RNA at the surface of phase-separated condensates impacts their size and number

Although it is now recognized that specific RNAs and protein families are critical for the biogenesis of ribonucleoprotein (RNP) condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells programmed to specifically recruit a single RNA species. Our bioengineered condensates are made of ArtiGranule scaffolds composed of an orthogonal protein that can bind to a specific heterologously expressed RNA. These scaffolds undergo liquid-liquid phase separation in cells and can be chemically controlled to prevent condensation or to trigger condensate dissolution. We found that the targeted RNAs localize at the condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. The recruitment of RNA changes the material properties of condensates by hardening the condensate body. Moreover, the condensate size scales with RNA surface density; the higher the RNA density is, the smaller and more frequent the condensates are. These results suggest a mechanism based on physical constraints, provided by RNAs at the condensate surface, that limit condensate growth and coalescence.