方面遇到障碍 迄今为止，全球对剪接的研究主要依赖于疾病遗传学研究。 对成熟胞质 RNA 亚型的分析，它代表转录、剪接、 核输出和降解，因此是一个非常不完整的观点，例如，至少 1/3 的剪接事件。 估计不会被检测到，因为所得的 RNA 产物很快就会被无义介导的靶向 由于当前基于测序的方法的灵敏度有限，许多其他因素未被检测到。 因此，快速绘制剪接事件发生的时间和地点至关重要。在这里，我们建议开发一种方法。 新方法将为人类选择性剪接提供直接、动态和全局的窗口 我们的方法能够同时测定 RNA 聚合酶的位置和剪接状态。 这将反过来打开一个新的剪接动力学窗口，该窗口将具有独特的作用。 对于基本绘图和机制研究以及表征和功能分析具有强大的实用性 影响剪接机制的突变在目标 1 中，我们开发了一种询问整个新生体的方法。 转录本，以链特异性方式同时绘制 3' 末端以及新生 RNA 的剪接状态 然后，我们将开发计算工具来量化 Pol II 转录的平均距离。 发生剪接，并将这些数据与平均转录率和高分辨率 Pol II 作图整合 揭示平均剪接时间——从而为剪接动力学打开了一个新的全局窗口。 数十毫秒的时间分辨率大大超过了其他测量剪接动力学的策略， 例如新生 RNA 的代谢标记，在目标 2 中，我们应用这种方法来表征全局效应。 癌症中发现的 2 个剪接调节因子的反复突变。 基因组测序研究已识别编码剪接的基因中的频繁突变 作为复杂生物应用的原理验证，我们建议应用我们的技术。 表征剪接调节因子突变并获得功能见解的方法 我们测量整体剪接动力学的综合方法具有以下特点： 能够快速发现和表征健康和疾病中的剪接调控的积极影响。