ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

Abstract

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

Keywords: DEAD box ATPases; RNA turnover; S. cerevisiae; biochemistry; cell biology; processing bodies.

Figure 1.. Loss of the ATPase activity of Dhh1 triggers bona fide processing body (PB) formation.

(A) Known PB components localize to Dhh1^DQAD foci. Cells co-expressing the indicated PB component were grown to exponential growth phase, then shifted to glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Scale bar: 5 μm (B) Constitutive PB formation by Dhh1^DQAD is rescued by the presence of wild-type Dhh1. Dhh1-GFP or Dhh1^DQAD-GFP was expressed from a CEN plasmid in DHH1 or dhh1∆ cells and were treated as in (A). Scale bar: 5 μm (C) Loss of ATPase activity mildly disrupts degradation of a Dhh1-tethered mRNA. Dhh1 or Dhh1^DQAD was co-expressed as a PP7CP fusion protein in dhh1∆ cells expressing FBA1-PP7L. FBA1 mRNA levels were measured by qPCR and normalized to ACT1 mRNA. Graphs show mean mRNA levels from three independent experiments of biological triplicate samples. Error bars represent SD. A student’s t-test comparing Dhh1 and Dhh1^DQAD is shown. Asterisks indicate p<0.005. (D) FBA1 mRNAs do not colocalize with PBs in Dhh1 or Dhh1^DQAD-expressing cells, suggesting functional mRNA decay, but enrich in PBs in xrn1∆ cells. The indicated strains were grown to exponential growth phase, shifted to either glucose-rich (2% glucose) or glucose starvation conditions for 20 min, fixed with paraformaldehyde and processed for smFISH. Depicted is a maximum projection of the central 10 planes of a 3D image. Insets show representative cells (1.67X magnification). The graph shows the quantification of a representative experiment (n = 2 biological replicates). Scale bar: 5 µm. (E) GFA1 mRNAs do not colocalize with PBs in Dhh1 or Dhh1^DQAD-expressing cells, suggesting functional mRNA decay, but enrich in PBs in xrn1∆ cells. The indicated strains were grown to exponential growth phase, shifted to either glucose-rich (2% glucose) or glucose starvation conditions for 20 min, fixed with paraformaldehyde and processed for smFISH as in (D). Insets show representative cells (1.67X magnification). The graph shows the quantification of a representative experiment (n = 2 biological replicates). Scale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.003

Figure 1—figure supplement 1.. Loss of ATPase activity of Dhh1 does not trigger stress granule formation.

(A) Dhh1^DQAD does not trigger constitutive stress granule formation. Cells co-expressing Pab1-GFP were grown to exponential growth phase, then shifted to glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Scale bar: 5 μm (B) PAT1 mRNAs do not colocalize with PBs in Dhh1 or Dhh1^DQAD-expressing cells, suggesting functional mRNA decay, but enrich in PBs in xrn1∆ cells. The indicated strains were grown to exponential growth phase, shifted to either glucose-rich or glucose starvation conditions for 20 min, fixed with paraformaldehyde and processed for smFISH. Depicted is a maximum projection of the central 10 planes of a 3D image. Insets show representative cells (1.67X magnification). Scale bar: 5 µm. (C) PGK1 mRNAs do not colocalize with PBs in Dhh1 or Dhh1^DQAD-expressing cells, suggesting functional mRNA decay, but enrich in PBs in xrn1∆ cells. The indicated strains were grown to exponential growth phase, shifted to either glucose-rich or glucose starvation conditions for 20 min, fixed with paraformaldehyde and processed for smFISH as in (A). Insets show representative cells (1.67X magnification). ale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.004

Figure 2.. ATP-bound, RNA-bound Dhh1 is required for robust PB formation.

(A) Disruption of ATP-binding activity of Dhh1 interferes with PB formation. Wild-type Dhh1 or Dhh1^Q-motif was co-expressed from a plasmid as a GFP fusion protein in dhh1∆ cells along with Dcp2-mCherry as a PB marker and grown to exponential growth phase, then shifted to either glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Images were also acquired using wide-field microscopy and PB formation was quantified using Diatrack 3.5 particle tracking software (see Materials and methods). Graphs represent average Dhh1-GFP or Dcp2-mCherry foci number per cell (n=3 biological replicates, >800 cells per experiment). Error bars represent SD. A student’s t-test comparing Dhh1 and Dhh1^Q-motif is shown. Asterisks indicate p<0.005. Scale bar: 5 µm. (B) Disruption of RNA binding activity of Dhh1 interferes with PB formation. Wild-type or mutant Dhh1 was co-expressed from a plasmid as a GFP fusion protein in dhh1∆ cells along with Dcp2-mCherry as a PB marker and treated as in (A). Scale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.005

Figure 2—figure supplement 1.. Loss of ATP binding and RNA binding by Dhh1 disrupts PB localization of other PB factors.

