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

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling JCB: ARTICLE Stress granules and processing bodies are dynamically linked sites of mRNP remodeling 1 1 1 2 3 4 Nancy Kedersha, Georg Stoecklin, Maranatha Ayodele, Patrick Yacono, Jens Lykke-Andersen, Marvin J. Fritzler, 5 5 2 1 Donalyn Scheuner, Randal J. Kaufman, David E. Golan, and Paul Anderson 1 2 Division of Rheumatology and Immunology and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Hematology Division, Brigham and Women’s Hospital, Boston, MA 02115 Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada University of Michigan Medical Center and Howard Hughes Medical Institute, Ann Arbor, MI 48109 tress granules (SGs) are cytoplasmic aggregates Fas-activated serine/threonine phosphoprotein, XRN1, of stalled translational preinitiation complexes that eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, Saccumulate during stress. GW bodies/processing eIF4G, and PABP-1 are restricted to SGs, whereas bodies (PBs) are distinct cytoplasmic sites of mRNA DCP1a and 2 are confined to PBs. SGs and PBs also can degradation. In this study, we show that SGs and PBs are harbor the same species of mRNA and physically associate spatially, compositionally, and functionally linked. SGs with one another in vivo, an interaction that is promoted and PBs are induced by stress, but SG assembly requires by the related mRNA decay factors TTP and BRF1. We eIF2 phosphorylation, whereas PB assembly does not. propose that mRNA released from disassembled poly- They are also dispersed by inhibitors of translational somes is sorted and remodeled at SGs, from which selected elongation and share several protein components, including transcripts are delivered to PBs for degradation. Introduction In response to environmental stress, eukaryotic cells reprogram sults suggest that SGs are sites of mRNA triage at which their translational machinery to allow the selective expression mRNP complexes are monitored for integrity and composition of proteins required for viability in the face of changing condi- and are then routed to sites of reinitiation, degradation, or stor- tions. During stress, mRNAs encoding constitutively expressed age (Anderson and Kedersha, 2002; Kedersha and Anderson, “housekeeping” proteins are redirected from polysomes to dis- 2002). During stress, mRNA continues to be directed to sites of Met crete cytoplasmic foci known as stress granules (SGs), a pro- reinitiation, but in the absence of eIF2–GTP–tRNA , it shut- cess that is synchronous with stress-induced translational arrest tles back to SGs, where it accumulates (Kedersha et al., 2000). (Anderson and Kedersha, 2002; Kedersha and Anderson, mRNAs within SGs are not degraded, making them available 2002). Both SG assembly (Kedersha et al., 1999) and transla- for rapid reinitiation in cells that recover from stress. The tional arrest (Krishnamoorthy et al., 2001) are initiated by the observation that labile mRNAs are stabilized during stress phosphorylation of translation initiation factor eIF2, which (Laroia et al., 1999; Bolling et al., 2002) suggests that some as- Met reduces the availability of the eIF2–GTP–tRNA ternary pect of the mRNA degradative process is disabled during the complex that is needed to initiate protein translation. Drugs that stress response. Thus, the accumulation of mRNA at SGs may stabilize polysomes (e.g., emetine) cause SG disassembly, be a consequence of both stress-induced translational arrest and whereas drugs that dismantle polysomes (e.g., puromycin) pro- stress-induced mRNA stabilization. mote the assembly of SGs, indicating that mRNA moves be- Although the process of stress-induced mRNA stabiliza- tween polysomes and SGs (Kedersha et al., 2000). These re- tion is poorly understood, it likely involves the inactivation of one or more mRNA decay pathways. Two major mechanisms of mRNA degradation are active in eukaryotic cells (Decker Correspondence to Nancy Kedersha: [email protected] and Parker, 2002). In the first pathway, deadenylated tran- Abbreviations used in this paper: ARE, adenine/uridine-rich destabilizing ele- scripts are degraded by a complex of 3–5 exonucleases ments; FAST, Fas-activated serine/threonine phosphoprotein; PB, processing known as the exosome. In vitro studies using cell extracts reveal body; SG, stress granule; siRNA, small interference RNA; TIA, T cell intracellular antigen; TIAR, TIA related; TTP, tristetraprolin. that some mRNAs bearing adenine/uridine-rich destabilizing el- The online version of this article contains supplemental material. ements (AREs) in their 3 untranslated regions are degraded by © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 169, No. 6, June 20, 2005 871–884 http://www.jcb.org/cgi/doi/10.1083/jcb.200502088 JCB 871 THE JOURNAL OF CELL BIOLOGY 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 M arsenite for 30 min (B and F); ex- posed to 20 M clotrimazole (Sigma Aldrich) for 1 h (C and G); or exposed to heat (44C) 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 clotrima- zole (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 repro- duced at the right as replicate views of the same field showing eIF4E, DCP1a, eIF3, and the merged view. this 3–5 exosome-dependent pathway (Jacobs et al., 1998; geted knockdown of XRN1 results in the accumulation of Chen et al., 2001; Mukherjee et al., 2002). The second path- poly(A) -containing mRNA at these sites, suggesting that this way entails the removal of the seven-methyl guanosine cap mRNA decay pathway is conserved in both lower and higher from the 5 end of the transcript by the DCP1–DCP2 complex eukaryotes. Although the composition of GW bodies/PBs is (Long and McNally, 2003; Jacobson, 2004), allowing 5–3 somewhat different in lower and higher eukaryotes, because exonucleolytic degradation by XRN1 (Stevens, 2001). In they share the ability to process mRNA, we will provisionally yeast, components of this 5–3 decay pathway are concen- refer to these foci as PBs. Interestingly, metabolic inhibitors trated at discrete cytoplasmic foci known as processing bodies that promote (e.g., puromycin) or inhibit (e.g., emetine) the (PBs; Sheth and Parker, 2003). Yeast genetic studies reveal assembly of SGs in mammalian cells have similar effects on that mRNA decay intermediates accumulate at PBs when nor- the assembly of both yeast and mammalian PBs. These results mal decay is blocked, suggesting that PBs are sites of decap- indicate that both SGs and PBs are sites at which mRNA accu- ping and 5–3 degradation (Sheth and Parker, 2003). Studies mulates after polysome disassembly. in mammalian cells have revealed similar structures that In this study, we catalog the protein composition of SGs contain DCP1/2, XRN1, GW182, and Lsm1–7 heptamer and PBs and report several links between these cytoplasmic (Eystathioy et al., 2002, 2003; Ingelfinger et al., 2002; Cougot subdomains. DCP1a/2 and GW182 are components of PBs but et al., 2004a,b; Yang et al., 2004). In mammalian cells, the tar- not of SGs, whereas most initiation factors (e.g., eIF3, eIF4G, 872 JCB • VOLUME 169 • NUMBER 6 • 2005 and PABP-1) are components of SGs but not of PBs. In con- trast, eIF4E, XRN1, Fas-activated serine/threonine phospho- protein (FAST), and tristetraprolin (TTP) are found in PBs in unstressed cells but partially or completely relocalize to SGs in stressed cells. A single class of reporter mRNA is found in both SGs and PBs, suggesting that individual transcripts at different stages of processing may localize in each structure. Pho- tobleaching studies reveal kinetically distinct classes of proteins within SGs and PBs: TTP, T cell intracellular antigen (TIA), and G3BP rapidly shuttle in and out of these structures, whereas