RNA phase transitions are entropically driven by phosphate backbones and modulated by nucleotide sequences

The biogenesis of ribonucleoprotein granules is thought to be driven by the reversible phase transitions of mixtures of RNA and proteins. Recently, GC-rich RNAs have been observed to form protein-independent foci in cells, which was conceptualized on the basis of an enthalpic model where base pairing and stacking interactions were postulated as drivers of RNA phase separation. Directly observing these transitions using temperature-controlled microscopy, we surprisingly discover that entropy rather than enthalpy drives RNA phase separation with a variety of RNA displaying system-specific Lower Critical Solution Temperatures (LCSTs) in the presence of Mg²⁺. This mode of phase separation cannot be ascribed to base-pairing and base-stacking, which are disfavored as temperature increases. Rather, desolvation entropy of the phosphate backbone as well as Mg²⁺ ion-mediated bridging interactions are the primary drivers of RNA phase separation. Further, we show that nucleobase composition tunes the intrinsic LCST by phosphate backbone as well as leads to a secondary percolation transition in the dense phase. Overall, our results suggest a framework to understand the molecular origin of RNA phase separation, opening new pathways to understand cellular responses to temperature variation.