Stress granules and processing bodies are dynamically linked sites of mRNP remodeling - PubMed (original) (raw)

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

Nancy Kedersha et al. J Cell Biol. 2005.

Abstract

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2alpha phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

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Figures

Figure 1.

Figure 1.

SGs and PBs in U2OS and HeLa cells. U2OS osteosarcoma (A–D) or HeLa (E–H) cells were untreated (A and E); exposed to 500 μM arsenite for 30 min (B and F); exposed to 20 μM clotrimazole (Sigma Aldrich) for 1 h (C and G); or exposed to heat (44°C) for 30 min (D and H). Cells were immediately fixed and stained for eIF4E, DCP1a, and eIF3. Yellow arrows indicate PBs; white arrowheads indicate SGs. In both cell lines, note that SGs are induced in cells lacking PBs upon clotrimazole (C and G) or heat shock treatment (D and H), whereas arsenite treatment induces both SGs and PBs that are juxtaposed (B and E). In each panel, the indicated inset is reproduced at the right as replicate views of the same field showing eIF4E, DCP1a, eIF3, and the merged view.

Figure 2.

Figure 2.

Distribution of proteins between G3BP-induced SGs and PBs. SGs were induced in DU145 cells by the transfection of GFP-G3BP and cells stained as indicated. In D, cells were cotransfected with FLAG-eIF4E and stained with anti-FLAG; (A) DCP1a and TIA-1; (B) XRN1 and eIF4E; (C) eIF4G and eIF4E; (D) eIF3b and FLAG-eIF4E; (E) PABP-1 and DCP1a; and (F) FAST and eIF4E. Yellow arrows indicate representative PBs; white arrowheads indicate SGs in the merged views.

Figure 3.

Figure 3.

Distribution of proteins between arsenite-induced SGs and PBs. SGs were induced in DU145 cells by arsenite treatment, and cells were triple stained for the indicated proteins: (A) eIF4E, DCP1a, and eIF3b; (B) PABP-1, XRN1, and TIA-1; (C) eIF4E, eIF4G, and eIF3b; (D) eIF4E, phospho-eIF2α, and eIF3b; (E) eIF4E, G3BP, and eIF3b; and (F) GW182, FAST, and TIA-1. Yellow arrows indicate representative PBs; white arrowheads indicate SGs in the merged views.

Figure 4.

Figure 4.

Role of eIF2α phosphorylation and Lsm4 expression in SG and PB formation. (A) Arsenite-treated wild-type (SS) and eIF2α S51A mutant (AA) MEFs stained for SG markers eIF3b, G3BP, and TIAR. (B) Arsenite-treated SS and AA MEFs stained for PB markers GW182 and DCP1a and the SG marker protein TIA-1. Yellow arrows indicate representative PBs; white arrowheads indicate SGs in the merged views. (C–E) DU145 or HT1080 cells were transfected with control siRNA or siRNA targeting Lsm4, processed for immunofluorescence, and examined for PBs and SGs. (C) Semiquantitative RT-PCR showing reduced expression of Lsm4 mRNA in Lsm4-siRNA–transfected HT1080 cells. (D) Percentage of cells containing visible PBs before (dark gray bars) or after (light gray bars) arsenite treatment. (E) Confocal micrographs of HT1080 cells stained for PB markers GW182 and DCP1a and SG marker TIA-1.

Figure 5.

Figure 5.

SG and PB assembly induced by different stresses. HeLa cells were subjected to different stresses and were stained for eIF4E, FAST, and eIF3. (A) Unstressed cells, some of which contain PBs (yellow arrow) but no SGs. (B) Arsenite (500 μM for 30 min) induces both SGs (white arrowhead) and PBs (yellow arrow). (C) Cells were treated with arsenite for 60 min, and 20 μg/ml emetine was added during the last 30 min. (D–F) Cells were subjected to heat shock (44°C) for 15 min (C), 30 min (D), or 60 min (E). Yellow arrows indicate representative PBs; white arrowheads indicate SGs in the merged views. In each panel, the indicated inset is reproduced at the bottom as replicate views of the same field showing eIF4E, FAST, and eIF3.

Figure 6.

Figure 6.

A single species of reporter mRNA is present in both SGs and PBs. (A) Schematic of the GFP-MS2–tethered mRNA reporter constructs used to visualize the subcellular localization of the globin-MS2 mRNA. (B and C) COS7 cells transiently transfected with both plasmids shown in A and counterstained for different SG and PB markers. (B) GFP-globin mRNA, PB marker DCP1a, and SG marker TIA-1. (C) GFP-globin mRNA, PB marker XRN1, and SG–PB marker eIF4E. Insets show enlargement of boxed areas with colors separated. Yellow arrows indicate representative PBs; white arrowheads indicate SGs in the merged views.

Figure 7.

Figure 7.

Dynamics of SGs and PBs in vivo_._ COS7 cells cotransfected with RFP-DCP1a and either (A) GFP–TIA-1, (B) YFP-TTP, (C) GFP-G3BP plus empty myc-vector, or (D) GFP-G3BP and TTP-myc. Cells were observed at 37°C in real time by using confocal microscopy. Images from 10-min intervals are shown; Videos 1–5 depicts animation of these series (available at

http://www.jcb.org/cgi/content/full/jcb.200502088.DC1

). Each image is volume rendered from 10 Z-sections. Yellow arrows indicate PBs; white arrowheads indicate SGs.

Figure 8.

Figure 8.

TTP and BRF1 promote fusion of SGs with PBs. COS7 cells triply transfected with GFP-G3BP as an SG marker, RFP-DCP1a as a PB marker, and one of the following: (A) vector; (B) FLAG-tagged FAST; (C) FLAG-XRN1; (D) FLAG-eIF4E; (E) TTP-myc; or (F) FLAG-BRF1. Yellow arrows indicate positions of representative PBs; white arrowheads indicate position of SGs.

Figure 9.

Figure 9.

FRAP analysis of SG and PB proteins. COS7 cells were transfected with GFP–TIA-1 (A), GFP-PABP1 (B), YFP-TTP (C), GFP-G3BP (D), FAST-YFP (E), YFP-DCP1a (F), and GFP-GW182 (G). A two-dimensional scan was taken of each field before photobleaching, and a target SG or PB was selected (red arrows). Fluorescence intensity was obtained by using a linear scan centered around the target region (vertical yellow line); the prebleach scans (pink tracing) represent the mean of three separate scans; the dark blue tracing represents the scan taken immediately after the 1-s bleach; and the aqua tracing represents the scan taken 30 s later. Images are shown pseudocolored as indicated by the key shown in the bottom right panel.

Figure 10.

Figure 10.

Hypothetical model of the relationship between SGs and PBs. Proteins found exclusively in SGs are shown in yellow; proteins found in both SGs and PBs are depicted in green; and proteins restricted to PBs are shown in blue type.

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