RNA sequence and structure control assembly and function of RNA condensates

Abstract

Intracellular condensates formed through liquid–liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+ concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.

RNAs undergo phase transitions with lower critical solution temperatures

“…7c). Based on our results, we propose a model that accounts for the formation of previously reported RNA condensates through annealing in vitro that employs a single heating-cooling ramp 23,25,31,32,36 . These condensates remain spherical after their preparation but behave as static aggregates without the characteristic dynamicity of phase-separated liquids.…”

“…RNA condensation in vitro , defined either as the compaction of single RNA molecules or phase separation at higher concentrations, depends on polyvalent cations (e.g., spermine, poly-lysine, cobalt (III) hexamine, Mg 2+ ions), crowding agents (e.g., polyethylene glycol), and/or RNA denaturation 22, 23, 25, 31, 32, 33 . Notably, in the presence of divalent cations such as Mg 2+ , GC-rich RNAs with trinucleotide repeats undergo a sol-gel transition, i.e ., percolation in vitro .…”

Concentrating and sequestering biomolecules in condensates: impact on plant biology

“…Yet, it is unclear what the conformation of proteins (and RNAs) that enter a condensed state is. In condensates, proteins may either get protected from degradation or get degraded more; this has also been suggested for RNA molecules (Poudyal et al, 2021). A less folded state may allow stickers to explore their surrounding space and find favourable partner stickers, thereby mediating condensation propensity.…”

The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability

“…The 25 mM free Mg 2+ condition exhibited higher degradation rates than the other conditions in the J2/3 junction (Figure 3C). This pattern was similar to ILP data published for another guanine riboswitch at a higher pH and a Mg 2+ concentration of 15 mM, 50 supporting that the increase in degradation rates in the 25 mM free Mg 2+ condition was dependent on the presence of Mg 2+ −OH − complexes (Figure S6).…”