A mechanism for MEX-5-driven disassembly of PGL-3/RNA condensates in vitro.

MEX-5 regulates the formation and dissolution of P granules in embryos, yet the thermodynamic basis of its activity remains unclear. Here, using a time-resolved in vitro reconstitution system, we show that MEX-5 dissolves preassembled liquid-like PGL-3/RNA condensates by altering RNA availability and shifting the phase boundary. We develop a microfluidic assay to systematically analyze how MEX-5 influences phase separation. By measuring the contribution of PGL-3 to phase separation, we show that MEX-5 reduces the free energy of PGL-3, shifting the equilibrium toward dissolution. Our findings provide a quantitative framework for understanding how RNA-binding proteins modulate condensate stability and demonstrate the power of microfluidics in precisely mapping phase transitions.