putative scaffold proteins DCP1a, GW182, and FAST are relatively stable constituents of these structures. We pro- pose a model wherein mRNA released from polysomes during stress is routed to SGs for triage, sorting, and mRNP remodeling, after which certain transcripts are selectively exported to asso- ciated PBs for degradation. Results SGs and PBs are induced by different stimuli Previous studies have shown that the composition of SGs var- ies with the stimulus used to elicit their assembly; e.g., heat shock–induced SGs contain HSP27, whereas arsenite-induced SGs do not (Kedersha et al., 1999), and SGs containing G3BP (Ras-GSP SH3 domain–binding protein) have been described as lacking TIA-1 (Tourriere et al., 2003). Therefore, we used a number of SG-inducing stimuli to survey SG and PB composi- tion. U2OS cells and HeLa cells were treated with arsenite (ox- idative stress), clotrimazole (mitochondrial stress), or heat shock, and were stained for SG markers eIF4E (Fig. 1) and eIF3 and PB marker DCP1a. As shown in Fig. 1 (A and E), some unstressed cells contain DCP1a-positive PBs (yellow ar- rows), whereas others do not. Remarkably, eIF4E appears Figure 2. Distribution of proteins between G3BP-induced SGs and PBs. present in PBs together with DCP1a. Arsenite treatment (Fig. SGs were induced in DU145 cells by the transfection of GFP-G3BP and 1, B and F) induces both SGs (Fig. 1, white arrowheads) 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; PBs in all cells, and the great majority of the PBs appear clus- (C) eIF4G and eIF4E; (D) eIF3b and FLAG-eIF4E; (E) PABP-1 and DCP1a; tered around SGs in both U2OS (Fig. 1 B) and HeLa (Fig. 1 F) and (F) FAST and eIF4E. Yellow arrows indicate representative PBs; white cells. In contrast, cells treated with the mitochondrial poison arrowheads indicate SGs in the merged views. clotrimazole (Fig. 1, C and G) or heat shock (Fig. 1, D and H) display SGs but do not show an increase in PBs, nor do PBs ap- cally irregular in shape and are frequently juxtaposed with PBs pear associated with SGs. We conclude that SGs and PBs are (Fig. 2, yellow arrows). GFP-G3BP transfectants were counter- coordinately induced by arsenite, but that other stress stimuli stained for the PB marker DCP1a and the SG marker TIA-1 induce SGs in cells lacking PBs. (Fig. 2 A). DCP1a is found in PBs but is largely excluded from Shared versus unique protein the SG, as shown by TIA-1 staining. This indicates that GFP- components of SGs and PBs G3BP and TIA-1 are present in SGs but are excluded from PBs, whereas DCP1a is present in PBs but not in SGs. Similar The presence of eIF4E in PBs was unexpected. Therefore, we analysis indicates that another PB component, XRN1 (Fig. 2 sought to confirm this result and determine whether other pre- B), is present in both PBs and G3BP-induced SGs. Consistent viously described SG components might also be present in with the data shown in Fig. 1, eIF4E (Fig. 2 C) is found in both PBs. We used DU145 cells, which had been previously used to SGs and PBs, whereas eIF4G is found in SGs but not in eIF4- analyze SG components (Kedersha et al., 2002), and induced positive PBs. Two approaches confirm that the eIF4E signal in SGs by the transient transfection of GFP-G3BP, an SG compo- PBs is not caused by antibody cross-reactivity with some PB nent whose expression induces the assembly of very large SGs protein: (1) a different eIF4E antibody gives identical results readily amenable to microscopic analysis (Tourriere et al., (unpublished data); and (2) transfected FLAG-tagged eIF4E re- 2003). GFP-G3BP (Fig. 2) induces the formation of large SGs veals the same PB–SG distribution when detected using anti- (1–5 m in diameter; Fig. 2, white arrowheads) that are typi- STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 873 eIF2 (Fig. 3 D), and endogenous G3BP (Fig. 3 E) are only in SGs, whereas GW182 (Fig. 3 F) and FAST (Fig. 2 F) predomi- nate in PBs. We conclude that G3BP, eIF4G, eIF3, phospho- eIF2, and PABP-1 are restricted to SGs, whereas DCP1a and 2 (unpublished data) are confined to PBs. GW182 autoantibody staining suggests that it is present in both PBs and SGs (Fig. 3 F, green); however, anti-GW182 is not monospecific by Western blot analysis, and a GFP-tagged construct encoding most of GW182 (aa 313–1709) is only found in PBs (Yang et al., 2004). Thus, GW182 localizes to PBs, whereas its association with SGs remains inconclusive. Of considerable interest is the finding that XRN1, FAST, and eIF4E are present in both PBs and SGs. The dual SG–PB localization of each of these pro- teins was confirmed by using tagged constructs in transient transfection assays (Fig. 2 D and see Fig. 8, B–D). FAST inter- acts with TIA-1 and antagonizes the translational silencing of TIA-1 (Li et al., 2004b). In unstressed COS7 cells, most FAST is nuclear and is associated with mitochondria (Li et al., 2004a). Its presence in PBs and its relocalization to SGs may reflect its function as a translational regulator of TIA proteins. PBs are present in AA cells that cannot phosphorylate eIF2 or assemble SGs Little is known about the signaling pathways and specific mo- lecular events that govern PB assembly, although their size and number increase when 5–3 mRNA decay is blocked (Sheth and Parker, 2003) and vary throughout the cell cycle (Yang et al., 2004). SG assembly requires the phosphoryla- tion of eIF2 (Kedersha et al., 1999) and is mediated by the aggregation of one of several RNA-binding proteins, includ- ing TIA proteins (Gilks et al., 2004), Fragile X Mental Retar- dation protein (Mazroui et al., 2002), G3BP (Tourriere et al., 2003), and the survival of motor neurons protein (Hua and Figure 3. Distribution of proteins between arsenite-induced SGs and PBs. Zhou, 2004). We therefore asked whether PBs are present in SGs were induced in DU145 cells by arsenite treatment, and cells were mutant AA cells, in which the normal eIF2 allele has been 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, replaced with a nonphosphorylatable mutant (S51A eIF2) phospho-eIF2, and eIF3b; (E) eIF4E, G3BP, and eIF3b; and (F) GW182, allele by homozygous replacement (Scheuner et al., 2001). As FAST, and TIA-1. Yellow arrows indicate representative PBs; white arrow- shown in Fig. 4 A, treatment of wild-type SS cells with arse- heads indicate SGs in the merged views. nite results in robust SG assembly (white arrowheads), as as- sessed using three independent SG markers (eIF3b; G3BP; FLAG (Fig. 2 D, blue). We conclude that eIF4E is present in and TIA related [TIAR]). In contrast, no SG assembly is seen both PBs and SGs. In contrast, eIF3b (Fig. 2 D) and PABP-1 with any of these SG markers in arsenite-treated AA mouse (Fig. 2 E) are restricted to SGs. The TIA-1–interacting protein cells (Fig. 4 A, right). Likewise, SGs are not induced in AA FAST (Fig. 2 F) exhibits a pattern similar to XRN1; i.e., it is cells by any other treatments, including heat shock, puromy- predominantly associated with PBs and is weakly associated cin treatment, or transfection with G3BP (unpublished data). with SGs. Only the enforced expression of the phosphomimetic form of To confirm that the SGs induced by G3BP overexpres- eIF2 generates SGs in AA cells (supplemental Fig. 1 in sion are compositionally similar to SGs induced by stress, we McEwen et al., 2005), demonstrating their competence to as- exposed DU145 cells to oxidative stress using arsenite and semble SGs given this essential trigger. stained for endogenous SG and PB markers (Fig. 3). Although Staining arsenite-stressed control SS cells and mutant arsenite-induced SGs are smaller than those induced by GFP- AA cells for PB marker proteins GW182 and DCP1a (Fig. 4) G3BP overexpression, the results are generally comparable. As reveals that both