DEAD-box ATPases are global regulators of phase-separated organelles

Abstract

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules^1,2. How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs)³ are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles.

Extended Data Fig. 1. Phase separation behavior of full-length and tail-less (core) Dhh1 in different pH conditions.

(a) Example images for the Dhh1 pH phase diagram. Reactions were assembled in 384 well plates. Each reaction contained 13.4 μl LSB-100, 2.1 μl 0.5 M KCl (final KCl concentration 100 mM), 1 μl CKM mix, 1.25 μl 100 mM ATP / MgCl₂ (final concentration 5 mM), 1 μl 10 mg/ml BSA, 2 μl 1 M Hepes of the respective pH, 1.25 μl 1 mg/ml polyU and 3 μl MH200G containing Dhh1 to achieve the final concentration as indicated. Reactions were incubated at room temperature for 20 minutes and imaged at room temperature on a Nikon widefield microscope using an automated script (4 images per well of one replicate). Dhh1 core = Dhh1 (residues 48 - 425), lacking the low complexity tails. Scale bar 50 μm. (b-c) For each well, individual droplets in each image were quantified using Diatrack (see MM) for their area and mean intensity. The sum of the product [area * mean fluorescence intensity] (arbitrary unit: [A*I]) of all droplets in one image was plotted against the Dhh1 concentration (μM). (Dotted) lines represent the mean, shaded area the SD of the four images recorded per well for one replicate. (b) FL = full-length Dhh1, (c) Dhh1 core = Dhh1 [48 - 425]. (d) Mean values of the [area * mean fluorescence intensity] (arbitrary unit: [A*I]) sum of the four images recorded per well are plotted against the Dhh1 concentration for all pH tested. Dhh1 core protein concentrations not tested are marked by a cross. FL = full-length Dhh1, Dhh1 core = Dhh1 [48 - 425].

Extended Data Fig. 2. Phase separation behavior of full-length and tail-less (core) Dhh1 in different salt concentrations.

(a) Example images for the Dhh1 salt phase diagram at pH 6.4 and 7.0. Reactions were assembled in 384 well plates. Each reaction contained 12.5 μl LSB-100, 3 μl 0.5 M KCl / water to achieve the final KCl concentration as indicated, 1 μl CKM mix, 1.25 μl 100 mM ATP / MgCl2 (final concentration 5 mM), 1 μl 10 mg/ml BSA, 2 μl 1 M Hepes of the respective pH, 1.25 μl 1 mg/ml polyU and 3 μl MH200G containing Dhh1 to achieve the final concentration as indicated. Reactions were incubated at room temperature for 20 minutes and imaged at room temperature on a Nikon widefield microscope using an automated script (9 images per well of one replicate). Dhh1 core = Dhh1 (residues 48 - 425), lacking the low complexity tails. Scale bar 50 μm. (b-c) For each well, individual droplets in each image were quantified using Diatrack (see MM) for their area and mean intensity. The sum of the product [area * mean fluorescence intensity] (arbitrary unit: [A*I]) of all droplets in one image was plotted against the Dhh1 concentration (μM). (Dotted) lines represent the mean, shaded area the SD of of the nine images recorded per well for one replicate. (b) FL = full-length Dhh1, (c) Dhh1 core = Dhh1 [48 - 425]. (d) Mean values of the [area * mean fluorescence intensity] (arbitrary unit: [A*I]) sum of the nine images recorded per well are plotted against the Dhh1 concentration for all conditions tested. Dhh1 core protein concentrations not tested are marked by a cross. FL = full-length Dhh1, Dhh1 core = Dhh1 [48 - 425].

Extended Data Fig. 3. Phase separation behavior of full-length and tail-less (core) Dhh1 in different ATP concentrations.

