RNA Processing Machines in Biology and Disease

生物学和疾病中的 RNA 加工机器

• 批准号: 9276460
• 负责人: ROBIN E. REED
• 金额: $ 64.5万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276460
• 关键词: Amyotrophic Lateral Sclerosis; Area; B-Lymphocytes; Biochemical; Biological Assay; Biology; Cell Line; Complex; Coupled; Defect; Disease; Functional disorder; Gene Expression; Genetic Transcription; Goals; Hela Cells; In Vitro; Investigation; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Methods; MicroRNAs; Molecular; Motor Neurons; Mutation; Neurodegenerative Disorders; Nuclear Extract; Pathogenesis; Pathway interactions; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reporting; Research; Resources; Role; Set protein; Stem cells; System; Therapeutic; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; Work; human disease; insight; mRNA Export; mutant; protein expression; protein function; transcriptome sequencing

Project Summary / Abstract The broad goal of our research is to understand the fundamental mechanisms of RNA processing and the machineries that carry out these essential steps in gene expression. It is becoming increasingly clear that defects in components of these machineries underlie numerous diseases, ranging from neurodegenerative disease to cancer. A complete biochemical understanding of the RNA processing machineries will provide a rational path forward for identifying therapeutic strategies for disease. Recently, we identified an important new area of investigation concerning the RNA binding proteins associated with Amyotrophic Lateral Sclerosis (ALS). We discovered that these proteins interact with each other and also with RNA polymerase II (RNAPII) and the splicing factor U1 snRNP. Moreover, we identified a set of proteins, which we designated PALs (Partners of ALS), that associate with the ALS RNA binding proteins and with RNAPII and U1 snRNP. We now plan to determine both the normal and ALS-causative roles of these proteins using a combination of CRSPR- edited proteins in conjunction with in vitro systems for RNA processing that we have developed. Among these are transcription-coupled splicing, transcription-coupled primary microRNA processing, and a robust method for preparing active small-scale nuclear extracts. These and other in vitro studies will be pursued in both HeLa cells and motor neurons, which will be differentiated from CRSPR-edited stem cells. RNA processing dysfunction and RNA targets of ALS/PALs proteins within the motor neurons will be identified by RNA-seq and iCLIP, respectively. The planned analysis of the ALS/PALs proteins will yield valuable information on the functions of these proteins and their associated molecular pathways, which may lead to new avenues for understanding ALS pathogenesis. Recently, several reports revealed that mutations in the U2 snRNP protein, SF3B1, are associated with a variety of cancers. Another key goal of our work is to determine the molecular mechanisms by which these mutations cause the widespread mis-splicing that was reported in RNA-seq studies. We plan to prepare active nuclear extracts from isogenic cell lines containing CRSPR-edited mutant or wild type SF3B1 for assays of U2 snRNP assembly and function. In addition, we plan to use a powerful quantitative mass spectrometry approach to examine protein expression alternations in the isogenic B cell lines. This analysis may reveal specific pathways that are disrupted due to SF3B1 mutation that might otherwise be difficult to detect at the RNA level. Finally, we recently discovered a protein, HNRNPUL1, which links the conserved TREX mRNA export machinery to the ALS-causative proteins. We now plan to determine the functional significance of these interactions via an analysis of both mRNA export and the pathways involving the RNAPII/U1 snRNP/ALS/PALs complex(es). Together, our proposed studies will provide important new insights into the biology of key proteins and pathways involved in diseases for which rapid progress is of the essence.

项目总结/摘要 我们研究的广泛目标是了解RNA加工的基本机制和RNA的表达。 在基因表达中执行这些重要步骤的机器。越来越清楚的是， 这些机制的组成部分的缺陷是许多疾病的基础， 从疾病到癌症。对RNA加工机制的完整的生物化学理解将提供一个 确定疾病治疗策略的合理途径。最近，我们发现了一个重要的新的 肌萎缩侧索硬化相关RNA结合蛋白的研究领域 （ALS）。我们发现，这些蛋白质相互作用，也与RNA聚合酶II（RNAPII） 和剪接因子U1 snRNP。此外，我们鉴定了一组蛋白质，我们将其命名为PAL （ALS的伴侣），其与ALS RNA结合蛋白以及与RNAPII和U1 snRNP缔合。我们现在 计划使用CRSPR- 编辑的蛋白质与我们开发的体外RNA加工系统相结合。其中 是转录偶联剪接，转录偶联初级microRNA加工，以及一种稳健的方法 用于制备活性小规模核提取物。这些和其他体外研究将在HeLa细胞中进行， 细胞和运动神经元，其将从CRSPR编辑的干细胞分化。rna加工 运动神经元内ALS/PAL蛋白的功能障碍和RNA靶将通过RNA-seq鉴定， iCLIP，分别。ALS/PALs蛋白的计划分析将产生关于ALS/PALs蛋白的有价值的信息。 这些蛋白质的功能及其相关的分子途径，这可能会导致新的途径， 了解ALS发病机制。最近，一些报道揭示了U2 snRNP蛋白的突变， SF3B1与多种癌症有关。我们工作的另一个关键目标是确定 这些突变导致RNA-seq中报道的广泛错误剪接的机制 问题研究我们计划从含有CRSPR编辑的突变体或突变体的等基因细胞系制备活性核提取物。 野生型SF3B1用于U2 snRNP组装和功能测定。此外，我们计划使用一个强大的 定量质谱法检测同基因B细胞中蛋白质表达的变化 线这种分析可能揭示由于SF3B1突变而被破坏的特定途径， 否则难以在RNA水平上检测。最后，我们最近发现了一种蛋白质，HNRNPUL1， 将保守的TREX mRNA输出机制与ALS致病蛋白联系起来。我们现在计划确定 通过分析mRNA输出和途径，这些相互作用的功能意义 涉及RNAPII/U1 snRNP/ALS/PAL复合物。总之，我们提出的研究将提供重要的 新的见解，生物学的关键蛋白质和途径，涉及疾病的快速进展， 本质