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 genera...