(a) Example images for the Dhh1 ATP phase diagram at pH 6.4 and pH 7.0. Reactions were assembled in 384 well plates. Each reaction contained 13.4 μl LSB-100, 2.1 μl 0.5 M KCl (final KCl concentration 100 mM), 1 μl CKM mix, 1.5 μl ATP / MgCl₂ (250 mM stock) and H₂O to achieve the final concentration as indicated, 1 μl 10 mg/ml BSA, 2 μl 1 M Hepes of the respective pH, 1.25 μl 1 mg/ml polyU and 3 μl MH200G containing Dhh1 to achieve the final concentration as indicated. Reactions were incubated at room temperature for 20 minutes and imaged at room temperature on a Nikon widefield microscope using an automated script (9 images per well of one replicate). Dhh1 core = Dhh1 (residues 48 - 425), lacking the low complexity tails. Scale bar 50 μm. (b-e) For each well, individual droplets in each image were quantified using Diatrack (see MM) for their area and mean intensity. The sum of the product [area * mean fluorescence intensity] (arbitrary unit: [A*I]) of all droplets in one image was plotted against the Dhh1 concentration (μM). (Dotted) lines represent the mean, shaded area the SD of the nine images recorded per well for one replicate. (b,c) FL = full-length Dhh1, (d,e) Dhh1 core = Dhh1 [48 - 425]. (b,d) pH 6.4, (c,e) pH 7.0. (f) Mean values of the [area * mean fluorescence intensity] (arbitrary unit: [A*I]) sum of the nine images recorded per well are plotted against the Dhh1 concentration for all conditions tested. Dhh1 core protein concentrations not tested are marked by a cross. FL = full-length Dhh1, Dhh1 core = Dhh1 [48 - 425].

Extended Data Fig. 4. Phase separation behavior of full-length and tail-less (core) Dhh1 in different polyU concentrations.

Example images for the Dhh1 polyU phase diagram at pH 6.4 and 7.0. Reactions were assembled in 384 well plates. Each reaction contained 13.4 μl LSB-100, 2.1 μl 0.5 M KCl (final KCl concentration 100 mM), 1 μl CKM mix, 1.25 μl 100 mM ATP/MgCl2 (final concentration 5 mM), 1 μl 10 mg/ml BSA, 2 μl 1 M Hepes of the respective pH, 1.25 μl water / 10 mg/ml polyU to achieve the final concentration as indicated, and 3 μl MH200G containing Dhh1 to achieve the final concentration as indicated. Reactions were incubated at room temperature for 20 minutes and imaged at room temperature on a Nikon widefield microscope using an automated script (9 images per well of one replicate). Dhh1 core = Dhh1 (residues 48 - 425), lacking the low complexity tails. Scale bar 50 μm. (b-c) For each well, individual droplets in each image were quantified using Diatrack (see MM) for their area and mean intensity. The sum of the product [area * mean fluorescence intensity] (arbitrary unit: [A*I]) of all droplets in one image was plotted against the Dhh1 concentration (μM). (Dotted) lines represent the mean, shaded area the SD of the nine images recorded per well for one replicate. (b) FL = full-length Dhh1, (c) Dhh1 core = Dhh1 [48 - 425]. (d) Mean values of the [area * mean fluorescence intensity] (arbitrary unit: [A*I]) sum of the nine images recorded per well are plotted against the Dhh1 concentration for all conditions tested. Dhh1 core protein concentrations not tested are marked by a cross. FL = full-length Dhh1, Dhh1 core = Dhh1 [48 - 425].

Extended Data Fig. 5. The unstructured LC-tails of Dhh1 are required for multivalent interactions underlying LLPS and PB formation.

(a) Larger field of view of the samples represented in Fig 1C. Scale bars 5 μm. Dcp2, the catalytic subunit of the Dcp1-Dcp2 mRNA decapping complex, is used as a marker of PBs. Representative images of > 4 independent experiments. (b) ClustalOmega sequence alignment of yeast DDXs and their human counterparts. The RecA core is displayed in green; asterisks indicate sequence identity, dots represent sequence similarity. (c) schematic representation of LC sequence distribution in the unstructured tails of A. thaliana DDXs. Clustal Omega alignment of Dhh1 with its three A. thaliana orthologues.

Extended Data Fig. 6. Three of the five E. coli DEAD-box ATPases harbor low complexity sequences, undergo phase separation in vitro and form foci in vivo.