cell lines display numerous PBs (Fig. 4 B, shown in Fig. 3 A, DCP1a is confined to PBs (yellow arrow), yellow arrows). In contrast, SGs (Fig. 4, white arrowheads) eIF3b is confined to SGs (white arrowhead), and eIF4E is are induced in SS cells, as shown by TIA-1 staining, but are present in both structures. PABP-1 and TIA-1 are restricted to absent in AA cells treated similarly. To verify that these ap- SGs, whereas XRN1 (Fig. 3 B) predominates in PBs, but a mi- parent PBs in both SS and AA cells behave normally, we con- nor amount is detectable in SGs. eIF4G (Fig. 3 C), phospho- firmed that they were abolished upon treatment of the cells 874 JCB • VOLUME 169 • NUMBER 6 • 2005 Figure 4. Role of eIF2 phosphorylation and Lsm4 expression in SG and PB formation. (A) Arsenite-treated wild-type (SS) and eIF2 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 trans- fected 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. Physical juxtaposition and transient with emetine or cycloheximide (unpublished data). We con- interactions between SGs and PBs clude that PBs, unlike SGs, do not require the phosphorylation We were struck by the observation that arsenite-induced SGs of eIF2 for their assembly. appear juxtaposed with PBs and contain eIF4E but no other PBs are induced by arsenite initiation factors (e.g., Figs. 1 and 3). Therefore, we investi- gated the kinetics of SG–PB assembly by using combinations As arsenite induces both PBs and SGs (Fig. 1), we asked of stress-inducing conditions. Fig. 5 shows HeLa cells sub- whether the knockdown of PBs would affect SG assembly in jected to different stresses and triple-stained for eIF3b (SG- response to arsenite. Several small interference RNAs (si- specific marker), FAST (PBs), and eIF4E (found in both SGs RNAs) were used to knockdown different PB components and PBs). Untreated cells (Fig. 5 A) display few PBs (Fig. 5, (unpublished data), but only siRNA against Lsm4 was mini- yellow arrows), which appear as yellow dots because of the mally effective in preventing PB assembly in response to merge of green (eIF4E) and red (FAST) signals. The treatment arsenite. DU145 and HT1080 cells were transfected with of cells with arsenite for 30 min (Fig. 5 B) resulted in a dra- control or Lsm4 siRNA, untreated or treated with arsenite, matic increase in the number of PBs coordinate with robust fixed and stained for PBs, scored microscopically, and SG assembly (Fig. 5, white arrowheads); remarkably, virtually counted. As shown in Fig. 4 C, RT-PCR reveals that efficient all PBs were found adjacent to SGs, as shown in Fig. 1. SG knockdown Lsm4 mRNA is obtained, which reduces PBs and PB formation appear synchronously in response to shorter (Fig. 4, D [dark gray bars] and E). However, upon arsenite arsenite treatments. treatment, the percentage of cells with PBs increases mark- Disassembly of both SGs and PBs is enforced by eme- edly despite knockdown for Lsm4. In HT1080 cells, Lsm4 tine and cycloheximide, which are drugs that inhibit transla- knockdown is able to reduce the percentage of PB-positive tional elongation and block the disassembly of polysomes, cells to 5% of control levels in the absence of stress (Fig. 4, D thereby preventing the translocation of mRNA into SGs and [right bars] and E). Arsenite treatment induces PBs in 75% PBs (Kedersha et al., 2000; Sheth and Parker, 2003; Cougot et of these cells, whereas 95% display SGs. As heat shock and al., 2004a). As the size of both PBs and SGs should be propor- clotrimazole also induce SGs in cells lacking PBs, the data tional to the amount of mRNA within each, we determined indicate that assembly of SGs and PBs is regulated by dis- whether adding emetine to arsenite-treated cells would cause tinct signaling pathways. STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 875 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 M for 30 min) induces both SGs (white arrowhead) and PBs (yellow arrow). (C) Cells were treated with arsenite for 60 min, and 20 g/ml emetine was added dur- ing the last 30 min. (D–F) Cells were subjected to heat shock (44C) for 15 min (C), 30 min (D), or 60 min (E). Yellow arrows indicate rep- resentative 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. the disassembly of SGs before PBs, or vice versa. Emetine ad- sembly. Cells that were exposed to heat shock (44C) for 15 dition to arsenite-treated cells followed by an additional 30- and 30 min (Fig. 5, D and E) displayed SGs in cells lacking min incubation (Fig. 5 C) resulted in partial SG disassembly PBs. Continued heat shock treatment for 1 h resulted in the (Fig. 5, white arrowheads) without affecting PBs. A longer disappearance of SGs and the appearance of PBs (Fig. 5 F). emetine treatment (1–2 h) completely dispersed SGs without Thus, heat shock appears to trigger a coordinate sequence of affecting PBs (unpublished data), but the treatment of cells for events: an early and transient induction of SGs followed by a 1 h with emetine in the absence of arsenite disassembled all late induction of PBs. Remarkably, eIF4E distribution appears the PBs (unpublished data). This indicates that emetine treat- to shift between the two compartments under these conditions, ment disassembles SGs before disassembling PBs and that suggesting that some eIF4E-bound mRNA may move from eIF4E is still present in PBs upon emetine-enforced SG disas- SGs to PBs during heat shock. 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 rep- resentative PBs; white arrowheads indicate SGs in the merged views. 876 JCB • VOLUME 169 • NUMBER 6 • 2005 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 37C 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. A single class of mRNA transcripts is with DCP1a and TIA-1 in Fig. 6 B and with XRN1 and eIF4E in present in both SGs and PBs Fig. 6 C. We conclude that a single class of mRNA localizes to SGs are thought to be sites of mRNA sorting rather than decay both SGs and PBs. (Kedersha and Anderson, 2002). As PBs are putative sites of SG–PB interactions in real time using 5–3 mRNA degradation (Sheth and Parker, 2003; Cougot et al., time-lapse microscopy 2004a), the juxtaposition of the two structures during arsenite treatment (Fig. 1) and their sequential assembly/disassembly dur- To investigate the physical interaction of PBs with SGs over ing heat shock (Fig. 5) suggests that mRNAs destined for decay time, we obtained a series of red (RFP) or green (GFP or YFP)- are sorted in SGs and are subsequently transported into PBs. If so, tagged proteins and transiently expressed various combinations a single class of mRNA transcripts should be detected in both in COS7 cells, which were subsequently viewed live using a SGs and PBs at different stages of its processing. To test this heated stage and inverted confocal microscope. The extremely prediction, we expressed a -globin mRNA containing the motile nature of PBs (Yang et al., 2004) required that each MS2-binding site in its 3 untranslated region (pEF-7B- frame be made from a volume-rendered image derived from MS2bs) together with a fusion protein comprised of GFP, MS2 10 z-axis sections (see Materials and methods) so as to visual- coat protein, and a nuclear localization signal (Fig. 6 A, GFP- ize all of the PBs within each cell. Cells that were cotransfected MS2-NLS). Transfection of GFP-MS2 alone or with a globin with RFP-DCP1a (PB marker) and GFP–TIA-1 (SG marker) reporter lacking the MS2-binding site resulted in a signal ex- display spontaneous SGs in 30–70% of the transfectants, and clusively localized to the nucleus (Rook et al., 2000; unpublished these SGs frequently associate with one or more PBs. When fol- data). When GFP-MS2 is cotransfected with the globin reporter lowed over time (Fig. 7 A and Video 1, available at http:// containing