α-proteobacterial RNA degradosomes assemble liquid-liquid phase separated RNP bodies

Al-Husini, Nadra; Tomares, Dylan T.; Childers, W. Seth; Schrader, Jared M.

doi:10.1101/272286

Cited by 54 publications

(176 citation statements)

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“…All in all, along with the few recently discovered cases of bacterial LLPS (Al-Husini et al, 2018;Heinkel et al, 2019;Monterroso et al, 2019), our findings highlight that LLPS is a fundamental, universally exploited mechanism that is governed by similar molecular mechanisms in bacteria and eukaryotes. The LLPS propensity predicted for human SSB homologs hSSB1/SOSB1 and hSSB2/SOSB2 suggests broad evolutionary conservation of this feature (Fig.…”

Section: Discussionsupporting

confidence: 60%

“…Whereas LLPS seems to be a heavily exploited process in eukaryotes and viruses (Heinrich et al, 2018;Nikolic et al, 2017Nikolic et al, , 2018Zhou et al, 2019), up until today only a few bacterial proteins were reported to undergo LLPS. These include Caulobacter crescentus Ribonuclease E (RNase E) that orchestrates RNA degradosomes (Al-Husini et al, 2018), Escherichia coli (E. coli) FtsZ that forms the division ring during cell divisions (Monterroso et al, 2019) and Mycobacterium tuberculosis Rv1747 that is a membrane transporter of cell wall biosynthesis intermediates (Heinkel et al, 2019). All in all, the hitherto accumulated observations imply that LLPS is a fundamental mechanism for the effective spatiotemporal organization of cellular space, with emerging but hardly explored functions in bacteria.…”

Section: Introductionmentioning

confidence: 99%

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Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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Bacterial single stranded (ss) DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL) and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the E. coli SSB protein forms liquid-liquid phase separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA and SSBinteracting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage. RESULTS SSB forms dynamic LLPS condensates in physiologically relevant conditions in vitroDue to the fact that SSB shares several features with hitherto described LLPS drivers, we sought to determine if LLPS could be a yet undiscovered capability of SSB facilitating its multifaceted roles in genome maintenance. We performed a bioinformatics analysis that revealed that E. coli SSB, in particular its IDL region, shows high propensity for LLPS, according to multiple dedicated sequencebased prediction methods (Bolognesi et al., 2016;Lancaster et al., 2014;Vernon et al., 2018) (Fig. 1B-D). This feature of SSB is universally conserved among bacteria, as the majority of analyzed SSBs (72.1 %) harbored by representative bacterial species from 15 large phylogenetic groups show similar LLPS propensities (Tables S1-2; Fig. 1E).In line with its predicted LLPS propensity, purified E. coli SSB (30 µM; SSB concentrations are expressed as those of tetramer molecules throughout the paper) forms an opaque, turbid solution at low NaCl concentration (50 mM) in vitro at room temperature and also at the native temperature of E. coli cells (37°C) ( Fig. 2A-B). The process is reversible as elevation of NaCl concentration to 200 mM leads to immediate loss of turbidity. The Clconcentration in E. coli cells depends on their environment (Schultz et al., 1962); however, while the exact value is not known, it can be assumed to be in a similar range (5-100 mM) as that measured for the cells of mammalian E. coli hosts, whereas the major metabolic anion in bacterial cells is glutamate (Glu; 100 mM) (Bennett et al., 2009). Strikingly, SSB forms a turbid solution even in 200 mM NaGlu ( Fig. 2A-B). (It must be noted that experiments using NaGlu always contained a fixed amount of 20 mM NaCl, for technical reasons.) Investigation of the protein solution by differential interference contrast (DIC) microscopy after diluting SSB into low-salt buffer revealed the formation of regular spherical drop...

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Section: Discussionsupporting

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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“…5 example, SGs contain multiple conserved helicases, including eIF4A/Tif1 and DDX3/Ded1, in both mammals and yeast, respectively (Jain et al, 2016). Similarly, RNA helicases are found localized in bacterial RNP granules as well (Taghbalout and Rothfield, 2008;Al-Husini et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Modulation of RNA condensation by the DEAD-box protein eIF4A

Tauber

Khong

et al. 2019

Preprint

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eTOC Blurb: The RNA helicase eIF4A limits stress granule formation by reducing RNA condensation. Highlights:•! RNA condensates promote intermolecular RNA-RNA interactions at their surfaces! •! eIF4A helicase activity limits the recruitment of RNAs to stress granules in cells! •! eIF4A helicase activity reduces the nucleation of stress granules in cells! •! Recombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner! Keywords: DEAD-box protein, RNA-RNA interactions, stress granule, ribonucleoprotein ! 2 SUMMARY:Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules are proposed to form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is not well understood. In vitro, we demonstrate that the minimization of surface free energy promotes the recruitment and interaction of RNAs on RNA or RNP condensate surfaces. We demonstrate that the ATPase activity of the DEAD-box RNA helicase eIF4A reduces RNA recruitment to RNA condensates in vitro and in cells, as well as limiting stress granule formation. This defines a new function for eIF4A, and potentially other RNA helicases, to limit thermodynamically favored intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function.

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“…Nevertheless, recent work suggests that bacteria may indeed contain biomolecular condensates (26). For example, the DEAD-box helicases DeaD, SrmB and RhlE form foci in E. coli (27) and RNase E forms foci in Caulobacter crescentus and other α-proteobacteria (28). Moreover, the disordered C-terminal domain of RNase E is necessary and sufficient for foci assembly in vivo.…”

Section: Introductionmentioning

confidence: 99%

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components.Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

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α-proteobacterial RNA degradosomes assemble liquid-liquid phase separated RNP bodies

Cited by 54 publications

References 50 publications

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Modulation of RNA condensation by the DEAD-box protein eIF4A

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

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