(a) Clustal Omega alignment of the five E. coli DEAD-box ATPases. The RecA core is displayed in green; asterisks indicate sequence identity, dots represent sequence similarity. (b) In vitro phase separation of E.coli SrmB-mCherry (6 µM) and DbpA-mCherry (6 µM) in the presence of ATP and RNA; scale bar 25 μM. Representative images of 3 independent experiments. (c) Individual imaging channels of the composite images presented in Fig 2e; representative images of >3 independent experiments. DDX-mCherry, GFP and a composite image for E. coli DeaD, SrmB, RhlE, and for RhlB as a negative control. Scale bars 2 μM. (d) Larger field of view of E. coli DDX-mCherry expression samples. Scale bar 15 µm. Representative images of 3 independent experiments. (e) Quantification of foci in E. coli samples for 4 images per construct. Cells were segmented and for each individual cell, the mean fluorescence intensity and number of foci was quantified. Cells with 0 or 1 foci were grouped for technical reasons (see Material and Methods). Cells were binned based on mean fluorescence intensity, representing their expression level, and the three highest bins excluded from further analysis since they contain cells where fluorescence intensity has reached saturation. The percentage of cells containing 0 / 1, 2, 3 or more than 3 foci are plotted for each bin. There is no correlation between expression levels and focus formation in the various strains.

Extended Data Fig. 7. The catalytically deficient mutant Ded1^DQAD forms constitutive stress granules, without being compromised for RNA or ATP binding.

(A) Ded1^DQAD-mCherry, but not wild-type Ded1-mCherry, forms SGs (marked with arrows) in unstressed cells. Representative images of >3 independent experiments. (B) Fluorescence polarization analysis to measure binding of MANT-ATP to either wild-type Ded1 or Ded1^DQAD. n = 3 technical replicates, mean and SD; nonlinear fit (on site binding curve) calculated using Prism (Graphpad). (C) Fluorescence polarization analysis to measure binding of a fluorescein-UTP labeled, 100bp-long RNA to wild-type either wild-type Ded1 or Ded1^DQAD. n = 3 technical replicates, mean and SD; nonlinear fit (on site binding curve) calculated using Prism (Graphpad).

Extended Data Fig. 8. The ATPase activity of Ded1 controls disassembly of stress granules.

Larger field of view, more time points and DMSO-treated control samples of the experiment presented in Fig 1B. Scale bars 5 μm. Representative images of 3 biological replicates.

Extended Data Fig. 9. DDX ATPase activity controls turnover of nuclear compartments in human and yeast.

(A, B) Depletion of the DDX ATPase UAP56 leads to an increase in nuclear speckle size. This is consistent with the model that UAP56, which does not contain LCDs and is not an essential ‘building block’ for nuclear speckles, is required for RNA turnover in speckles and its absence would thus lead to an increased residence time of RNA in the compartment and a subsequent increase in the size of preexisting compartments. (A) A549 cells were transfected with control siRNA or UAP56 siRNA. After 48 h, cells were infected with influenza virus (WSN) at a MOI of 10 for 6 h. Cells were subjected to smRNA-FISH to label viral M mRNA and immunofluorescence to stain nuclear speckles with SON antibody. ‘Viral M RNA’ is an influenza virus transcript that has been described to traffic through nuclear speckles and is used as a model to represent poly-adenylated, spliced cellular transcripts. Insets: enlargements of the marked white squares showing nuclear speckles. Scale bar 10 μm. Data are representative of three independent experiments. (B) The percentage of M mRNA at nuclear speckles was plotted against the nuclear speckle volume (423 nuclear speckles for each condition - control and UAP56 siRNA). (C) Stably transfected A549 cells expressing wild-type (WT) or UAP56 mutant (E197A) were treated with siRNA targeting 3’-UTR to knockdown endogenous UAP56. Cells were subjected to immunofluorescence microscopy with SON antibody. Scale bar 10 μm. Data are representative of three independent experiments. (D, E) Selection and characterization of stably transfected A549 cells expressing WT or E197A UAP56. For gel source data, see Supplementary Figure 1. Data are representative of three independent experiments. (D) Several cell clones stably expressing WT or E197A UAP56 were tested by western blot using anti-Flag antibody. Clone 2 of WT and clone 3 of E197A were selected for further studies. (E) Immunofluorescence with anti-Flag antibody shows similar expression levels of exogenous UAP56 or UAP56 (E197A) in the selected stable cell lines. Scale bar, 10 μm. (F) Merged images with nuclear rim staining for experiments in Fig 3F. Representative images of 5 (Sub2 degron) or 6 (Sub2) biological replicates. (G) Cells were treated as in Figure 4G. At time point t=60 min after reporter RNA induction cells were treated with either water or 5% 1.6 hexanediol for 20 min. Representative images of 3 biological replicates. (H) Quantification of percentage of cells displaying either distinct nuclear RNA foci (transcription foci, TF; up to 2 to account for mitotic cells) or diffuse nuclear RNA signal in Sub2-depleted cells. Representative images of 5 biological replicates with n>380 cells per replicate. Unpaired t-test (two-tailed) with *** p=0.0009 and **** p<0.0001. Mean and SEM, dots represent mean of individual replicates.

