Understanding the Starvation Induced Selective Autophagy of Specific mRNAs and lncRNAs

了解饥饿诱导的特定 mRNA 和 lncRNA 的选择性自噬

• 批准号: 9754581
• 负责人: Graham Jordan Ray
• 金额: $ 4.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754581
• 关键词: Affect; Affinity; Alternative Splicing; Autophagocytosis; Biology; Cell Line; Cells; Complex; Cytoskeleton; Data; Defect; Elements; FRAP1 gene; Genetic; Genetic Transcription; Growth Factor; Immunoprecipitation; Interphase Cell; Link; Longevity; Lysosomes; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Metabolism; Molecular; Neurodegenerative Disorders; Nutrient; Oncogenes; Population; Proteins; Proteome; Proteomics; RNA; RNA-Binding Proteins; Recycling; Regulator Genes; Research; Ribosomes; Role; Signal Pathway; Signal Transduction; Spliceosomes; Starvation; Stress; Techniques; Testing; Time; Translations; Untranslated RNA; Work; base; cell growth; detection of nutrient; experimental study; interest; loss of function; macromolecule; misfolded protein; protein aggregate; protein complex; response; stress granule; transcriptome sequencing

Project Summary/Abstract The lysosome is an essential element of cellular metabolism, functioning as both a recycling center and signaling hub. Autophagy targets macromolecules to the inside of the lysosome to be broken-down. Specifically, selective autophagy uses autophagic adapters to target specific macromolecules to the autophagosome1. The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway converges at the lysosomal membrane to integrate nutrient, growth factor, and stress signals to ultimately control cell growth and metabolism2. The signaling and recycling functions of the lysosome work in concert; mTORC1 senses the nutrient state of the cell and induces autophagy when nutrients are low. To date, research has emphasized the role of proteins at the lysosome; autophagy targets misfolded proteins and protein aggregates to lysosomes and protein complexes such as mTORC1 mediates lysosomal signaling. It is now recognized that there is a role for autophagy in degrading both protein-RNA complexes, such as ribosomes and stress granules, and RNA, such as RNA viruses3. For this reasons we are interested in the identifying and characterizing messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) that are targeted to the lysosome by selective autophagy. Our lab has developed a technique, termed LysoIP, for the rapid isolation of intact lysosomes using an affinity-tag expressed on the lysosomal membrane. LysoIP has been used to establish metabolite concentrations in the lysosome4 as well as a lysosomal proteome (unpublished). Based on the hypothesis that specific mRNAs or lncRNAs are targeted for lysosomal degradation by selective autophagy, we used LysoIP to purify RNA at lysosomes. Our preliminary data reveals a population of mRNAs and lncRNAs that increase in abundance at the lysosome when cells are starved of nutrients. The top RNAs in this population include mRNAs for spliceosome and cytoskeleton regulators, a lncRNA characterized as a transcriptional regulator and oncogene, and mRNAs and lncRNAs of unknown function. While we appreciate that some of these RNAs may localize to the lysosomal membrane, we hypothesize that most of this population includes RNAs targeted for selective autophagy as a cell starvation response. To understand how and why selective autophagy targets this RNA population to the lysosome we propose the following aims: 1. Identify the mechanism by which the starvation-induced RNA population localizes to the lysosome. 2. Determine the signaling mechanism necessary to localize starvation-induced RNA to lysosomes. 3. Investigate the function of localizing starvation-induced spliceosome regulator mRNA to the lysosome.

项目总结/摘要 溶酶体是细胞代谢的基本要素，既作为回收中心， 信号集线器自噬将大分子靶向到溶酶体内部以被分解。 具体地，选择性自噬使用自噬适配器将特定的大分子靶向细胞， 自噬体1.雷帕霉素复合物1（mTORC 1）信号通路的机制靶点会聚在 溶酶体膜整合营养素、生长因子和应激信号以最终控制细胞生长 新陈代谢2溶酶体的信号传导和再循环功能协同工作; mTORC1感知溶酶体的信号传导。 细胞的营养状态，并诱导自噬时，营养低。 迄今为止，研究强调了蛋白质在溶酶体中的作用;自噬靶点错误折叠 蛋白质和蛋白质聚集体与溶酶体和蛋白质复合物如mTORC 1介导溶酶体 发信号。现在认识到，自噬在降解蛋白质-RNA复合物， 例如核糖体和应激颗粒，以及RNA，例如RNA病毒3。因此，我们感兴趣的是 识别和表征信使RNA（mRNA）和长链非编码RNA（lncRNA）， 通过选择性自噬作用靶向溶酶体。 我们的实验室已经开发了一种称为LysoIP的技术，用于快速分离完整的溶酶体， 亲和标签在溶酶体膜上表达。LysoIP已用于确定代谢产物 溶酶体中的浓度4以及溶酶体蛋白质组（未发表）。基于这样的假设， 特异性mRNA或lncRNA通过选择性自噬被靶向用于溶酶体降解，我们使用LysoIP， 在溶酶体纯化RNA。我们的初步数据揭示了一个mRNAs和lncRNAs的群体， 当细胞缺乏营养物质时，溶酶体中的蛋白质会大量增加。这个群体中的顶级RNA包括 剪接体和细胞骨架调节因子的mRNA，一种以转录调节因子为特征的lncRNA 和癌基因，以及功能未知的mRNA和lncRNA。虽然我们意识到这些RNA中的一些 可能定位于溶酶体膜，我们假设这一群体的大部分包括RNA靶向 作为细胞饥饿反应的选择性自噬。为了理解选择性自噬如何以及为什么靶向 我们提出以下目标： 1.确定饥饿诱导的RNA群体定位于溶酶体的机制。 2.确定将饥饿诱导的RNA定位于溶酶体所必需的信号传导机制。 3.探讨饥饿诱导的剪接体调节因子mRNA定位于溶酶体的作用。