Modulation of RNA Condensation by the DEAD-Box Protein eIF4A - PubMed (original) (raw)

Modulation of RNA Condensation by the DEAD-Box Protein eIF4A

Devin Tauber et al. Cell. 2020.

Abstract

Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.

Keywords: DEAD-box protein; RNA chaperone; RNA-RNA interaction; biomolecular condensate; ribonucleoprotein; stress granule.

Copyright © 2020 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.

Figure 1.. RNAs Are Recruited to and Self-Organize on RNA Condensate Surfaces

(A) Pairwise combinations of homopolymers were condensed together and visualized with fluorescent antisense oligos. (B) PolyA assemblies (labeled by oligoU) were condensed with fluorescent in vitro transcribed RNAs. (C) Purified SG cores containing GFP-G3BP1 were incubated with fluorescent luc RNA. Scale bars, 500 nm. (D) Line profile of (C) along the line denoted by the white arrow. (E) A fluorescent myotonic dystrophy repeat RNA (reRNA) containing exons 5–11 of DMPK and ~590 CUG repeats was condensed with fluorescent luc. Scale bars, 2 μm. (F) Fluorescent pgc was condensed with polyC and polyU (and the corresponding fluorescent antisense oligos), localizing to the polyC/polyU interface. Scale bars, 5 μm. See also Figure S1.

Figure 2.

Figure 2.. RNA Condensate Surface Localization Stabilizes Intermolecular RNA-RNA Interactions

(A) Fluorescent pgc was condensed with polyA and fluorescent U19, and the condensates were subjected to 1:10 dilution in TE buffer. Scale bar, 20 μm. (B) Quantification of (A) as an index of dispersion, showing the persistence of pgc shell assemblies over time. Dashed lines are 95% confidence intervals. n = 6 replicates. (C) Images of the crosslinking conditions showing pgc on droplets, in solution, and as gels. (D) Representative fluorescence denaturing gel. (E) Quantification of (D), showing that RNA condensate surfaces enhance intermolecular RNA interactions compared to solvated RNA or RNA gels alone. X represents the mean. *p < 0.05, **p < 0.01, ****p < 10−4. n = 4 replicates. See also Figure S2.

Figure 3.

Figure 3.. eIF4A Limits RNA Recruitment to SGs

(A) Scatterplot of SG-associated helicase abundance in U2OS versus HeLa cells. (B) IF of eIF4A1 in SGs. eIF4A1 extends past the SG periphery as compared to DDX1, which co-extends with G3BP1. Scale bars, 2 μm. n = 3 replicates. (C) smFISH images and quantification of SG enrichment for TFRC and POLR2A mRNAs in U2OS cells treated with 60 min arsenite, then 30 min DMSO, Hipp, or 2DG and CCCP. Gray boxes denote the SGs in the insets. SGs are visualized by anti-PABPC1 IF. Scale bars, 20 μm. n ≥ 5 frames per condition. x represents the mean in our quantifications. (D) NORAD lncRNA smFISH images and quantification in U2OS cells treated with arsenite, Hipp, or PatA. SGs are visualized by anti-G3BP1 IF. Scale bars, 5 μm. n = 3 replicates. *p < 0.05, **p < 0.01, ***p < 10−3, ****p < 10−4. x represents the mean in our quantifications. See also Figure S3.

Figure 4.

Figure 4.. eIF4A Limits SG Formation

(A) Images displaying SG formation (assessed by PABPC1 IF) in both WT and ΔΔG3BP1/2 U2OS cells with the indicated treatments. Hipp treatment or ATP depletion following arsenite induces SG formation in ΔΔG3BP1/2 cells. (B) Quantification of images in (A). n = 3. Error bars, SD. (C) siRNA knockdown of eIF4A1 in ΔΔG3BP1/2 cells restores SGs upon addition of arsenite. n = 3. Error bars, SD. (D) Quantification of (C) as fold change. (E) Overexpression of eIF4A1 in WT U2OS cells prevents SG formation in cells expressing Myc-tagged eIF4A1, compared to non-transfected neighbor cells or control transfections. ATPase mutant E183Q was able to prevent SG formation when overexpressed, but RNA binding mutants R362Q and T158Q were not. (F) Quantifications of (E) as %SG area/cell area of Myc-eIF4A1 expressing (TF) or non-transfected (NTF) cells. x represents the mean of all cells analyzed. n = 3 replicates with all replicates pooled. (G) Quantifications of translation between NTF and TF WT and mutant versions of Myc-eIF4A1 as assessed by puromycin intensity. Translation is repressed equally in all conditions when arsenite is added, indicating that effects on SG formation are not due to increased translation. *p ≤ 0.05. Error bars, SD with n ≥ 3 replicates. See also Figures S4 and S5.

Figure 5.

Figure 5.. eIF4A Limits the Docking of P-bodies with SGs

(A) The number of PB/SG interfaces increase in the presence of arsenite in combination with Hipp compared to combinations of arsenite with PatA or arsenite alone. (B–E) This effect holds true when interface quantities are normalized to the total amount or area of PBs (B and D) or SGs (C and E). PBs and SGs are visualized by EDC3 and G3BP1 IF, respectively. n ≥ 5 replicates. x represents the mean. (F and G) Hipp addition produces a slight, but not significant, increase in PB number and area (F) but not in SG area (G).

Figure 6.

Figure 6.. Recombinant eIF4A Is Sufficient to Limit RNA Condensation In Vitro

(A) Formation of fluorescent total RNA droplets was monitored over a period of 20 min and is inhibited by eIF4A + ADPNP and more drastically by eIF4A + ATP. Hipp inhibits eIF4A1 catalytic function and restores droplet formation, while PatA does not. (B and C) Quantification of RNA condensation kinetics comparing ATP + eIF4A1 and ADPNP + eIF4A1 (B) and Hipp addition compared to PatA (C), as assessed by the percent of total frame area occupied by droplets (%Droplet area) over time. n = 3 replicates. SB, protein storage buffer. Error bars, SD. See also Figure S6.

Figure 7.

Figure 7.. eIF4A Limits RNA Condensation as an ATP-Dependent RNA Chaperone, Analogous to Heat Shock Proteins

(A) The general mechanism utilized by chaperones like HSP70 to resolve aberrant protein-protein interactions. Misfolded proteins are bound by HSP70·ATP, which limits aggregation while ATP hydrolysis liberates the protein. (B) Model of eIF4A’s function to resolve aberrant RNA-RNA interactions. ATP-dependent binding of eIF4A to RNA limits the multivalent RNA-RNA interactions driving RNA condensation, while ATP hydrolysis facilitates eIF4A release to re-enter the catalytic cycle.

Comment in

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