the MS2-binding site, nuclear export of the tethered www.jcb.org/cgi/content/full/jcb.200502088/DC1), some “at- GFP signal is observed in 2–10% of transfected cells; only in tached” PBs (Fig. 7 A, white arrowheads) remain stably bound cells expressing high amounts of globin-MS2 is the tethered GFP to SGs. Other PBs (Fig. 7 A, yellow arrows) appear intermit- exported from the nucleus. Cytoplasmic GFP signal is found in tently attached to SGs or move freely in the cytoplasm without SGs and PBs, as shown in Fig. 6 (B and C). The RNA-tethered interacting with SGs. “Free” or unbound PBs exhibit greater signal is equally distributed between PBs (Fig. 6, yellow arrows) motility than SG-associated PBs, even when both types of PBs and SGs (Fig. 6, white arrowheads), as shown by colocalization are present in the same cell. SGs exhibit fission, fusion, and STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 877 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. occasional dispersal, which are properties consistent with on- sought to verify the ability of TTP to induce SG–PB fusion by going sorting and export of their contents. Similar results are using GFP-G3BP to induce SGs and test whether the coexpres- obtained when GFP-G3BP is used to induce and detect SG–PB sion of nonfluorescent TTP would alter the interaction of interactions. The expression of FAST-YFP (Fig. 7, green) with SGs with PBs. As shown in Video 4 (available at http:// RFP-DCP1a (Fig. 7, red) resulted in the incorporation of FAST www.jcb.org/cgi/content/full/jcb.200502088/DC1) and Fig. 7 C, into SGs in some cells but into PBs in other cells (Video 2, avail- the coordinate expression of GFP-G3BP, RFP-DCP1a, and able at http://www.jcb.org/cgi/content/full/jcb.200502088/DC1). myc-tagged vector does not alter the relationship between These data are consistent with the distribution of endogenous GFP-G3BP SGs and PBs; both free and interacting structures FAST, as shown in Figs. 1 and 2, and suggest that FAST (like are observed. However, cells expressing myc-tagged TTP with eIF4E) may be present in both structures. Unfortunately, YFP- GFP-G3BP and RFP-DCP1a (Fig. 7 D and Video 5) display a eIF4E constructs fail to recapitulate the localization of endoge- nearly complete recruitment of PBs to SGs. Similar results nous or FLAG-tagged eIF4E, so we are unable to examine its were seen using GFP–TIA-1 as the SG inducer/marker, as the distribution between SGs and PBs in real time. coexpression of GFP–TIA-1 with TTP promotes interactions We reasoned that SG–PB interaction may be influenced between SGs and PBs (unpublished data). by the amount of mRNA being transported from the SG into We then asked whether FAST, XRN1, eIF4E, or the TTP- the PB and hypothesized that the expression of TTP, an SG- related protein BRF1 would promote interactions between GFP- associated protein that promotes mRNA decay (Stoecklin et al., G3BP SGs and PBs. Fig. 8 depicts GFP-G3BP and RFP-DCP1a 2004), might increase SG–PB interactions by increasing the cotransfected with one of the following: empty vector (Fig. 8 A), amount of mRNA routed from SGs into PBs. As shown in Fig. FAST-myc (Fig. 8 B), FLAG-XRN1 (Fig. 8 C), FLAG-eIF4E 7 B and Video 3 (available at http://www.jcb.org/cgi/content/ (Fig. 8 D), TTP-myc (Fig. 8 E), or FLAG-BRF1 (Fig. 8 F). Only full/jcb.200502088/DC1), the expression of YFP-TTP with TTP (Fig. 8 E) and its close homologue BRF1 (Fig. 8 F) are RFP-DCP1a results in the quantitative and stable association of found to induce SG–PB fusion. Remarkably, BRF1 promotes the PBs with SGs. Remarkably, the YFP-TTP SGs appear to en- complete engulfment of large PBs by SGs, whereas in TTP capsulate single or multiple PBs. Although fusion events be- transfectants, smaller, more numerous PBs are embedded in a tween these conglomerate SG–PB structures were observed, single SG. Although not affecting the SG–PB relationship, fission events were rare. The data indicate that both the number eIF4E overexpression appears to reduce the size of PBs but in- and duration of SG–PB interactions is stabilized by the expres- creases their number (Fig. 8 D, red), whereas XRN1 expression sion of TTP. As YFP-TTP is also diffusely present in the cyto- results in fewer, larger PBs (Fig. 8 C, red). Altogether, the data plasm, making the borders of the SG difficult to determine, we indicate that the expression of different SG–PB components 878 JCB • VOLUME 169 • NUMBER 6 • 2005 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. affects their size and interaction. Most important, the expression selected SG (Fig. 9 A, arrow) was obtained before bleaching of TTP and BRF1, which functionally accelerate mRNA decay (Fig. 9, pink tracing) and was subsequently bleached at the po- in these cells, also promote the interaction of SGs with PBs. sition indicated by the vertical yellow line. The dark blue line indicates the scan intensity taken immediately after bleaching. Dynamics of different SG–PB A scan taken 30 s later (Fig. 9, aqua tracing) reveals the com- components in real time using FRAP plete recovery of GFP–TIA-1 fluorescence. The FRAP be- The TTP- and BRF1-induced fusion of SGs and PBs could either havior of GFP–PABP-1, shown in Fig. 9 B, also recapitulates be direct (i.e., a physical linkage between SG–PB structural previous findings (Kedersha et al., 2000). GFP–PABP-1 exhibits components) or indirect (i.e., by shunting more substrate slower and less complete recovery than does GFP–TIA-1 be- mRNA destined for decay through SGs into PBs). To analyze cause only 60% of SG-associated GFP–PABP-1 fluorescence the dynamic nature of SG and PB components within these recovers after 30 s. This suggests that TIA-1 and PABP-1 are structures, we used FRAP using GFP and/or YFP-tagged ver- not quantitatively present in the same mRNP complexes. sions of different SG–PB-associated proteins. Previous studies TTP overexpression generates spontaneous SGs, and ar- (Kedersha et al., 2000) demonstrated that GFP–TIA-1 rapidly senite treatment induces TTP to leave SGs (Stoecklin et al., moves in and out of SGs. In these experiments, 90% recovery 2004) but not PBs (unpublished data), which is an effect depen- of the bleached signal occurred within 10 s. We used this sys- dent on TTP phosphorylation that mediates its binding to 14-3-3 tem to analyze the FRAP kinetics of representative members of (Stoecklin et al., 2004). YFP-TTP SG bleaching (Fig. 9 C) is the “SG-only proteins” GFP–PABP-1 and GFP-G3BP, the followed by rapid and complete recovery; this result was consis- “SG–PB shared proteins” YFP-TTP and FAST-YFP, and the tently obtained in 10 cells and was the same when either large “PB-only proteins” GFP-GW182 and YFP-DCP1a. As shown (probable SGs) or small foci (probable PBs) were bleached. in Fig. 9 A, GFP–TIA-1 forms large, distinct SGs in response to Cells coexpressing RFP-DCP1a were used to verify the rapid arsenite treatment. A linear scan of the region containing a kinetics of YFP-TTP that was unambiguously localized to STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 879 PBs (Fig. S1, available at http://www.jcb.org/cgi/content/full/ of poly(A) RNA at PBs, supporting the contention that these jcb.200502088.DC1). Thus, YFP-TTP rapidly moves in and out foci are sites of mRNA degradation (Cougot et al., 2004a). of both PBs and SGs, suggesting that TTP constitutes a transient Recently, dual immunofluorescence using antibodies tether between mRNA and the decay machinery. against the SG marker TIA-1 and the PB marker DCP1a SG-associated GFP-G3BP also displays rapid, complete re- clearly showed that SGs and GW bodies/PBs are distinct and covery (Fig. 9 D) that is unaltered by arsenite treatment (unpub- independent cytoplasmic structures (Cougot et al., 2004a), but lished data). FAST-YFP induces spontaneous SGs in 30–70% of the relationship between them has not been addressed. Our re- transfectants and is localized to PBs in most of the remaining sults confirm that SGs and PBs are compositionally and mor- transfectants. As shown in Fig. 9 E, its FRAP kinetics are very phologically distinct entities, each of which can be assembled slow, and recovery is minimal in all cells that were tested (n  in the absence of the other and are compositionally distinct. 