RNA Processing Machines in Biology and Disease

生物学和疾病中的 RNA 加工机器

• 批准号: 9893724
• 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/9893724
• 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; System; Therapeutic; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; Work; human disease; insight; mRNA Export; mutant; protein expression; protein function; stem cells; 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.

项目摘要/摘要 我们研究的广泛目标是了解RNA加工的基本机制和 在基因表达中执行这些基本步骤的机器。越来越清楚的是， 这些机器部件的缺陷导致了许多疾病，从神经退行性疾病 从疾病到癌症。对RNA加工机制的全面生化理解将提供 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 肌萎缩侧索硬化症相关RNA结合蛋白的研究领域 (ALS)。我们发现这些蛋白质相互作用，并与RNA聚合酶II(RNAPII)相互作用。 剪接因子U1和SnRNP。此外，我们鉴定了一组蛋白质，我们将其命名为PALS (ALS的伙伴)，与ALS RNA结合蛋白以及RNAPII和U1 SnRNP相关。我们现在 计划使用CRSPR-组合来确定这些蛋白质的正常和ALS致病作用 编辑的蛋白质与我们开发的用于RNA加工的体外系统相结合。其中包括 是转录偶联剪接、转录偶联初级microRNA处理和一种健壮的方法 用于制备活性小规模的核提取物。这些和其他体外研究将在两个HeLa中进行 细胞和运动神经元，这些细胞将从CRSPR编辑的干细胞分化出来。RNA加工 运动神经元内ALS/PALS蛋白的功能障碍和RNA靶标将通过RNA-seq和 ICLIP。对ALS/PALS蛋白的计划分析将产生关于 这些蛋白质及其相关的分子途径的功能，这可能会导致新的途径 了解肌萎缩侧索硬化的发病机制。最近，几份报告显示，U2SnRNP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分子 这些突变导致rna-seq中报道的广泛错误剪接的机制。 学习。我们计划从含有CRSPR编辑的突变体或 野生型SF3B1用于检测U2SnRNP的组装和功能。此外，我们计划使用强大的 定量质谱法检测同基因B细胞蛋白质表达的变化 台词。这一分析可能揭示由于SF3B1突变而中断的特定通路 否则很难在RNA水平上检测到。最后，我们最近发现了一种蛋白质，HNRNPUL1，它 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 从信使核糖核酸输出和途径分析这些相互作用的功能意义 涉及RNAPII/U1 SnRNP/ALS/PALS复合体(ES)。总之，我们提议的研究将提供重要的 对关键蛋白质的生物学和涉及疾病的快速进展的途径的新见解 精华所在。