Understanding the Starvation Induced Selective Autophagy of Specific mRNAs and lncRNAs

项目摘要/摘要 溶酶体是细胞新陈代谢的基本元素，既是循环中心，也是 信号集线器。自噬作用的目标是要分解的溶酶体内部的大分子。 具体地说，选择性自噬使用自噬适配器将特定的大分子靶向 自噬1。雷帕霉素复合体1(MTORC1)信号通路的机制靶点 溶酶体膜整合营养、生长因子和压力信号，最终控制细胞生长 代谢率为2。溶酶体的信号传递和循环功能协同工作；mTORC1感觉到 细胞的营养状态，并在营养不足时诱导自噬。 到目前为止，研究强调了蛋白质在溶酶体中的作用；自噬靶标被错误折叠 蛋白质和蛋白质聚集到溶酶体和蛋白质复合体，如mTORC1介导溶酶体 发信号。现在已经认识到，自噬在降解蛋白质-RNA复合体方面具有作用， 如核糖体和应激颗粒，以及RNA，如RNA病毒3。出于这个原因，我们对此感兴趣 识别和表征信使RNA(MRNAs)和长非编码RNA(LncRNAs)是 通过选择性的自噬作用靶向溶酶体。 我们实验室开发了一种名为LysoIP的技术，用于快速分离完整的溶酶体 表达于溶酶体膜上的亲和标签。LysoIP已用于建立代谢物 溶酶体4和溶酶体蛋白质组中的浓度(未发表)。基于这样的假设 通过选择性自噬，特定的mRNAs或lncRNAs被定向为溶酶体降解，我们使用LysoIP 提纯溶酶体处的RNA。我们的初步数据显示，mRNAs和lncRNAs数量在 当细胞缺乏营养时，溶酶体的丰度。这个群体中排名靠前的RNA包括 剪接体和细胞骨架调节因子的mRNAs，一种转录调节因子 和癌基因，以及未知功能的mRNAs和lncRNAs。虽然我们很欣赏其中一些RNA 可能定位于溶酶体膜，我们假设这个群体中的大多数包括靶向的RNA。 作为细胞饥饿反应的选择性自噬。了解选择性自噬靶标的方式和原因 对于溶酶体这种RNA群体，我们提出了以下目标： 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定位到溶酶体所需的信号机制。 3.研究饥饿诱导的剪接体调节基因对溶酶体的定位作用。

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 LysoIP has been used to establish metabolite concentrations in the lysosome4 as well as a lysosomal proteome (unpublished).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.