20) and unaltered by arsenite (unpublished data). The slow kinet- However, there are strong spatial and functional links be- ics of FAST and its presence in both SGs and PBs suggests that it tween SGs and PBs. First, oxidative stress induces the assem- may play a scaffolding role in organizing SGs and PBs. bly of both SGs and PBs and promotes interactions between The overexpression of YFP-DCP1a induces very large them. Second, time-lapse microscopy reveals that a subset of PBs in many cells and more normal-sized PBs in others. PBs PBs is stably tethered to SGs, whereas another subset is inde- normally exhibit size variation depending on metabolic state pendent and highly mobile within the cytoplasm. Third, sev- (Sheth and Parker, 2003) and cell cycle (Yang et al., 2004). eral proteins (i.e., FAST, XRN1, eIF4E, and TTP) and mRNAs Fig. 9 F shows photobleaching of a medium-sized YFP-DCP1a (i.e., globin-MS2 reporter) are found in both SGs and PBs. PB, which exhibits kinetics similar to those of GFP–PABP-1. Fourth, SGs and PBs are induced to fuse by the overexpres- However, YFP-DCP1a FRAP kinetics is variable: the ex- sion of TTP or BRF1, which are RNA-binding proteins that change rate is rapid in small PBs but is slower in larger PBs. promote mRNA decay and are components of both SGs and GFP-GW182 exhibits very slow FRAP recovery (Fig. 9 G), PBs. Finally, pharmacologic inhibitors of translational elon- similar to that of FAST. The transit time of PABP-1 and gation promote the disassembly of both structures, suggesting DCP1a is intermediate between that of TIA-TTP-G3BP and that both PBs and SGs are assembled from translationally GW182-FAST, suggesting that PABP-1 and DCP1a either competent mRNA. shuttle in and out of SGs and PBs independently of the RNA The SG–PB fusion induced by TTP and BRF1 suggests substrates or are removed from the transcripts during mRNP that these proteins regulate the dynamic interactions between remodeling that occurs in tandem with their movement. SGs and PBs. Both TTP and BRF1 promote the degradation of mRNAs bearing ARE in their 3 untranslated regions. TTP has been proposed to direct these transcripts to exosomes, which Discussion are degradative machines that promote 3–5 exonucleolytic Stress-induced phosphorylation of eIF2 results in stalled degradation of deadenylated transcripts (Chen et al., 2001). translational initiation such that actively translating ribosomes The ability of TTP to promote interactions between PBs and “run off” their transcripts, resulting in polysome disassembly SGs suggests that this class of destabilizing factor might also concurrent with SG assembly (Anderson and Kedersha, 2002; promote 5–3 mRNA degradation at the SG, which is consis- Kedersha and Anderson, 2002). SG assembly is regulated by tent with recent data suggesting that TTP and BRF1 comprise one or more RNA-binding proteins, including TIA-1 (Gilks et molecular links between ARE-containing mRNAs and mRNA al., 2004), G3BP (Tourriere et al., 2003), Fragile X Mental Re- decay enzymes present in PBs (Lykke-Andersen and Wagner, tardation protein (Mazroui et al., 2002), survival of motor neu- 2005). Our data indicate that this molecular link has morpho- rons protein (Hua and Zhou, 2004), and/or TTP (Stoecklin et logical as well as functional consequences. It is important to al., 2004). Another cytoplasmic mRNP domain termed the note that the SGs observed in the real-time experiments are in- GW body was first visualized by using a patient-derived au- duced by the overexpression of either TTP or G3BP. As such, toantisera reactive with GW182, a 182-kD RNA-binding pro- they may not have the same composition and function as arse- tein (Eystathioy et al., 2002). GW bodies contain RNA, but nite or heat-induced SGs. Nevertheless, the TTP- and BRF-1– unlike SGs, GW bodies are prominent in actively growing un- induced stabilization of PB–SG interactions reveals a unique stressed cells (Eystathioy et al., 2002). Convergent studies mechanism whereby this class of protein might regulate from several laboratories have shown that GW bodies contain mRNA metabolism. proteins involved in mRNA degradation, including the decap- The presence of eIF4E in PBs is somewhat surprising, as ping enzymes DCP1a and 2, a heptamer of Lsm proteins re- it binds to the seven-methyl guanosine cap and is thought to quired for mRNA decapping, and the exonuclease XRN1 protect the integrity of the cap (Ramirez et al., 2002; Liu et al., (Eystathioy et al., 2002, 2003; Ingelfinger et al., 2002; Cougot 2004). In S. cerevisiae, eIF4G and eIF4E are removed from et al., 2004a,b; Yang et al., 2004). In Saccharomyces cerevisiae, mRNA before the recruitment of DCP1 and decapping the accumulation of nondegradable mRNAs at compositionally (Tharun and Parker, 2001). Our data show that eIF4G, PABP, similar cytoplasmic foci (PBs) implicated these phylogeneti- and eIF3 are present in SGs but not in PBs, suggesting that cally conserved foci in the process of mRNA degradation eIF4G is removed from the cap before its transit into PBs, (Sheth and Parker, 2003). In mammalian cells, the interference whereas eIF4E remains bound to the cap. Because the rate at RNA–mediated knockdown of XRN1 enhances the accumulation which eIF4E dissociates from capped mRNA is accelerated in 880 JCB • VOLUME 169 • NUMBER 6 • 2005 the absence of eIF4G (Haghighat and Sonenberg, 1997), In the model shown in Fig. 10, we posit that SGs contain capped mRNA may be liberated within the PB, allowing transcripts routed from disassembling polysomes in accord DCP1a/2-mediated decapping. with the absolute requirement for eIF2 phosphorylation in SG We have proposed that SGs are sites of mRNA triage in assembly. This idea is in agreement with the studies of Thomas which individual transcripts are sorted for storage, reinitia- et al. (2005), who demonstrated that newly synthesized mRNAs tion, or degradation (Anderson and Kedersha, 2002; Kedersha are not present in SGs. Error-containing transcripts selected for and Anderson, 2002). This model predicts that those mRNAs nonsense-mediated decay during the pioneer round of transla- targeted for decay will be exported from the SG to sites of tion (before polysome assembly) may contribute to free PBs, as mRNA decay such as PBs. The aforementioned interactions nonsense-mediated decay occurs via decapping and 5–3 de- between SGs and PBs may allow mRNA to move from the cay (Maquat, 2002; Neu-Yilik et al., 2004) and is inhibited by SG to the PB. Two lines of evidence suggest the direction of cycloheximide. SGs induced by stress are likely to contain a this process. First, arsenite induces the formation of juxta- mixture of transcripts, but SGs induced by the overexpression posed SGs and PBs, and subsequent emetine treatment forces of different RNA-binding proteins (e.g., TIA, G3BP, and TTP) the disassembly of SGs before the disassembly of PBs. Sec- are likely to differ in their mRNA composition. For example, ond, heat shock induces SG formation before PB formation. TIA-induced SGs are likely enriched for TIA-bound transcripts Initially, eIF4E is concentrated at SGs in cells lacking PBs, that are targeted for translational silencing, whereas TTP- but in the continued presence of heat, SGs are disassembled, induced SGs may be enriched for TTP-bound transcripts