Global control of co-transcriptional splicing

共转录剪接的全局控制

基本信息

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
项目类别：
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

项目摘要

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) 十分普遍，极大地扩展了蛋白质和 RNA 的种类来自人类基因组的产品。 AS 对于细胞分化和身份至关重要，其失调与一系列广泛且不断扩大的人类疾病存在因果关系，包括肌肉疾病营养不良、神经退行性疾病和癌症。然而，目前我们对这一问题的了解还很有限。由于缺乏提供AS的工具，因此在局部（基因）和全球（全基因组）水平上对AS进行调控拼接过程的直接、高分辨率和定量视图。这种赤字反过来又阻碍了在理解如何调节剪接以赋予细胞身份和控制分化方面取得进展流程。每个人类基因平均有八个内含子通过剪接体进行共转录处理与新生 RNA 中特定序列结合的亚基和调节因子。这些顺式元件通常是内含子内，因此仅从它们从 RNA 聚合酶中出现到它们被剪接时发挥作用。因此，为了剖析剪接调节机制，我们需要确定剪接发生的速度以及新生转录本中内含子切除的顺序。我们最近开发了 CO- 的纳米孔分析转录处理（nano-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 直接测序获得长读的平台。我们发现剪接动力学受到内含子长度和邻近性的影响选择性剪接的外显子，该剪接顺序不遵循转录顺序，并且相邻的外显子内含子具有同时协调剪接的倾向。这笔赠款的目标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、剪接顺序和剪接协调。具体目标 1：确定反式作用因子如何影响剪接动态。我们将学习八个通过我们的分析或其他研究，RNA 结合蛋白与剪接调节相关。减少次要影响，我们将使用诱导降解系统在数小时内降解目标因子。我们将执行 subRNA-seq 和 nano-COP 以研究每个因子丢失后的剪接动态。具体目标2：确定顺式作用元件在决定剪接动力学中的作用。我们将确定如何改变拼接位点序列和其他顺式元件改变剪接动力学和选择性剪接。我们将使用 CRISPR-Cas9 并利用自然遗传变异来研究顺式元素的扰动。具体目标 3：确定人类肌生成过程中剪接动力学与 AS 之间的关系。我们假设关键交易因素控制剪接动力学，进而影响 AS。我们将研究剪接动力学如何变化使用纳米 COP 进行肌生成。肌生成剪接调节因子在控制剪接动力学中的作用将也受到调查。总之，剪接动力学的变化将与 AS 结果相关联，以确定剪接如何受剪接动力学调节的模型。