Extended Data Fig. 10. DDX ATPase activity regulates transfer of RNA molecules between phase-separated compartments in vivo and in vitro.

(A, B) In vivo, SG assembly upon treatment with 0.5% sodium azide was monitored by Ded1-yEGFP in cells expressing untagged Dhh1 WT or Dhh1^DQAD as the sole copy, and in a dhh1Δ background. Quantification of SGs per cell was performed using Diatrack. 4 biological replicates, at least 855 (WT), 755 (dhh1Δ) or 106 (Dhh1^DQAD) cells per replicate. Ded1 and Pab1 (polyA-binding protein) are bona fide markers of stress granules. Mean and SD, unpaired t-test (two-tailed), *** p = 0.0003 (dhh1Δ) respectively p = 0.0001 (Dhh1^DQAD). Dots represent mean of individual replicates. (C, D) RNA transfer between Dhh1 and Ded1 droplets. (C) Forward reaction: Dhh1-mCherry droplets were assembled with Cy5-labeled RNA and added to Ded1-GFP droplets. Upon Not1^MIF4G addition, Dhh1 droplets dissolve and the Cy5-RNA accumulates in the Ded1 droplets. (D) Inverse reaction: Ded1-mCherry droplets were assembled with Cy5-labeled RNA and added to Dhh1-GFP droplets. Upon eIF4G^C-terminus addition, Ded1 droplets dissolve, but the Cy5-RNA does not accumulate in the Dhh1 droplets. In contrast to the reaction presented in the main Figure 4, for the reactions shown in C and D, no stabilizing agents such as BSA or PEG were added in order to make the results in the forward and inverse reaction comparable. The fluorescence intensity scaling was adjusted for the first image (before Not1 / eIF4G addition) to account for the sample dilution upon the addition of Not1 or eIF4G, respectively. However, scaling of the Cy5 channel in the first image, and in the subsequent frames [20s – 180s], is identical for the forward and the inverse reaction to enable a direct visual comparison. (E) Quantification of the reactions presented in (C) and (D). For each experiment, the mean Cy5-RNA intensity accumulating in six Dhh1-GFP droplets is plotted over time after addition of Not1 or eIF4G, respectively. For background correction, six identically sized areas outside of Dhh1-GFP droplets were quantified and subtracted from the intensity measured inside the Dhh1-GFP droplets, mean and SD. These experiments were repeated at least three times, with comparable results. Mean (line) and SD (shaded area) of 6 large droplets per movie and forward and inverse reaction. At t= 180 s, 16.7 +/- 2.7% of the Cy5-RNA is enriched in Ded1-GFP droplets occupying 5-7% of the surface area (n = 3 movies, mean and SD). (F) Line scan of the Cy5 channel (raw data), at timepoint t = 180 s through Ded1 droplets shown in Extended Data Fig. 6c. In the ‘forward’ reaction, Ded1 droplets enrich Cy5-RNA 2-3 fold over background.

Fig. 1. The RNA-binding core and the unstructured tails of Dhh1 are required for LLPS and PB formation.

(A) Domain organization of Dhh1: RecA core and LCD tails. (B) In vitro phase separation of recombinant Dhh1-mCherry variants in the presence of ATP and RNA. Full-length (FL) and a truncation construct lacking both tails (core) were imaged at 10.5 μM protein. Representative images of at least 3 independent experiments. Scale bars: 25 μm. (C) Images of yeast cells expressing Dhh1-yEGFPs variants after 30 min glucose starvation. PBs are marked with arrows. Scale bars 5 μm. (D) Quantification of the number of PBs per cell using Diatrack. 3 (FL) or 4 (core) biological replicates of at least 417 (FL) or 505 (core) cells. Mean and SD; unpaired t-test (two-tailed), ** p-value = 0.0032. Dots represent the mean of individual replicates. (E) Phase separation behavior of full-length (FL) Dhh1 and the RecA core in response to changes in pH, salt, ATP and polyU concentration. Sum of the mean fluorescence intensity * area (arbitrary unit [A*I]) for all droplets per field of view, mean of 9 images (pH: 4 images).