Xrn1-GFP (A), Dcp1-GFP (B), or Edc3-GFP (C) was co-expressed in dhh1∆ cells along with pDHH1-TAP, pDHH1^Q-motif-TAP, or pDHH1^3X-RNA-TAP and Dcp2-mCherry as a PB marker and treated as in Figure 2A. A student’s t-test comparing DHH1 and either dhh1^Q-motif or dhh1^3X-RNA is shown. Asterisks indicate p<0.005 (***), or p<0.05 (*). Scale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.006

Figure 2—figure supplement 2.. Disruption of RNA-binding, but not ATP-binding, affects the ability of tethered Dhh1 to promote mRNA decay.

(A) Dhh1^Q-motif is functional in mRNA decay when tethered to an mRNA. Dhh1 or Dhh1^Q-motif was co-expressed as a PP7CP fusion protein in dhh1∆ cells expressing FBA1-PP7L. FBA1 mRNA levels were measured by qPCR and normalized to ACT1 mRNA. Graphs show mean mRNA levels from three independent experiments of biological triplicate samples. Error bars represent SD. A student's t-test comparing Dhh1 and Dhh1^Q-motif is shown. (B) Loss of RNA-binding activity disrupts degradation of a Dhh1-tethered mRNA. Dhh1 or Dhh1^3X-RNA was co-expressed as a PP7CP fusion protein in dhh1∆ cells expressing FBA1-PP7L. FBA1 mRNA levels were measured by qPCR and normalized to ACT1 mRNA. Graphs show mean mRNA levels from three independent experiments of biological triplicate samples. Error bars represent SD. A student’s t-test comparing Dhh1 and Dhh1^3X-RNA is shown. Asterisks indicate p<0.005. (C) Wild-type and Dhh1 mutant proteins are expressed to similar levels. Western blot of Dhh1 and Dhh1 mutant protein expression from cells in exponential growth phase from Figure 2A and B. Dhh1 was detected using an anti-Dhh1 antibody. Hxk1 was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.18746.007

Figure 3.. Loss of the ATPase activity of Dhh1 disrupts PB dynamics.

(A) Loss of the ATPase activity of Dhh1 does not alter the dynamics of known PB components. Fluorescence recovery after photobleaching experiments (FRAP) were performed on cells expressing the indicated GFP-tagged PB component. Cells were glucose starved for 20 min to allow PBs to form, then PBs were bleached and recovery of GFP fluorescence to the PB was followed over time. Recovery of PB components is presented as an averaged data plot of FRAP recovery curves from three independent experiments (n > 8 PBs per experiment, typically ~12 PBs per experiment). Error bars represent SD. (B) The ATPase activity of Dhh1 is required for proper PB disassembly. dhh1∆ cells expressing Dhh1-GFP or Dhh1^DQAD-GFP were glucose starved for 30 min to allow PBs to form and then treated with either 50 μg/mL cycloheximide or solvent only (DMSO) for 2 hr and disappearance of Dhh1-GFP or Dhh1^DQAD-GFP foci per cell was monitored for 2 hr. Each time point image is a maximum-projection of 8 z-stacks at a distance of 0.4 µm. The graph shows foci number per cell measurements for Dhh1 and Dhh1^DQAD normalized to 1 to account for differences in PB formation between Dhh1 and Dhh1^DQAD (n = 3 biological replicates, >100 cells). Error bars represent SEM. Scale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.008

Figure 3—figure supplement 1.. Loss of ATPase activity of Dhh1 disrupts the PB dynamics of other PB components.

(A) Loss of ATPase activity of Dhh1 affects disassembly of Dcp2 foci. Dcp2-mCherry was expressed in dhh1∆ cells co-expressing Dhh1-GFP or Dhh1^DQAD-GFP from a plasmid, and cells were glucose starved for 30 min to allow PBs to form, followed by treatment with either 50 μg/mL cycloheximide or solvent only (DMSO) for 2 hr. Disappearance of Dcp2-mCherry foci over time is monitored (n = 3 biological replicates). Each time point image is a maximum-projection of 8 z-stacks at a distance of 0.4 µm. Scale bar: 5 µm (B) Two-hour cycloheximide treatment does not disrupt cell viability. The indicated cells were glucose starved for 30 min, and then treated with or without 50 μg/mL cycloheximide and 5-fold serial dilutions were plated on SD (-URA) + 2% dextrose media. DOI: http://dx.doi.org/10.7554/eLife.18746.009

Figure 4.. The ATPase activity of Dhh1 is stimulated in vitro and in vivo by Not1.

