Cochard, A. | Search bioRxiv

bioRxiv2022

Condensate functionalization with motors directs their nucleation in space and allows manipulating RNA localization

The localization of RNAs in cells is critical for many cellular processes. Whereas motor-driven transport of RNP condensates plays a prominent role in RNA localization in cells, their studies remain limited by the scarcity of available tools allowing to manipulate condensates in a spatial manner. To fill this gap, we reconstitute in cellula a minimal RNP transport system based on bioengineered condensates which were functionalized with kinesins and dynein-like motors, allowing for their positioning at either the cell periphery or centrosomes. This targeting mostly occurs through the active transport of the condensate scaffolds, which leads to localized nucleation of phase-separated condensates. Then, programming the condensates to recruit specific mRNAs is able to shift the localization of these mRNAs towards the cell periphery or the centrosomes. Our method opens novel perspectives to examine the role of RNA localization as a driver of cellular functions.

cell biology↗

bioRxiv2021

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

Membrane-less organelles, by localizing and regulating complex biochemical reactions, are ubiquitous functional subunits of intracellular organization. They include a variety of nuclear and cytoplasmic ribonucleoprotein (RNP) condensates, such as nucleoli, P-bodies, germ granules and stress granules. While is it now recognized that specific RNA and protein families are critical for the biogenesis of 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 with controlled RNA and protein composition. Our bioengineered condensates are made of ArtiGranule scaffolds undergoing liquid-liquid phase separation in cells and programmed to specifically recruit a unique RNA species. We found that RNAs localized on condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. This simplified system allowed us to demonstrate that the size of the condensates scales with RNA surface density, the higher the RNA density is, the smaller and more frequent the condensates are. Our observations suggest a mechanism based on physical constraints, provided by RNAs localized on condensate surface, that limit condensate growth and coalescence.

cell biology↗