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 病原体释放病原体相关分子模式(PAMP)，线粒体释放新奇 损伤相关分子模式(DAMP)，包括脂类、多肽和线粒体DNA(MtDNA)； 这表明线粒体处于压力状态。线粒体双链rna(Mtdsrna)是一类新的核糖核酸。 这是当mtRNA中的非编码链没有有效降解并积累时产生的，使得 与编码链进行碱基配对。在正常情况下，解旋酶SUV3解离mtRNAs并 多核苷酸磷酸化酶(PNPase)降解它们。然而，SUV3的击倒会导致 线粒体内mtdsRNAs的积累和PNPase的敲除导致线粒体释放 MtdsRNA进入细胞质。一旦进入细胞质，mtdsRNA就会被dsRNA传感器MDA5和 RIG-I，导致I型干扰素途径的诱导。MtdsRNA的出口可能很重要，因为 在PNPT1、编码PNPase的突变患者的胞浆中发现了mtdsRNAs，并在 疾病包括癌症、心脏病、与酒精相关的肝病和自身免疫性疾病。 MtdsRNAs代表线粒体功能障碍的新生物标记物的假设将得到检验。 由于这是一种新的途径，在理解分子规则和机制方面存在着严重的差距。 MtdsRNAs穿过线粒体内膜和外膜进行胞质输出。我们的研究目标是 包含在三个独立但主题相连的具体目标中。在目标1中，mtdsRNA是 从线粒体输出的序列将在大小和序列特异性方面进行表征。此外, 将对RNA修饰酶进行测试，以确定哪些成分是产生 MtdsRNA。目标2将集中于识别外膜和内膜通道以及PNPase在 线粒体外的线粒体DNA的运输。第三个目标将定义生理参数，以导致 线粒体dsRNA的产生和胞质dsRNA感受器在 这一过程。由于PNPase的突变会导致线粒体疾病，突变的人将被表征为 确定mtdsRNA降解和/或输出的步骤是否可以分开。 我们的研究小组一直在广泛地研究PNPase及其在线粒体中的功能。独一无二 可用于我们的工作的模型系统包括一个鼠标模型，在该模型中，可以通过 Cre重组酶，以及来源于该模型的小鼠胚胎成纤维细胞。这项提案的结果将是 详细定义线粒体外mtdsRNA的运输途径，并为 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.