that and PBs containing eIF4E are concomitantly assembled. are targeted for decay. Thus, TTP-induced SG–PB fusion occurs These results imply (but do not mandate) that eIF4E is first because TTP-induced SGs are assembled from mRNAs se- incorporated into SGs and later translocates into PBs. As lected by TTP binding for rapid decay. Given the very rapid eIF3, eIF4G, PABP-1, small ribosomal subunits, and G3BP flux of TTP within SGs and PBs assessed by photobleaching, are found in SGs but not in PBs, these proteins must be re- it is unlikely that TTP itself constitutes a stable component of moved from mRNA before its export from the SG. Because either compartment. It is more likely that TTP serves to de- eIF4G and PABP-1 are directly involved in mRNA circular- liver its mRNA cargo to PBs by interacting with stable com- ization, it is probable that mRNAs exported from SGs into ponents of these particles (Lykke-Andersen and Wagner, PBs are decircularized before translocation, which is concur- 2005). However, FAST has the properties of a putative scaf- rent with their deadenylation (the activation step for mRNA fold protein that might stabilize SG–PB interactions; it dis- decay by both mRNA decay pathways). Finally, as eIF4E and plays a very slow exchange rate, as measured by photobleach- TTP are components of both SGs and PBs, these RNA-bind- ing, lacks known RNA binding motifs, nucleates both SGs ing proteins may remain with mRNA as it moves from the SG and PBs upon overexpression, and interacts with TIA-1. Pos- to the PB. sibly, TTP or TTP-associated proteins promote SG–PB fusion Figure 10. Hypothetical model of the rela- tionship between SGs and PBs. Proteins found exclusively in SGs are shown in yellow; pro- teins found in both SGs and PBs are depicted in green; and proteins restricted to PBs are shown in blue type. STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 881 For pcDNA3-Flag-BRF1, the human BRF1 cDNA was excised as a by interacting either directly or indirectly with FAST to re- BamHI/blunt–XbaI fragment from bsdHis-BRF1 (Stoecklin et al., 2002) model the SG–PB scaffold. and inserted into the BamHI/blunt–XbaI sites of pcDNA3-Flag-Bak (a gift The data presented in this study establish that SGs and from T. Chittenden, ImmunoGen, Inc., Cambridge, MA). For pcDNA3- Flag-eIF4E, eIF4E was amplified from plasmid pcDNA3-eIF4E (a gift from PBs are discrete cytoplasmic structures that share some protein D. Dixon, University of South Carolina, Columbia, SC) using primers 5- and mRNA components as well as some functional properties. TTTGAATTCGCGACTGTCGAACCG-3 and 5-TGTTCTAGATTAAACAA- Both structures are induced by stress but are regulated by dis- CAAACCTATTTTTAG-3, digested with EcoRI and XbaI, and ligated into the EcoR–XbaI sites of pCDNA3-Flag. GFP-G3BP was a gift from J. Tazi tinct signaling events, and each can exist without the other. PBs (Centre National de la Recherche Scientifique, Montpellier, France). and SGs exhibit a high degree of motility when independent pGFP-GWaa313-1709 (Eystathioy et al., 2002) and FLAG-FAST (Li et al., but appear less motile when they are tethered together, and 2004b) were described previously. For pEF-FAST-myc, FAST was ampli- fied using primers 5-CCACCATGGAATAGCCACCATGAGGAGGC- their interaction is promoted by the mRNA-destabilizing pro- CGCGGGGGGAA-3 and 5-ATAAGAATGCGGCCGCGCCCCCT- tein TTP. The Janus-like juxtapositioning of SGs and PBs is TCAGGCCCCCAGCG-3, digested with NcoI and NotI, and ligated into reminiscent of the relationship between the nuclear gemini of the NcoI–NotI sites of pEF-myc. To make FAST-YFP, the coding region of FAST was amplified using primers 5-TGTGAGATCTAGTAGGAGGC- coiled bodies and Cajal bodies (Dundr et al., 2004), a case in CGCGGGGG-3 and 5-CCGAAGCTTGCCCCCTTCAGGCCC-3, di- which the morphology of linked compartments arises from or- gested with BglII and HindIII, and ligated into pEF-YFP-N1 vector (CLON- dered, compartmentalized stages in nuclear small nuclear RNP TECH Laboratories) that was digested with the same enzymes. biogenesis. The dynamic relation between SGs and PBs reiter- siRNA transfection ates the importance of compartmentalization in regulating the Du145 and HT1080 cells were transfected with 1.25 l/ml of Lipo- fate of cytoplasmic mRNA. fectamine 2000 and 100 nM siRNA duplexes for 48 h. Subsequently, cells were reseeded and, after 8 h, were transfected again with siRNA for another 40–44 h. siRNAs were designed using published recommenda- Materials and methods tions (Reynolds et al., 2004) and were purchased from Ambion. The fol- lowing target sequences (sense strand) were chosen: control siRNA (D0), Cell lines 5-GCAUUCACUUGGAUAGUAA-3; and Lsm4 siRNA (L4), 5-ACA- COS7, HeLa, and DU145 cells were obtained from the American Type ACUGGAUGAACAUUAA-3. Culture Collection, and U2OS cells were obtained from J. Blenis (Harvard Medical School, Boston, MA). HT1080 cells were obtained from C. Mo- RT-PCR roni (University of Basel, Basel, Switzerland). Cells were maintained in HT1080 cells were transfected with siRNA D0 or L4. Total cytoplasmic DME containing 10% FBS at 7.0% CO RNA was extracted, and 5 g RNA was used for reverse transcription us- ing oligo-dT and MMLV-RT (Promega). cDNA was purified with the Antibodies Qiaquick PCR purification kit (QIAGEN), and one tenth was used per PCR Antibodies against eIF4E (monoclonal and rabbit polyclonal), eIF4G, reaction using Taq polymerase (2.5 U/50 l) and solution Q (QIAGEN). eIF3b, myc, TIA-1, FXR1, and TIAR were obtained from Santa Cruz Bio- Annealing was performed at 56C using primers 5-CCTTGTCACTGCT- -GAGACTGTGGAGCGGAATC-3 for the amplifica- GAAGACG-3 and 5 technology, Inc. Phospho-specific anti-eIF2 was obtained from StressGen tion of Lsm4 and 5-GGTGGTCGGAAAGCTATC-3 and 5-GAGCTTCT- Biotechnologies. Human autoantisera against GW182 was an index se- TATAGACACCAG-3 for the amplification of ribosomal protein S7 as a rum from a 48-yr-old female with mixed motor and sensory neuropathy, control. Parallel reactions were performed using 15, 20, 25, 30, and 35 which was obtained from Advanced Diagnostics Laboratory. Antibodies PCR cycles, and the products were resolved by 1.5% agarose gel electro- against DCP1a and XRN-1 were previously described (Lykke-Andersen phoresis and were stained with ethidium bromide. and Wagner, 2005). Antisera against FAST (anti–FAST-N) were de- scribed previously (Li et al., 2004a). Monoclonal anti-myc was a gift from L. Klickstein (Brigham and Woman’s Hospital, Boston, MA). Anti-HA was Fluorescence microscopy obtained from Covance Research Products. Anti–PABP-1 was a gift from Cells were stained and processed for fluorescence microscopy as previ- G. Dreyfuss (University of Pennsylvania, Philadelphia, PA). Anti-G3BP was ously described (Gilks et al., 2004). Conventional fluorescence micros- a gift from I. Gallouzi (McGill University, Montreal, Canada). Anti- copy was performed using a microscope (model Eclipse E800; Nikon) Dcp2was a gift of B. Seraphin (Centre de Genetique Moleculaire, Gif-sur- with epifluorescence optics and a digital camera (model CCD-SPOT RT; Yvette, France) and M. Kildejian (Rutgers University, Piscataway, NJ). Diagnostic Instruments). The images were compiled using Adobe Photo- shop software (v7.0). Plasmids Plasmids encoding FLAG-DCP1a, FLAG-XRN1, and FLAG-DCP2 were pre- Confocal microscopy viously described (Lykke-Andersen and Wagner, 2005). To make pEYFP- Cells transfected with combinations of GFP- and RFP-tagged vectors were DCP1a, the human DCP1a cDNA was amplified from plasmid pcDNA3- viewed live at 37C using an inverted microscope (model TE2000-U; Ni- Flag-DCP1a by using primers 5-GTGCTCGAGCTGAGGCGCTGAGT-3 kon) equipped with a 60 oil objective Cfi planapo lens (NA 1.40; Ni- and 5-GTGGAATTCTCATAGGTTGTGGTTG-3 and was ligated