Living cells contain many membrane-less organelles that are assembled based on liquid-liquid phase separation. Different proteins and RNAs can be recruited into these cellular granules to facilitate spatiotemporal regulation. However, the effect of these granule formations on the cellular functions of specific RNAs or proteins is still largely unknown. Herein, we introduce a genetically encoded RNA tag that could be used to recruit almost “any” RNA of interest into membrane-less granules both <i>in vitro</i> and in living bacterial cells. In our system, different lengths of CAG triple-ribonucleotide repeats were used as a scaffold to form multivalent interactions and drive granule formation. Our results indicated that after conjugating with these CAG repeat tags, target RNAs (including small noncoding RNAs and mRNAs) with lengths even &gt;3,000 nucleotides can be recruited into granules in the absence of any proteins. With the help of a fluorogenic RNA aptamer Broccoli, we have further systematically studied the dependence of the granule size and density on the RNA and magnesium ion concentration and the length of CAG repeats and target RNA. The kinetics and cellular distributions of granule formation have also been monitored in live <i>E. coli</i> cells. We believe these modular and genetically encodable CAG-repeat tags can be widely used to study the regulation function of granules on the gene expression, RNA-RNA and RNA-protein interactions.

Triplet repeats mediated RNA phase transitions in live cells

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ACS Fall 2022

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