Mitochondrial-Encoded Regulators of the Nucleus and Cellular Homeostasis

摘要 我们的细胞有两个基因组，每个基因组在细胞核和线粒体中分隔。双基因组细胞 通过早期内共生细菌基因组和原核细菌基因组的共同进化， 我们祖先细胞的基因组。线粒体和线粒体之间长期而密切的关系反映了这一点。 细胞核之间积极沟通，以协调各种细胞功能。这样的mitteles 通信对细胞健康和衰老至关重要，并且越来越多地被认为是高度复杂的， 复杂.然而，尽管> 1，000个核编码蛋白直接调节线粒体，但没有线粒体- 已知编码因子积极调节细胞核。我们最近发表了一流的 调节核基因组的寡核苷酸编码肽（即MOTS-c）。在这里，我们提出了一个未发表的 一种新的与MOTS-c基因连锁的基因，我们将其命名为MOTS-b。特别是，MOTS- 通过免疫共沉淀偶联蛋白质组学确定B和MOTS-c在细胞核中相互作用 (mass光谱法）。与MOTS-c一样，MOTS-b的核转位似乎是作为一种适应性的调节机制而被调节的。 反应例如，MOTS-b和MOTS-c都在时间上动态地易位到细胞核。 单核细胞分化的协调方式。此外，MOTS-b在纯化的核染色质中富集 基于我们的体外进化研究，可以直接结合DNA，以鉴定特异性MOTS-b靶向 核苷酸序列。在功能水平，MOTS-b处理调节细胞增殖和代谢， 这也与MOTS-c一致。 在这里，我们建议的特点和验证MOTS-b的核作用。这个最重要的假设是 提出MOTS-b是一种新的细胞编码基因，它可以易位到细胞核中， 与MOTS-c协同调节适应性基因表达。首先，我们将描述分子和 MOTS-b的细胞机制使用多管齐下的方法，包括诱变，免疫共沉淀， 邻近标记辅助蛋白质组学和DNA结合测定。我们将绘制的功能肽结构域 MOTS-b与DNA结合和肽相互作用有关，我们假设这对它的核 作用我们还将确定触发MOTS-b易位到细胞核的细胞背景/事件。然后， 我们将通过有和没有应激的全局无偏RNA-seq来确定MOTS-b诱导的转录组， 将通过MOTS-b结合的染色质位点（ChIP-seq）的全基因组定位来补充， 染色质（ATAC-seq）。然后我们将筛选出介导MOTS-b对细胞凋亡影响的候选基因。 增殖和代谢。 了解线粒体基因组中编码的调节因子的贡献将提供更多的信息。 全面的基因组视角，增加了生物学意义。如果成功，我们预测我们的研究将 对（i）衰老的基础生物学产生广泛而持久的影响，通过鉴定一个新的由蛋白质编码的家族， 核基因组的调节器，这为我们共同进化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年龄相关疾病中的作用，以及（ii）通过 将mtDNA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 Here, we present an unpublished novel mitochondrial-encoded gene that is genetically linked to MOTS-c, which we named MOTS-b. Notably, MOTS- b and MOTS-c interact with each other in the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.