Analysis of mRNP granule clearance, vacuolar RNA decay and TDP-43 turnover

分析 mRNP 颗粒清除、液泡 RNA 衰减和 TDP-43 周转

• 批准号: 10444527
• 负责人: John Ross Buchan
• 金额: $ 31.09万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10444527
• 关键词: ALS pathology; Address; Affect; Amyotrophic Lateral Sclerosis; Autophagocytosis; Biochemical; Biological Assay; Biology; Cell Survival; Cell model; Cell physiology; Cells; Cellular Stress; Cellular biology; Coupled; Cytoplasmic Granules; Cytoplasmic Protein; Data; Disease; Event; Excision; Failure; Functional disorder; Gene Expression; Genetic; Genetic Screening; Goals; Grant; Growth; Homeostasis; Human; Image; Impairment; Individual; Knowledge; Label; Link; Lysosomes; Malignant Neoplasms; Maps; Measures; Membrane; Messenger RNA; Methods; Microscopy; Modeling; Molecular Chaperones; Multivesicular Body; Mutagenesis; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; Pathology; Pathway interactions; Phenotype; Physiologic pulse; Process; Proteins; RNA; RNA Decay; RNA Stability; RNA-Binding Proteins; Regulation; Reporting; Ribonucleases; Role; Signal Pathway; Signal Transduction; Signaling Protein; Specificity; Stress; Testing; Therapeutic; Toxic effect; Ubiquitin; Ubiquitination; Vacuole; Validation; Viral Cancer; Virus Diseases; Work; Yeasts; activation-induced cytidine deaminase; base; innovation; molecular imaging; multicatalytic endopeptidase complex; mutant; novel; novel therapeutic intervention; prevent; protein TDP-43; protein complex; proteostasis; screening; single molecule; stress granule; therapeutic target; trafficking; transcriptome; transcriptome sequencing

Clearance of cytoplasmic RNA, protein and mRNA-protein (mRNP) granules maintains homeostasis and prevents the accumulation of toxic species. Stress granules (SGs) and P-bodies (PBs) are mRNP granules enriched in mRNAs, RNA binding proteins and signaling proteins, that often aid cell survival during stress. This may reflect regulation of the transcriptome and signaling pathways. Aberrant SG clearance is implicated in many cancers, viral infections, and Amyotrophic Lateral Sclerosis (ALS), where SGs may promote cytoplasmic mis- localization and aggregation of TAR DNA-binding protein 43 (TDP-43); this is toxic to neurons. SGs are likely cleared by various disassembly and degradative means, with roles for chaperones, the proteasome, and a selective autophagic pathway termed granulophagy. In contrast, PB clearance has barely been studied. Recently, cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomal trafficking pathway (distinct from autophagy), which, when induced, suppresses TDP-43 toxicity. Understanding of the mechanisms and consequences for SG, PB and TDP-43 clearance remains at an early stage. It is also known that large amounts of RNA decay occur in vacuoles and lysosomes, though the RNA molecules targeted, trafficking mechanisms used and impacts of such decay on gene expression are unknown. Key gaps in understanding include determining how different clearance pathways function, co-operate and affect the degradation or disassembly of mRNP granules, cytoplasmic RNA and TDP-43. The impact of such clearance pathways on cell function and disease also requires elucidation. The aims of this grant are: 1.) define the usage, importance and co-operativity of reported SG and PB clearance mechanisms under disease-relevant stress, and identify the mechanism of granulophagy; 2.) determine the extent, specificity and trafficking mechanism(s) underlying vacuolar/lysosomal RNA decay; 3.) mechanistically assess TDP-43 endolysosomal degradation and evaluate consequences to neuronal and TDP-43-related RNA phenotypes. Using genetic, biochemical and cell biology assays, a granulophagy model based on a prior unbiased yeast screen will be tested. These efforts will be aided by a novel SG purification method, which will identify SG-localized granulophagy effectors. RNA-sequencing and vacuole isolation will be combined to quantify the vacuolar RNA degradome, while genetics and single-molecule imaging will identify RNA vacuolar decay trafficking mechanism(s). Finally, supported by an unbiased yeast screen identifying regulators of TDP-43 abundance, a model of TDP-43 degradation involving endosomal membrane invagination will be tested. Yeast, human, and neuronal cell models will be used. This proposal is innovative in that it will generate basic understanding of how novel vacuolar/lysosomal trafficking mechanisms affect RNA and protein homeostasis. The value of this work is that the knowledge obtained will offer paradigms for clearance of similar cellular substrates and globally reveal targets of an unappreciated RNA decay pathway. Finally, understanding clearance of SGs and cytoplasmic TDP-43 may identify therapeutic targets in ALS and cancer.

细胞质RNA、蛋白质和mRNA-蛋白质（mRNP）颗粒的清除维持体内平衡， 防止有毒物质的积累。应激颗粒（SG）和P体（PB）是mRNP颗粒 富含mRNA，RNA结合蛋白和信号蛋白，这些蛋白通常有助于细胞在压力下存活。这 可能反映了转录组和信号通路的调节。异常SG清除与许多 癌症、病毒感染和肌萎缩侧索硬化症（ALS），其中SG可能会促进细胞质错误- TAR DNA结合蛋白43（TDP-43）的定位和聚集;这对神经元有毒。SG很可能 通过各种分解和降解手段清除，分子伴侣、蛋白酶体和 选择性自噬途径称为颗粒吞噬。相比之下，PB清除率几乎没有研究。 最近，细胞质TDP-43被证明是通过一种新的内溶酶体运输途径降解的（独特的 自噬），其在诱导时抑制TDP-43毒性。对机制的理解， SG、PB和TDP-43清除的结果仍处于早期阶段。也知道大量的 的RNA衰变发生在液泡和溶酶体，虽然RNA分子的目标，运输机制 这种衰变对基因表达的影响是未知的。理解方面的主要差距包括 确定不同的清除途径如何发挥作用、相互配合和影响降解或拆卸， mRNP颗粒、细胞质RNA和TDP-43。这种清除途径对细胞功能和细胞周期的影响 疾病也需要解释。该补助金的目的是：1.）定义的用途，重要性和合作性 的SG和PB的清除机制，在疾病相关的压力，并确定机制， 嗜粒细胞性; 2.）确定空泡/溶酶体的程度、特异性和运输机制 RNA衰变; 3.）机械地评估TDP-43内溶酶体降解并评价 神经元和TDP-43相关的RNA表型。使用遗传、生物化学和细胞生物学分析， 将测试基于先前无偏酵母筛选的粒细胞吞噬模型。这些努力将得到一本小说的帮助。 SG纯化方法，其将鉴定SG定位的颗粒吞噬效应物。RNA测序和空泡 将结合分离来定量液泡RNA降解组，而遗传学和单分子成像 将确定RNA空泡衰变运输机制。最后，通过无偏见的酵母筛选， 鉴定TDP-43丰度的调节剂，涉及内体膜的TDP-43降解模型 将测试内陷。将使用酵母、人和神经元细胞模型。这一建议具有创新性， 它将产生新的液泡/溶酶体运输机制如何影响RNA的基本理解， 蛋白质稳态这项工作的价值在于，所获得的知识将为清除 类似的细胞基质，并全面揭示了未被重视的RNA衰变途径的目标。最后， 了解SGs和胞质TDP-43的清除可以确定ALS和癌症的治疗靶点。