Abstract

Previously, we reported the discovery of mesoscale membraneless droplets in bacteria called aggresomes which form through liquid-liquid phase separation (LLPS) from a plethora of key proteins implicated in cell resuscitation following stress [1, 2]. Here, I will discuss new findings which integrate time-resolved single-molecule live-cell microscopy with new biophysical modelling, that highlight the crucial role aggresomes play as an analogue of eukaryotic stress granules (SGs) in safeguarding mRNA during stress. We find that upon stress onset, mobile mRNA molecules selectively incorporate into individual proteinaceous SGs based on length-dependent enthalpic gain over entropic loss. As stress prolongs, SGs undergo compaction facilitated by stronger non-specific RNA -protein interactions, thereby promoting recruitment of shorter RNA chains. Remarkably, mRNA ribonucleases are repelled from bacterial SGs, potentially due to the influence of protein surface charge. This exclusion mechanism ensures the integrity and preservation of mRNA within SGs during stress conditions, providing valuable insights into the intricate processes involved in mRNA storage within membraneless droplets. Following stress removal, SGs facilitate mRNA translation, thereby enhancing cell fitness in changing environments. These droplets maintain mRNA physiological activity during storage, making them potentially valuable for mRNA therapeutics manufacturing. 1            Pu, Y. et al. ATP-Dependent Dynamic Protein Aggregation Regulates Bacterial Dormancy Depth Critical for Antibiotic Tolerance. Mol Cell 73, 143-156 e144 (2019). https://doi.org:10.1016/j.molcel.2018.10.022 2            Jin, X. et al. Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness. Sci Adv 7, eabh2929 (2021). https://doi.org:10.1126/sciadv.abh2929