Fig. 2. Phase separation by DDXs is wide-spread and evolutionary conserved.

(A) Graph illustrating occurrence of LC domains in yeast, human and E. coli DDXs. (B-D) Representative images of at least 3 independent experiments: in vitro phase separation in the presence of ATP and RNA of select S. cerevisiae (B), human (C) and E. coli DDXs (D); scale bars 25 μm; for details see SI 2 Table 4. (E) Images of E. coli cells co-expressing mCherry-tagged DDXs and GFP. Subcellular DDX foci are marked with arrows. Scale bars 2 μm. (F) Droplets formed from Ded1-mCherry, ATP and polyU dissolve upon addition of recombinant eIF4G^C-terminus, but not buffer. Scale bars 25 μm, representative images of > 3 independent experiments.

Fig. 3. The catalytic activity of DDXs regulates compartment turnover and RNA accumulation in phase-separated organelles.

(A) Ded1-mCherry labeled SGs were imaged after addition of 50 μg/ml cycloheximide (CHX). The ATP-deficient variants do not alter ATP and RNA-binding (Extended Data Fig. 3b,c) (B) Quantification of the percentage of SGs per cell normalized to t = 0 min; mean (solid line) and SEM (shaded area), n=3 biological replicates, at least 150 cells per replicate and strain. (C) A549 cells expressing WT or E179A mutant UAP56 were infected with influenza virus. After 6h, viral M mRNA was detected by smFISH and SON by immunofluorescene. Insets: enlargements of the marked white squares. Data are representative of three independent experiments. (D) Quantification of nuclear speckle volume (885 speckles per condition). (E) Quantification of relative M mRNA intensity at nuclear speckles (32 WT cells / 938 speckles and 34 E179A cells / 885 speckles). Mean and SD; two-sided t-test, *** p = 6.4*10^-9. (F) Sub2 depletion leads to accumulation of a PP7CP-yEGFP labeled reporter mRNA in nuclear foci. Scale bar 5 μm. (G) Quantification of (F). Mean and SEM of 5 biological replicates with n>78 cells per time point. (H) Quantification of nuclear focus intensity, 5 (Sub2 degron) or 6 (WT) biological replicates; at least 13 / 33 cells (60 min WT / degron) or 14 / 60 (90 min WT / degron) cells per replicate. Mean and SEM; unpaired two-tailed t-test with *** p = 0.0002 and **** p = <0.0001. Dots represent the mean of individual replicates.

Fig. 4. DDX ATPase activity controls RNA partitioning between phase-separated compartments in vivo and in vitro.

(A) Pab1-yEGFP labeled SGs are induced in an untagged Dhh1 WT or Dhh1^DQAD or dhh1Δ background. 3 biological replicates, at least 383 (WT), 93 (dhh1Δ) or 657 (Dhh1^DQAD) cells per replicate. (B) Quantification of (A), mean and SD. Unpaired t-test (two-tailed), * p = 0.0224. Dots represent mean of individual replicates. (C) Schematic representation of in vitro RNA transfer experiment (D-F) Droplets were assembled from Dhh1-mCherry (mCh) with Cy5-labelled RNA (red circles), and from Ded1-yEGFP (green circles). Cy5-RNA, Dhh1-mCh and Ded1-GFP were monitored upon addition of buffer (D) or Not1^MIF4G (F). Representative images of > 3 independent experiments. (E) Quantification of Cy5 intensity inside Ded1 areas, normalized to the intensity value at t=0 sec. Mean (line) and SD (shaded area) of 17 (“Not1 addition”) and 12 (“buffer addition”) large droplets per movie. At t = 2 min, 17.4 +/- 3.3% of the Cy5-RNA is enriched in Ded1-GFP droplets upon Not1 addition (n = 3 movies). Scale bars 5 μm. (G) Concept how DDXs could regulate multivalency, phase separation and compartment formation (I), ATPase-controlled compartment turnover and RNA release (II), and RNA transfer (III).