The Nucleolar Detention Center: A Hub of Long Noncoding RNA that Imprison Proteins during Stress

项目摘要 细胞适应各种应激条件的能力在各种生理和病理过程中起着关键作用。 病理环境，包括发育、癌症和神经系统疾病。我们的小组报告说， 胁迫诱导低复杂性二核苷酸重复序列非编码RNA的鉴定 核糖体基因间间隔区（rIGSRNA）的基因座;历史上人类基因组的一个神秘区域 被认为是"垃圾" DNA我们发现，低复杂性的rIGSRNA启动生理性淀粉样蛋白生成， 将核仁转化为淀粉样体的程序：可逆的无纤维膜细胞器，由 具有淀粉样特征的固定化蛋白质。虽然已经描述了许多无膜隔室， 作为液体状（例如，应激颗粒、P体、生殖细胞颗粒），淀粉样体的发现提供了 淀粉样蛋白生成过程的证据，该过程可以在生理上将生物物质转变为固体样状态。 这种相当不寻常的翻译后调节途径使一系列蛋白质的快速和可逆储存成为可能。 淀粉样体中的内源性蛋白抑制细胞对严重环境的反应 侮辱。我们建议将2015年资助并于2018年续签的NIGMS R01赠款转换为MIRA， 统一主题“应激过程中低复杂性rIGSRNA的功能”。我们的研究计划包括深入 研究以了解（i）rIGSRNA激活生理性淀粉样蛋白生成以构建 淀粉样体和（ii）功能的rIGSRNA和淀粉样体在压力。本文概述的研究 一项提案将涉及分离的淀粉样体，活性翻译位点的多色单分子成像 在应激、蛋白质动力学、非模板RNA的长读序、rRNA生物学和体外原纤化期间 分析，以了解低复杂性RNA的细胞和生物化学功能的细胞从事厌氧 代谢，以及其他条件。这项由NIGMS资助的研究使我们的实验室能够 我们对基因组中简单二核苷酸低复杂性重复序列的理解的概念性进展。第一、 rIGRSNA构建淀粉样体发现提供了细胞可以激活生理活性的证据 液-固相转变以组装具有淀粉样性质的缩合物。这些特点 将淀粉样体与哺乳动物细胞中大量的液体凝聚物区分开来， 通常不显示淀粉样蛋白生成特征。我们提出的工作不仅将阐明适应性 同时也为病理性淀粉样变性的研究提供了新的思路 与一系列人类神经系统疾病有关第二，低复杂性RNA序列 是rIGSRNA的功能决定子，可能会刺激对长二核苷酸生理作用的研究。 在整个基因组中观察到的基因间重复序列，但通常被认为是无用的DNA/RNA。所以这 该项目将普遍感兴趣的科学家感兴趣的细胞反应的压力，长非编码RNA 生物学、核/胞质结构、翻译和生理/病理淀粉样蛋白生成。

Project Summary The ability of cells to adapt to a wide variety of stress conditions plays a critical role in various physiological and pathological settings, including development, cancer and neurological disorders. Our group reported the identification of stress-induced low complexity dinucleotide repeat noncoding RNA derived from stimuli-specific loci of the ribosomal intergenic spacer (rIGSRNA); an enigmatic region of the human genome historically dismissed as “junk” DNA. We showed that low complexity rIGSRNA initiate physiological amyloidogenic programs that convert nucleoli into Amyloid-bodies: reversible fibrous membrane-less organelles composed of immobilized proteins with amyloid-like features. While many membrane-less compartments have been described as liquid-like (e.g., stress granules, P-bodies, germ cell granules), the discovery of Amyloid-bodies provided evidence of an amyloidogenic process that can physiologically transition biological matter to a solid-like state. This rather unusual post-translational regulatory pathway enables the rapid and reversible storage of an array of endogenous proteins in Amyloid-bodies to suppress metabolism in cells responding to severe environmental insults. We propose to convert our NIGMS R01 grant funded in 2015 and renewed in 2018 to a MIRA under the unifying theme “Function of low complexity rIGSRNA during stress”. Our research program includes in-depth studies to understand (i) the mechanisms by which rIGSRNA activate physiological amyloidogenesis to construct Amyloid-bodies and (ii) the function of rIGSRNA and Amyloid-bodies during stress. Studies outlined in this proposal will involve isolated Amyloid-bodies, multi-color single molecule imaging of active translation sites during stress, protein dynamics, long read sequencing of untemplated RNA, rRNA biology, and in vitro fibrillation assays to understand the cellular and biochemical functions of low complexity RNA in cells engaging in anaerobic metabolism, amongst other conditions. This NIGMS-funded research has enabled our laboratory to make conceptual advances in our understanding of simple dinucleotide low complexity repeats in the genome. First, the discovery that rIGRSNA construct Amyloid-bodies provided evidence that cells can activate physiological liquid-to-solid phase transitions to assemble condensates with amyloid-like properties. These characteristics distinguish Amyloid-bodies from the multitude of liquid condensates that populate mammalian cells, which typically do not display amyloidogenic features. Our proposed work will not only shed light on adaptive mechanisms to stressors but also provide alternative insights for the study of pathological amyloidogenesis involved in an array of human neurological disorders. Second, the finding that low complexity RNA sequences are functional determinants of rIGSRNA may stimulate research on the physiological role of long dinucleotide intergenic repeats observed across the genome, but generally dismissed as useless DNA/RNA. Hence, this project will be of general interest to scientists interested in cellular response to stressors, long noncoding RNA biology, nuclear/cytoplasmic structures, translation and physiological/pathological amyloidogenesis.