RNA trafficking in mitochondria

摘要 线粒体有许多信号通路将压力传递到细胞的其他部分。类似于 释放病原体相关分子模式（PAMP）的病原体，线粒体释放新的 损伤相关分子模式 (DAMP)，包括脂质、肽和线粒体 DNA (mtDNA)， 表明线粒体应激。线粒体双链 RNA (mtdsRNA) 是一类新的 DAMP 当 mRNA 中的非编码链没有被有效降解并积累时就会产生，从而允许 与编码链碱基配对。正常情况下，解旋酶 SUV3 解旋 mtRNA， 多核苷酸磷酸化酶 (PNPase) 会降解它们。然而，SUV3 的淘汰导致 mtdsRNA 在线粒体内积累，PNPase 的敲低会导致 mtdsRNA 的释放 mtdsRNA 进入细胞质。一旦进入细胞质，mtdsRNA 就会被 dsRNA 传感器 MDA5 感知并 RIG-I，导致 I 型干扰素途径的诱导。 mtdsRNA 的输出可能很重要，因为 已在编码 PNPase 的 PNPT1 突变患者的细胞质中鉴定出 mtdsRNA 疾病包括癌症、心脏病、酒精相关肝病和自身免疫性疾病。 mtdsRNA 代表线粒体功能障碍的新生物标志物的假设将得到检验。 由于这是一条新途径，因此在理解其分子规则和机制方面存在重大差距 mtdsRNA 穿过线粒体内膜和外膜进行胞质输出。我们的学习目标是 包含三个独立但主题相关的具体目标。在目标 1 中，mtdsRNA 是 从线粒体输出的产物将在大小和序列特异性方面进行表征。此外， 将测试 RNA 修饰酶以确定哪些成分对于生成 mtdsRNAs.目标 2 将重点关注外膜通道和内膜通道的识别以及 PNPase 在 mtdsRNA 从线粒体中运输出来。第三个目标将定义导致 mtdsRNA 的生成和胞质 dsRNA 传感器的测定，这些传感器在 这个过程。由于 PNPase 突变会导致线粒体疾病，因此突变体的特征是 确定 mtdsRNA 的降解和/或输出步骤是否可以分开。 我们的研究团队一直在广泛表征 PNPase 及其在线粒体中的功能。独特 可用于我们工作的模型系统包括一个小鼠模型，其中 floxxed PNPT1 可以通过 Cre 重组酶和源自该模型的小鼠胚胎成纤维细胞。该提案的结果将 详细定义 mtdsRNA 从线粒体运输出来的途径，并提供一个平台 了解 PNPase 突变如何导致疾病。从长远来看，这些研究可能会导致 建立 mtdsRNA 作为线粒体功能障碍的新生物标志物。

SUMMARY Mitochondria have numerous signaling pathways for conveying stress to the rest of a cell. Similar to pathogens that release pathogen-associated molecular patterns (PAMPs), mitochondria release novel damage-associated molecular patterns (DAMPs), including lipids, peptides, and mitochondrial DNA (mtDNA), that indicate mitochondrial stress. Mitochondrial double-stranded RNA (mtdsRNA) is a new class of DAMPs that is generated when the noncoding strand in mtRNA is not degraded efficiently and accumulates, allowing base-pairing with the coding strand. Under normal conditions, the helicase SUV3 unwinds the mtRNAs and polynucleotide phosphorylase (PNPase)ndegrades them. However, knockdown of SUV3 results in the accumulation of mtdsRNAs within mitochondria, and knockdown of PNPase leads to the release of the mtdsRNAs into the cytosol. Once in the cytosol, the mtdsRNAs are sensed by dsRNA sensors MDA5 and RIG-I, leading to the induction of the type I interferon pathway. The export of mtdsRNA is likely important as mtdsRNAs have been identified in the cytosol of patients with mutations in PNPT1, encoding PNPase, and in diseases including cancer, cardiac disease, alcohol-associated liver disease, and autoimmune diseases. The hypothesis that mtdsRNAs represent a new biomarker for mitochondrial dysfunction will be tested. As this is a new pathway, there is a critical gap in understanding the molecular rules and mechanisms by which mtdsRNAs cross the mitochondrial inner and outer membranes for cytosolic export. Our study goals are contained within three independent, but thematically connected, specific aims. In Aim 1, mtdsRNAs that are exported from mitochondria will be characterized with respect to size and sequence specificity. In addition, RNA modifying enzymes will be tested to determine which components are essential for the generation of mtdsRNAs. Aim 2 will focus on identification of outer and inner membrane channels and the role of PNPase in the trafficking of mtdsRNAs out of mitochondria. The third aim will define physiologic parameters that lead to the generation of mtdsRNAs and determination of the cytosolic dsRNA sensors that become activated during this process. Because mutations in PNPase lead to mitochondrial disease, mutants will be characterized to determine whether steps in the degradation and/or export of mtdsRNAs can be separated. Our study team has been characterizing PNPase and its function in mitochondria extensively. Unique model systems available for our work include a mouse model in which floxxed PNPT1 can be removed by the Cre recombinase, and mouse embryonic fibroblasts derived from this model. Results from this proposal will define the pathway for mtdsRNA trafficking out of mitochondria in detail and provide a platform for understanding how mutations in PNPase contribute to disease. Long-term, these studies may lead to establishing mtdsRNA as a new biomarker for mitochondrial dysfunction.