Mitochondrial-Encoded Regulators of the Nucleus and Cellular Homeostasis

摘要 我们的细胞有两个基因组，每个基因组都划分在细胞核和线粒体中。双基因组细胞 通过早期内共生细菌基因组和原核的共同进化建立了系统 我们祖先细胞超过十亿年的基因组。反映了它们长期而密切的关系，线粒体和 细胞核之间积极沟通，协调各种细胞功能。这样的有丝分裂核 沟通对细胞健康和衰老至关重要，而且越来越被认为是高度复杂和 很复杂。然而，尽管&gt；1000核编码蛋白直接调节线粒体，但没有线粒体- 已知的编码因子可以主动调节细胞核。我们最近发表了一流的 线粒体编码的多肽(即MOTS-c)，调节核基因组。在这里，我们呈现一份未发表的 新的线粒体编码基因，与mots-c基因连锁，我们将其命名为mots-b。值得注意的是，MOTS- 免疫共沉淀偶联蛋白质组学研究表明，b和mots-c在细胞核内相互作用。 (质谱学)。与mots-c一样，mots-b的核转位似乎是作为一种适应性调节。 回应。例如，MOTS-b和MOTS-c都在时间上动态地移位到原子核 单核细胞分化的协调方式。此外，MOTS-b还富含纯化的核染色质。 根据我们的体外进化研究，可以直接与DNA结合以识别特定的MOTS-b靶向 核苷酸序列。在功能水平上，MOTS-B治疗调节细胞的增殖和代谢， 这再次与MOTS-C一致。 在这里，我们建议表征和验证MOTS-b的核心作用。其中最重要的假设是 有观点认为，mots-b是一种新的线粒体编码基因，它易位到细胞核，并直接 与mots-c协同调节适应性基因的表达。首先，我们将描述分子和 多管齐下的MOTS-b的细胞机制，包括诱变，免疫共沉淀， 邻近标记辅助蛋白质组学和DNA结合分析。我们将绘制其功能多肽结构域 MOTS-b与DNA结合和多肽相互作用有关，我们假设这对其核很重要 角色。我们还将确定触发MOTS-b移位到细胞核的细胞上下文/事件。然后, 我们将通过全球无偏见的rna-seq来确定mots-b诱导的转录组，包括应激和非应激。 将通过基因组范围内开放的mots-b结合染色质位点的图谱(芯片-序列)来补充 染色质(ATAC-seq)。然后我们将筛选介导mots-b对细胞的影响的候选基因。 增殖和新陈代谢。 了解线粒体基因组中编码的调节子的作用将提供更多 具有更多生物学意义的全面基因组学观点。如果成功，我们预测我们的研究将 通过识别一个新的线粒体编码家族，对(I)衰老的基础生物学产生广泛和持久的影响 核基因组的调节者，这为我们共同进化中的基因调控提供了另一层复杂性 双基因组细胞系统及其在衰老和老年相关疾病中的作用，以及(Ii)通过 将线粒体DNA描述为新的治疗/诊断靶点的来源。

ABSTRACT Our cells have two genomes, each compartmentalized in the nucleus and mitochondria. The bi-genomic cellular system was established through co-evolution of the early endosymbiotic bacterial genome and the proto-nuclear genome of our ancestral cell over a billion years. Reflecting their long and close relationship, mitochondria and the nucleus actively communicate with each other to coordinate various cellular functions. Such mitonuclear communication is vital to cellular fitness and aging, and increasingly appreciated to be highly sophisticated and complex. However, whereas >1,000 nuclear-encoded proteins directly regulate the mitochondria, no mitochondrial- encoded factors have been known to actively regulate the nucleus. We recently published the first-in-class mitochondrial-encoded peptide (i.e. MOTS-c) that regulates the nuclear genome. Here, we present an unpublished novel mitochondrial-encoded gene that is genetically linked to MOTS-c, which we named MOTS-b. Notably, MOTS- b and MOTS-c interact with each other in the nucleus, determined by co-immunoprecipitation-coupled proteomics (mass spectrometry). Like MOTS-c, the nuclear translocation of MOTS-b appears to be regulated as an adaptive response. For instance, MOTS-b and MOTS-c both dynamically translocate to the nucleus in a temporally coordinated manner upon monocyte differentiation. Further, MOTS-b is enriched in purified nuclear chromatin samples and can directly bind DNA based on our in vitro evolution studies to identify specificMOTS-b-targeted nucleotide sequences. At the functional level, MOTS-b treatment regulates cellular proliferation and metabolism, which again is consistent with MOTS-c. Here, we propose to characterize and validate the nuclear role of MOTS-b. The overarching hypothesis of this proposal is that MOTS-b is a novel mitochondrial-encoded gene that translocates to the nucleus and directly regulates adaptive gene expression in coordination with MOTS-c. First, we will characterize the molecular and cellular mechanisms of MOTS-b using a multipronged approach including mutagenesis, co-immunoprecipitation, proximity-labeling assisted proteomics, and DNA-binding assays. We will map the functional peptide domains of MOTS-b pertinent to DNA binding and peptide interaction, which we hypothesize to be important for its nuclear role. We will also determine the cellular context/event that triggers MOTS-b to translocate to the nucleus. Then, we will determine the MOTS-b-induced transcriptome by global unbiased RNA-seq with and without stress, which will be complemented by genome-wide mapping of MOTS-b-bound chromatin sites (ChIP-seq) that are within open chromatin (ATAC-seq). We will then screen candidate genes that mediate the effects of MOTS-b on cellular proliferation and metabolism. Understanding the contributions of regulators encoded in the mitochondrial genome will provide a more comprehensive genomic perspective with added biological significance. If successful, we predict that our study will have broad and lasting impact on (i) basic biology of aging by identifying a novel family of mitochondrial-encoded regulators of the nuclear genome, which provides another layer of complexity to gene regulation in our co-evolved bi-genomic cellular system and their role in aging and age-related diseases, and (ii) translational development by describing the mtDNA as a source of novel therapeutic/diagnostic targets.