RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome

Abstract

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein-protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein-protein and RNA-RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA-RNA assembly in cells could lead to disease.

RNAs, Phase Separation, and Membrane‐Less Organelles: Are Post‐Transcriptional Modifications Modulating Organelle Dynamics?

“…Of note, while most studies addressed the role of particular phase‐separating proteins in both LLPS and BioMC formation, only very recently did RNA itself move into focus of phase transition research. Indeed, latest insights revealed profound roles for RNA in the regulation of LLPS processes …”

“…In contrast to transcription‐dependent seeding of particular nuclear BioMCs, the formation of cytoplasmic BioMCs is probably and primarily governed by the availability of existing RNPs, proteins, or RNAs serving structural functions. However, regardless of whether RNA itself scaffolds the formation of BioMCs or becomes recruited into an existing proteinaceous network, RNA content impacted and even regulated the material properties of assembled BioMCs both in vitro and in vivo . More specifically, RNA facilitated LLPS of particular IDR‐containing proteins by reducing the protein concentration needed for their phase‐separation .…”

“…Of note, particular repeat‐containing RNAs form structures that have been connected to the formation of BioMCs . For instance, biochemically purified SG cores were enriched in antisense RNAs capable of forming intramolecular duplexes, and these SG cores were resistant to high salt or aliphatic alcohol treatment, which disrupt protein‐protein but not RNA‐RNA interactions .…”

“…Of note, particular repeat‐containing RNAs form structures that have been connected to the formation of BioMCs . For instance, biochemically purified SG cores were enriched in antisense RNAs capable of forming intramolecular duplexes, and these SG cores were resistant to high salt or aliphatic alcohol treatment, which disrupt protein‐protein but not RNA‐RNA interactions . Interestingly, some products of stress‐induced tRNA metabolism, tsRNAs, can form homo‐ and heterodimers but also particular structures such as tetramers through G‐quads, which are important for SG formation.…”

mRNA structure determines specificity of a polyQ-driven phase separation

“…4F). This mechanism is likely relevant for the sorting of specific RNAs to other RNA-granules such as stress and P granules, and P-bodies (17, 18). Future work will address the timing and location of how mRNA secondary structure influences selective uptake of cellular constituents into droplets.…”