(A) ATPase activity of Dhh1 is stimulated by Not1. The ATPase activity of full-length Dhh1 or Dhh1^DQAD was measured with increasing concentrations of Not1^MIF4G. Graphs represent average ATPase activity (n=3). Error bars represent SD. (B) Disruption of Dhh1 interaction with the MIF4G region of Not1 by mutation of conserved residues in Dhh1. TAP-tagged Dhh1, Dhh1^5X-Not, or untagged Dhh1 were purified from cells in exponential growth phase using IgG-coupled magnetic beads and co-purifying Not1-3HA was detected by Western blot. Quantification of Not1 to Dhh1 ratio is plotted with SEM (n=5 biological replicates). A representative Western blot is shown. A student’s t-test comparing Dhh1 and Dhh1^5X-Not is shown. Asterisks indicate p<0.01. (C) Mutations in the Not1-binding surface of Dhh1 trigger constitutive PB assembly. Wild-type or mutant Dhh1 was co-expressed from a plasmid as a GFP fusion protein in dhh1∆ cells along with Dcp2-mCherry as a PB marker and grown to exponential growth phase, then shifted to either glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Images were also acquired using wide-field microscopy and PB formation was quantified using Diatrack 3.5 particle tracking software. Graphs represent the average Dhh1-GFP foci or Dcp2-mCherry foci number per cell (n=3 biological replicates, >800 cells per experiment). Error bars represent SD. Scale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.014

Figure 4—figure supplement 1.. Not1 is a specific activator of the ATPase activity of Dhh1.

(A) ATPase activity of Dhh1 is weakly stimulated by RNA. ATPase activity of full-length Dhh1 was measured with increasing concentrations of polyU RNA. Graphs represent average ATPase activity (n=3 biological replicates). Error bars represent SD.(B) Not1 is a specific activator of Dhh1. ATPase activity of full-length Dhh1 or Dbp5 was measured in the presence of the indicated protein. Graphs represent average ATPase activity (n=3 biological replicates). Error bars represent SD. (C) Not1 binding is diminished in a ATP-binding mutant of Dhh1. TAP-tagged Dhh1 or Dhh1^Q-motif were purified from cells in exponential growth phase using IgG-coupled magnetic beads and co-purifying Not1-3HA was detected by Western blot. Quantification of Not1 to Dhh1 ratio is plotted with SEM (n=4 biological replicates). A representative Western blot is shown. A student’s t-test comparing Dhh1 and Dhh1^Q-motif is shown. Asterisks indicate p<0.01. (D–F) Known PB components localize to Dhh1^5X-Not foci. Xrn1-GFP (D), Dcp1-GFP (E), or Edc3-GFP (F) was co-expressed in DHH1 or dhh1^5X-Notcells, along with Dcp2-mCherry as a PB marker. Cells were grown to exponential growth phase, then shifted to glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Images were also acquired using wide-field microscopy and PB formation was quantified using Diatrack 3.5 particle tracking software. Graphs represent the average foci number per cell (n = 3 biological replicates, >800 cells per experiment). Error bars represent SD. Scale bar: 5 μm. DOI: http://dx.doi.org/10.7554/eLife.18746.015

Figure 4—figure supplement 2.. Tethered Dhh1 does not require ATPase activation by Not1 to promote mRNA decay.

(A) Dhh1^5X-Not does not show a significant defect in mRNA decay when tethered to an mRNA. Dhh1 or Dhh1^5X-Not was co-expressed as a PP7CP fusion protein in dhh1∆ cells expressing FBA1-PP7L. FBA1 levels were measured by qPCR and normalized to ACT1 mRNA. Graphs show mean mRNA levels from three independent experiments of biological triplicate samples. Error bars represent SD. (B) Wild-type and Dhh1 mutant proteins are expressed to similar levels. Western blot of Dhh1 and Dhh1 mutant protein expression from cells in exponential growth phase. Dhh1 was detected using an anti-GFP antibody. Hxk1 was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.18746.016

Figure 5.. Mutations in Dhh1-binding surface of Not1 trigger constitutive PB assembly.

