DEAD-box ATPases as master regulators of phase-separated compartments to control cellular RNA flux and the remodeling of RNA-protein complexes

Life ultimately depends on the tight control of gene expression, which requires an ordered and efficient processing of various RNA molecules.

Messenger RNAs (mRNAs) bound by a constantly changing coat of passenger proteins - transit from transcription in the nucleus to translation and ultimately decay in the cytoplasm.

Similarly, ribosomal rRNAs migrate through the nucleolus where they gradually en-counter ribosomal proteins to assemble functional ribosomes.

Still, we know very little about the pro-cesses that orchestrate this flux of RNA in a temporal and spatial manner.Intriguingly, many RNA processing steps occur in membraneless organelles formed by liquid-liquid phase separation, e.g. nuclear speckles or the nucleolus, but the function of condensate formation in RNA processing is not known.

I have discovered that the family of DEAD-box ATPases (DDXs) are master regulators of RNA-containing membraneless organelles, from bacteria to man.

DDXs use their low-complexity domains and ATPase activity to regulate condensate dynamics and RNA flux through these compartments.I propose that cells use DDX-controlled condensate stations to establish an RNA transit map to reg-ulate the cellular flux of mRNA and rRNA molecules and to spatially and temporally control RNA pro-cessing.

In three work packages, I will (1) characterize central DDXs that control mRNA flux and use DDX mutants as unique tools to map passenger protein changes along the life of an mRNA; (2) charac-terize how DDXs regulate the formation of the phase-separated nucleolar environment and facilitate the flux of rRNA during ribosome assembly; (3) dissect how DDX condensates function as biomolecular filters to selectively enrich or exclude proteins, and how selectivity contributes to the remodeling of the RNA protein coat and directional RNA flux.Our research will provide key novel insight into our understanding of RNA processing and uncover novel layers of gene expression regulation.