Global control of co-transcriptional splicing

共转录剪接的全局控制

• 批准号: 10549312
• 负责人: Lee Stirling Churchman
• 金额: $ 52.04万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10549312
• 关键词: Affect; Alternative Splicing; Binding; Biological Assay; CRISPR/Cas technology; Cells; Chromatin; Computer Analysis; Cytoplasm; DNA-Directed RNA Polymerase; Data; Defect; Diagnosis; Disease; Elements; Excision; Exons; Frequencies; Genes; Genetic; Genetic Transcription; Genomics; Goals; Grant; Hour; Human; Human Genome; Immunoprecipitation; In Vitro; Individual; Introns; Kinetics; Knowledge; Length; Link; Malignant Neoplasms; Measures; Methods; Mission; Modeling; Muscle; Muscle Fibers; Muscular Dystrophies; Myoblasts; Neurodegenerative Disorders; Nucleoplasm; Nucleotides; Outcome; Positioning Attribute; Process; Processed Genes; Proteins; Public Health; RNA; RNA Sequences; RNA Splicing; RNA purification; RNA-Binding Proteins; Regulation; Research; Resolution; Role; Site; Spliceosomes; System; Techniques; Time; Tissues; Trans-Activators; Transcript; United States National Institutes of Health; cis acting element; crosslink; experience; experimental study; genetic variant; genome-wide; human disease; in vivo; insight; myogenesis; nanopore; nervous system disorder; novel therapeutic intervention; tool; transcriptome sequencing

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 critical for cellular differentiation and identity, and its dysregulation has been causally linked with a broad and expanding array of human diseases, including muscular dystrophies, neurodegenerative disorders and cancers. However, we currently have limited insight into the regulation of AS at both the local (gene) and global (genome-wide) levels, due 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 understanding how splicing is regulated to confer cellular identity and to control differentiation processes. The eight introns per average human gene are processed co-transcriptionally through spliceosomal subunits and regulatory factors binding to specific sequences in nascent RNA. These cis-elements are typically within introns and thus act only from when they emerge from RNA polymerase to when they are spliced out. Consequently, in order to dissect splicing regulation mechanisms, we need to determine how fast splicing occurs and the order of intron excision across nascent transcripts. We recently developed nanopore analysis of CO- transcriptional Processing (nano-COP) that measures the kinetics, order and coordination of splicing of endogenous genes in vivo. Nascent RNA is purified and then directly sequenced using the Oxford Nanopore platform to obtain long reads. We found that splicing kinetics is influenced by intron length and proximity to alternatively spliced exons, that splicing order does not follow the order of transcription and that neighboring introns have the propensity to be spliced coordinately at the same time. The goal of this grant is to determine how cis-acting elements and trans-acting factors impact human splicing kinetics, splicing order and splicing coordination. Specific Aim 1: Determine how trans-acting factors impact splicing dynamics. We will study eight RNA-binding proteins that are connected to splicing regulation by our analysis or other studies. To diminish secondary effects, we will use an inducible degradation system to degrade target factors within hours. We will perform subRNA-seq and nano-COP to study splicing dynamics after the loss of each factor. Specific Aim 2: Determine the role of cis-acting elements in dictating splicing dynamics. We will determine how changes to splice site sequences and other cis-elements alter splicing kinetics and alternative splicing. We will use CRISPR-Cas9 and leverage natural genetic variants to study perturbations to cis-elements. Specific Aim 3: Determine the relationship between splicing dynamics and AS during human myogenesis. We hypothesize that key trans-acting factors control splicing kinetics that in turn affect AS. We will study how splicing dynamics change during myogenesis using nano-COP. The roles of myogenesis splicing regulators in controlling splicing dynamics will also be investigated. In sum, changes in splicing kinetics will be associated with AS outcomes to determine models of how splicing is regulated by splicing dynamics.

人类基因的选择性剪接（AS）是普遍存在的，极大地扩展了蛋白质和RNA的库 人类基因组产生的产物。AS对于细胞分化和身份是至关重要的，其 调节异常与一系列广泛且不断扩大的人类疾病有因果关系，包括肌肉疾病， 营养不良、神经退行性疾病和癌症。然而，我们目前对 AS在局部（基因）和全局（全基因组）水平上的调控，由于缺乏工具， 直接、高分辨率和定量地观察拼接过程。这种赤字反过来阻碍了 了解剪接是如何被调节以赋予细胞身份和控制分化的进展 流程.每个人类基因平均有8个内含子通过剪接体共转录处理， 亚基和调节因子与新生RNA中的特定序列结合。这些顺式元件通常是 在内含子内，因此只有当它们从RNA聚合酶中出现时才起作用，直到它们被剪接出来。 因此，为了剖析剪接调控机制，我们需要确定剪接发生的速度 以及内含子在新生转录物中切除的顺序。我们最近开发了CO的纳米孔分析- 转录处理（纳米COP），测量剪接的动力学，顺序和协调， 体内内源基因。纯化新生RNA，然后使用Oxford Nanopore 平台，以获得长时间的阅读。我们发现剪接动力学受内含子长度和邻近内含子的影响。 选择性剪接的外显子，剪接顺序不遵循转录顺序，并且邻近的 内含子具有同时协调剪接的倾向。这项补助金的目的是确定 顺式作用元件和反式作用因子如何影响人类剪接动力学、剪接顺序和剪接 协同具体目标1：确定反式作用因子如何影响剪接动力学。我们将学习八 通过我们的分析或其他研究与剪接调控有关的RNA结合蛋白。减少 二次效应，我们将使用诱导降解系统在数小时内降解靶因子。我们将 进行subRNA-seq和nano-COP以研究每个因子丢失后的剪接动力学。具体目标二： 确定顺式作用元件在剪接动力学中的作用。我们将决定如何改变拼接 位点序列和其它顺式元件改变剪接动力学和可变剪接。我们将使用CRISPR-Cas9 并利用天然遗传变异来研究顺式元件的扰动。具体目标3：确定 剪接动力学与AS在人肌发生中的关系。我们假设关键的交互作用 这些因素控制剪接动力学，而剪接动力学又影响AS。我们将研究剪接动力学如何在 使用纳米COP的肌生成。肌生成剪接调节子在控制剪接动力学中的作用将 也被调查。总之，剪接动力学的变化将与AS结局相关，以确定 剪接如何受剪接动力学调控的模型。