Not1 or Not1^9X-Dhh1 was co-expressed with Dhh1-GFP and Dcp2-mCherry and grown to exponential growth phase, then shifted to either glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Images were also acquired using wide-field microscopy and PB formation was quantified using Diatrack 3.5 particle tracking software (see Materials and methods). Graphs represent average Dhh1-GFP foci or Dcp2-mCherry foci number per cell (n=3 biological replicates, >800 cells per experiment). Error bars represent SD. Scale bar: 5 µm. DOI: http://dx.doi.org/10.7554/eLife.18746.017

Figure 5—figure supplement 1.. Not19X-Dhh1 triggers PB assembly.

(A–C) Known PB components localize to Not1^9X-Dhh1 foci. Xrn1-GFP (A), Dcp1-GFP (B), or Edc3-GFP (C) was co-expressed in NOT1, or not1^9X-Dhh1cells, along with Dcp2-mCherry as a PB marker. Cells were grown to exponential growth phase, then shifted to glucose-rich or glucose starvation conditions for 20 min and observed by confocal microscopy. Images were also acquired using wide-field microscopy and PB formation was quantified using Diatrack 3.5 particle tracking software. Graphs represent average foci number per cell (n=3 biological replicates, >800 cells per experiment). Error bars represent SD. A student's t-test comparing localization between Not1 and Not1^9x-Dhh1 is shown. Asterisks indicate p<0.005 (***), or p<0.05 (*). Scale bar: 5 μm (D) Not1^9X-Dhh1 mutant is expressed to wild-type Not1 levels. Western blot of Not1 or Not1^9X-Dhh1 mutant protein expression from cells in exponential growth phase. Not1 was detected using Rabbit IgG, and Dhh1 was detected using an anti-Dhh1 antibody. Hxk1 was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.18746.018

Figure 6.. Dhh1 PB dynamics can be recapitulated in vitro.

(A) Formation of liquid Dhh1-droplets depends on the presence of the RNA analog polyuridylic acid (polyU) and increases with increasing protein concentration. Recombinant mCherry-tagged Dhh1 was diluted into a low salt buffer and incubated at 4°C for 1 hr. Formation of liquid droplets was observed by fluorescence microscopy. Scale bar: 10 μm. (B) Dhh1 liquid droplet formation requires ATP. Dhh1 liquid droplets were formed as in (A), in the presence or absence of ATP and the creatine kinase ATP regeneration system. Scale bar: 20 μm. (C) Addition of Not1^MIF4G or RNase A, but not buffer alone, dissolves pre-formed Dhh1 liquid droplets. Dhh1 liquid droplets were pre-formed for 20 min at 4°C, followed by the addition of 5 µM Not1^MIF4G or RNase A. Scale bar: 10 μm. (D, E) Pre-incubation with Not1^MIF4Gprevents formation of Dhh1, but not Dhh1^DQAD liquid droplets. Reactions were imaged after 1h incubation at 4°C. Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.18746.019

Figure 6—figure supplement 1.. Single point mutants in the ATP binding site of Dhh1 affect PB assembly and liquid droplet formation.

(A) Single mutations in the Q-motif of Dhh1 show minor defects in PB formation. Wild-type Dhh1, Dhh1^F66R or Dhh1^Q73A were expressed from a plasmid as a GFP fusion protein in dhh1∆ cells along with Dcp2-mCherry as a PB marker and grown to exponential growth phase, then shifted to either glucose-rich or glucose starvation conditions for 20 min and observed by wide-field microscopy. PB formation was quantified using Diatrack 3.5 particle tracking software. Graphs represent average Dhh1-GFP or Dcp2-mCherry foci number per cell (n=3 biological replicates, >300 cells per experiment). Error bars represent SD. A student’s t-test comparing Dhh1^F66R or Dhh1^Q73A with Dhh1 is shown. Scale bar: 5 µm (B) Dhh1 liquid droplet formation requires ATP binding. Recombinant mCherry-tagged Dhh1, Dhh1^DQAD or Dhh1^F66R were diluted into a low salt buffer and incubated at 4°C for 1 hr in the presence of ATP and 0.075 mg/mL polyU. Formation of liquid droplets was observed by fluorescence microscopy. Scale bar: 10 μm DOI: http://dx.doi.org/10.7554/eLife.18746.020

Figure 7.. Model: The ATPase cycle of Dhh1 controls PB assembly and disassembly.

An ATP- and RNA-bound conformation of Dhh1 nucleates PB formation, while stimulation of Dhh1’s ATPase activity by Not1 promotes granule disassembly. DOI: http://dx.doi.org/10.7554/eLife.18746.025