as an kon) and a confocal system (model C-1; Nikon). Each image was volume XhoI–EcoRI fragment into the Xho–EcoRI sites of pEYFP-C1 (CLONTECH rendered from 10 Z-stacks of 0.85-m thickness using EZ-C1 software (Ni- Laboratories). To make mRFP-DCP1a, monomeric RFP (provided by R.Y. kon). Timed series were acquired at a rate of 1 min per frame; each frame Tsien, Howard Hughes Medical Institute, University of California, San Di- represents a volume-rendered image. Videos are shown in the supplemen- ego, La Jolla, CA; Campbell et al., 2002) was amplified using primers tal material; frames taken 10 min apart are shown in Fig. 7. Videos were 5-ATTCATACCGGTCCACCATGGCCTCCTCCG-3 and 5-TAAATTCTC- made using Adobe Image Ready software (v7.0) to animate the volume- GAGAGGCGCCGGTGGAG-3 and was ligated as an AgeI–XhoI frag- rendered TIF images exported from the EZ-C1 software (Nikon). ment into the AgeI–XhoI sites of pEYFP-DCP1a, thereby replacing YFP. GFP-MS2-NLS was a gift from K. Kosik (University of California, Santa FRAP photobleaching analysis Barbara, Santa Barbara, CA) and was previously described (Rook et al., Fluorescently tagged constructs of SG and PB proteins were tested to de- 2000). For pEF-7B-MS2bs, the T7-tagged rabbit -globin gene containing termine whether they exhibited localization that was compatible with their a sixfold repeat of the MS2bs was excised from plasmid pcDNA3-7B- endogenous or (in the case of TTP) FLAG-tagged counterparts; those fail- MS2bs as a HindIII/blunt–XbaI fragment and ligated into the NcoI/blunt– ing to meet this criterion were not used. COS7 cells were transiently trans- XbaI sites of pEF/myc/cyto (Invitrogen). The plasmid pcDNA3-TTP-mycHis fected with the indicated constructs using SuperFect (QIAGEN), were re- was made as described previously (Stoecklin et al., 2004). For YFP-TTP, plated onto glass coverslips after 10 h of transfection, and were analyzed murine TTP cDNA was amplified from plasmid pcDNA3-TTP-mycHis by us- 38–46 h posttransfection. Transfectants were viewed using a 60 oil ob- ing primers 5-TATCAAGCTTATGAATTCCGTTCC-3 and 5-TCAGATC- jective (NA 1.40) on an interactive laser cytometer (model Ultima; Merid- CTCTTCTGAGATG-3 digested with HindIII and XbaI and inserted into the ian Instruments). Appropriate cells were located, and images were taken HindIII–XbaI sites of pEYFP-C1 (CLONTECH Laboratories). using a two-dimensional scanning mode before bleaching. Selected SGs 882 JCB • VOLUME 169 • NUMBER 6 • 2005 or PBs (Fig. 9, arrows) were photobleached for 1 s at 0.5 mW of power yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3 to 5 exonucleases of the exosome using a beam radius of 0.7 m and an excitation wavelength of 488 nm. complex. EMBO J. 17:1497–1506. Fluorescence emission was detected at 530 15 nm. The results shown Jacobson, A. 2004. Regulation of mRNA decay: decapping goes solo. Mol. Cell. were representative of three independent transfections in which a total of 15:1–2. 10 different cells were analyzed. In some cases (see Fig. S1), two-color scans were obtained by simultaneously exciting both fluorophores at 488 Kedersha, N., and P. Anderson. 2002. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. Biochem. Soc. Trans. nm and separating the two emissions using a 575-nm dichroic filter and 30:963–969. the appropriate emission filters (green emission 530 15 nm; red emis- sion 630 nm). Kedersha, N.L., M. Gupta, W. Li, I. Miller, and P. Anderson. 1999. RNA-bind- ing proteins TIA-1 and TIAR link the phosphorylation of eIF-2 to the assembly of mammalian stress granules. J. Cell Biol. 147:1431–1441. Online supplemental material Fig. S1 shows photobleaching of PB-localized YFP-TTP. Video 1 shows the Kedersha, N., M.R. Cho, W. Li, P.W. Yacono, S. Chen, N. Gilks, D.E. Go- lan, and P. Anderson. 2000. Dynamic shuttling of TIA-1 accompanies dynamics of GFP–TIA-1 SGs and PBs; Video 2 shows the dynamics of the recruitment of mRNA to mammalian stress granules. J. Cell Biol. FAST-YFP and RFP-DCP1a PBs; Video 3 shows YFP-TTP and RFP-DCP1 PBs; 151:1257–1268. and Video 4 shows GFP-G3BP SGs and RFP-DCP1 PBs, all in real time. Kedersha, N.L., S. Chen, N. Gilks, W. Li, I.J. Miller, J. Stahl, and P. Anderson. Video 5 shows that TTP coexpression promotes fusion between GFP-G3BP 2002. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-defi- SGs and RFP-DCP1 PBs. Online supplemental material is available at http: cient preinitiation complexes are core constituents of mammalian stress //www.jcb.org/cgi/content/full/jcb.200502088.DC1. granules. Mol. Biol. Cell. 13:195–210. We thank R. Tsien for the mRFP construct, K. Kosik for GFP-MS2-NLS, J. Tazi Krishnamoorthy, T., G.D. Pavitt, F. Zhang, T.E. Dever, and A.G. Hinnebusch. 2001. Tight binding of the phosphorylated alpha subunit of initiation fac- for GFP-G3BP, M. Kiledjian and B. Serephin for antibodies, and H. Gilbert tor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide ex- and M. Gurish for help with the confocal microscopy. We thank W. Li change factor eIF2B is required for inhibition of translation initiation. (Brigham and Women’s Hospital, Boston, MA) for the FAST-myc construct and Mol. Cell. Biol. 21:5018–5030. N. Gilks and members of the Anderson lab for lively discussions. Laroia, G., R. Cuesta, G. Brewer, and R.J. Schneider. 1999. Control of mRNA de- This work was supported by the National Institutes of Health grants cay by heat shock-ubiquitin-proteasome pathway. Science. 284:499–502. AI50167, AR051472, and AI33600 (to P. Anderson); DK42394 and Li, W., N. Kedersha, S. Chen, N. Gilks, G. Lee, and P. Anderson. 2004a. FAST HL52173 (to R.J. Kaufman); GM 066811 (to J. Lykke-Andersen); HL32854 is a BCL-X(L)-associated mitochondrial protein. Biochem. Biophys. Res. and HL070819 (to D.E. Golan) and the Canadian Institutes for Health Re- Commun. 318:95–102. search Grant MOP-38034 (to M.J. Fitzler). M.J. Fitzler holds the Arthritis Soci- Li, W., M. Simarro, N. Kedersha, and P. Anderson. 2004b. FAST is a survival ety Chair at the University of Calgary. J. Lykke-Andersen is a Pew Scholar. protein that senses mitochondrial stress and modulates TIA-1-regulated changes in protein expression. Mol. Cell. Biol. 24:10718–10732. Submitted: 14 February 2005 Liu, S.W., X. Jiao, H. Liu, M. Gu, C.D. Lima, and M. Kiledjian. 2004. Functional Accepted: 16 May 2005 analysis of mRNA scavenger decapping enzymes. RNA. 10:1412–1422. Long, R.M., and M.T. McNally. 2003. mRNA decay: x (XRN1) marks the spot. Mol. Cell. 11:1126–1128. References Lykke-Andersen, J., and E. Wagner. 2005. 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Jacobs, J.S., A.R. Anderson, and R.P. Parker. 1998. The 3 to 5 degradation of MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule as- STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 883 sociation and ARE-mRNA decay. EMBO J. 23:1313–1324. Tharun, S., and R. Parker. 2001. Targeting an mRNA for decapping: displace- ment of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Mol. Cell. 8:1075–1083. Thomas, M.G., L.J. Tosar, M. Loschi, J.M. Pasquini, J. Correale, S. Kindler, and G.L. Boccaccio. 2005. Staufen recruitment into stress granules does not affect early mRNA transport in oligodendrocytes. Mol. Biol. Cell. 16:405–420. Tourriere, H., K. Chebli, L. Zekri, B. Courselaud, J.M. Blanchard, E. Bertrand, and J. Tazi. 2003. The RasGAP-associated endoribonuclease G3BP as- sembles stress granules. J. Cell Biol. 160:823–831. Yang, Z., A. Jakymiw, M.R. Wood, T. Eystathioy, R.L. Rubin, M.J. Fritzler, and E.K. Chan. 2004. GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation. J. Cell Sci. 117: 5567–5578. 884 JCB • VOLUME 169 • NUMBER 6 • 2005 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

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

