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

细胞质RNA、蛋白质和mRNA-蛋白质(MRNP)颗粒的清除维持动态平衡和 防止有毒物种的积累。应激颗粒(SGS)和P小体(PBS)是mRNP颗粒 富含mRNAs、RNA结合蛋白和信号蛋白，通常有助于细胞在应激状态下存活。这 可能反映了转录组和信号通路的调节。异常的SG清除与许多 癌症、病毒感染和肌萎缩侧索硬化症(ALS)，在这些疾病中，SGS可能促进细胞质错误- TDP-43(TDP-43)的定位和聚集；对神经元有毒性。SGS很可能 被各种拆解和降解手段清除，具有伴侣、蛋白酶体和 选择性自噬途径称为颗粒吞噬。相比之下，对铅的清除几乎没有人研究过。 最近，细胞质中的TDP-43被证明是通过一种新的内溶酶体转运途径(独特的 来自自噬)，当诱导时，抑制TDP-43的毒性。对机制的理解和 对SG、PB和TDP-43清除的后果仍处于早期阶段。还有一点是已知的，大量的 的RNA衰变发生在液泡和溶酶体中，尽管RNA分子具有靶向、运输机制 这种衰变对基因表达的影响尚不清楚。理解方面的主要差距包括 确定不同的清除途径如何起作用、协同作用和影响降解或分解 MRNP颗粒、胞质RNA和TDP-43。这种清除途径对细胞功能和 疾病也需要澄清。这笔赠款的目的是：1.)定义用法、重要性和协作性 已报道的疾病相关应激下SG和PB的清除机制，并确定其机制 食肉癖；2.)确定空泡/溶酶体的范围、特异性和转运机制(S) RNA衰变；3.机械地评估TDP-43内溶酶体降解并评估其后果 神经元和TDP-43相关的RNA表型。利用遗传、生化和细胞生物学分析，一种 将测试基于先前无偏酵母菌筛选的颗粒吞噬模型。这些努力将得到一本小说的帮助 SG纯化方法，用于鉴定SG定位的吞噬粒细胞效应子。RNA测序与液泡 分离将结合量化空泡RNA降解穹顶，而遗传学和单分子成像 将确定核糖核酸空泡衰变的转运机制(S)。最后，由无偏酵母筛支撑 识别TDP-43丰度的调节因子，一个涉及内膜的TDP-43降解模型 内陷将受到考验。将使用酵母、人类和神经细胞模型。这项建议在以下方面具有创新性 它将产生对新的空泡/溶酶体运输机制如何影响RNA和 蛋白质动态平衡。这项工作的价值在于，所获得的知识将为清除 相似的细胞底物和全球揭示了未被认识的RNA衰退途径的目标。最后， 了解SGS和胞浆TDP-43的清除可以确定ALS和癌症的治疗靶点。

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.