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JCB: ARTICLE Stress granules and processing bodies are dynamically linked sites of mRNP remodeling 1 1 1 2 3 4 Nancy Kedersha, Georg Stoecklin, Maranatha Ayodele, Patrick Yacono, Jens Lykke-Andersen, Marvin J. Fritzler, 5 5 2 1 Donalyn Scheuner, Randal J. Kaufman, David E. Golan, and Paul Anderson 1 2 Division of Rheumatology and Immunology and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Hematology Division, Brigham and Women’s Hospital, Boston, MA 02115 Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada University of Michigan Medical Center and Howard Hughes Medical Institute, Ann Arbor, MI 48109 tress granules (SGs) are cytoplasmic aggregates Fas-activated serine/threonine phosphoprotein, XRN1, of stalled translational preinitiation complexes that eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, Saccumulate during stress. GW bodies/processing eIF4G, and PABP-1 are restricted to SGs, whereas bodies (PBs) are distinct cytoplasmic sites of mRNA DCP1a and 2 are confined to PBs. SGs and PBs also can degradation. In this study, we show that SGs and PBs are harbor the same species of mRNA and physically associate spatially, compositionally, and functionally linked. SGs with one another in vivo, an interaction that is promoted and PBs are induced by stress, but SG assembly requires by the related mRNA decay factors TTP and BRF1. We eIF2 phosphorylation, whereas PB assembly does not. propose that mRNA released from disassembled poly- They are also dispersed by inhibitors of translational somes is sorted and remodeled at SGs, from which selected elongation and share several protein components, including transcripts are delivered to PBs for degradation. Introduction In response to environmental stress, eukaryotic cells reprogram sults suggest that SGs are sites of mRNA triage at which their translational machinery to allow the selective expression mRNP complexes are monitored for integrity and composition of proteins required for viability in the face of changing condi- and are then routed to sites of reinitiation, degradation, or stor- tions. During stress, mRNAs encoding constitutively expressed age (Anderson and Kedersha, 2002; Kedersha and Anderson, “housekeeping” proteins are redirected from polysomes to dis- 2002). During stress, mRNA continues to be directed to sites of Met crete cytoplasmic foci known as stress granules (SGs), a pro- reinitiation, but in the absence of eIF2–GTP–tRNA , it shut- cess that is synchronous with stress-induced translational arrest tles back to SGs, where it accumulates (Kedersha et al., 2000). (Anderson and Kedersha, 2002; Kedersha and Anderson, mRNAs within SGs are not degraded, making them available 2002). Both SG assembly (Kedersha et al., 1999) and transla- for rapid reinitiation in cells that recover from stress. The tional arrest (Krishnamoorthy et al., 2001) are initiated by the observation that labile mRNAs are stabilized during stress phosphorylation of translation initiation factor eIF2, which (Laroia et al., 1999; Bolling et al., 2002) suggests that some as- Met reduces the availability of the eIF2–GTP–tRNA ternary pect of the mRNA degradative process is disabled during the complex that is needed to initiate protein translation. Drugs that stress response. Thus, the accumulation of mRNA at SGs may stabilize polysomes (e.g., emetine) cause SG disassembly, be a consequence of both stress-induced translational arrest and whereas drugs that dismantle polysomes (e.g., puromycin) pro- stress-induced mRNA stabilization. mote the assembly of SGs, indicating that mRNA moves be- Although the process of stress-induced mRNA stabiliza- tween polysomes and SGs (Kedersha et al., 2000). These re- tion is poorly understood, it likely involves the inactivation of one or more mRNA decay pathways. Two major mechanisms of mRNA degradation are active in eukaryotic cells (Decker Correspondence to Nancy Kedersha: [email protected] and Parker, 2002). In the first pathway, deadenylated tran- Abbreviations used in this paper: ARE, adenine/uridine-rich destabilizing ele- scripts are degraded by a complex of 3–5 exonucleases ments; FAST, Fas-activated serine/threonine phosphoprotein; PB, processing known as the exosome. In vitro studies using cell extracts reveal body; SG, stress granule; siRNA, small interference RNA; TIA, T cell intracellular antigen; TIAR, TIA related; TTP, tristetraprolin. that some mRNAs bearing adenine/uridine-rich destabilizing el- The online version of this article contains supplemental material. ements (AREs) in their 3 untranslated regions are degraded by © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 169, No. 6, June 20, 2005 871–884 http://www.jcb.org/cgi/doi/10.1083/jcb.200502088 JCB 871 THE JOURNAL OF CELL BIOLOGY 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 M arsenite for 30 min (B and F); ex- posed to 20 M clotrimazole (Sigma Aldrich) for 1 h (C and G); or exposed to heat (44C) 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 clotrima- zole (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 repro- duced at the right as replicate views of the same field showing eIF4E, DCP1a, eIF3, and the merged view. this 3–5 exosome-dependent pathway (Jacobs et al., 1998; geted knockdown of XRN1 results in the accumulation of Chen et al., 2001; Mukherjee et al., 2002). The second path- poly(A) -containing mRNA at these sites, suggesting that this way entails the removal of the seven-methyl guanosine cap mRNA decay pathway is conserved in both lower and higher from the 5 end of the transcript by the DCP1–DCP2 complex eukaryotes. Although the composition of GW bodies/PBs is (Long and McNally, 2003; Jacobson, 2004), allowing 5–3 somewhat different in lower and higher eukaryotes, because exonucleolytic degradation by XRN1 (Stevens, 2001). In they share the ability to process mRNA, we will provisionally yeast, components of this 5–3 decay pathway are concen- refer to these foci as PBs. Interestingly, metabolic inhibitors trated at discrete cytoplasmic foci known as processing bodies that promote (e.g., puromycin) or inhibit (e.g., emetine) the (PBs; Sheth and Parker, 2003). Yeast genetic studies reveal assembly of SGs in mammalian cells have similar effects on that mRNA decay intermediates accumulate at PBs when nor- the assembly of both yeast and mammalian PBs. These results mal decay is blocked, suggesting that PBs are sites of decap- indicate that both SGs and PBs are sites at which mRNA accu- ping and 5–3 degradation (Sheth and Parker, 2003). Studies mulates after polysome disassembly. in mammalian cells have revealed similar structures that In this study, we catalog the protein composition of SGs contain DCP1/2, XRN1, GW182, and Lsm1–7 heptamer and PBs and report several links between these cytoplasmic (Eystathioy et al., 2002, 2003; Ingelfinger et al., 2002; Cougot subdomains. DCP1a/2 and GW182 are components of PBs but et al., 2004a,b; Yang et al., 2004). In mammalian cells, the tar- not of SGs, whereas most initiation factors (e.g., eIF3, eIF4G, 872 JCB • VOLUME 169 • NUMBER 6 • 2005 and PABP-1) are components of SGs but not of PBs. In con- trast, eIF4E, XRN1, Fas-activated serine/threonine phospho- protein (FAST), and tristetraprolin (TTP) are found in PBs in unstressed cells but partially or completely relocalize to SGs in stressed cells. A single class of reporter mRNA is found in both SGs and PBs, suggesting that individual transcripts at different stages of processing may localize in each structure. Pho- tobleaching studies reveal kinetically distinct classes of proteins within SGs and PBs: TTP, T cell intracellular antigen (TIA), and G3BP rapidly shuttle in and out of these structures, whereas putative scaffold proteins DCP1a, GW182, and FAST are relatively stable constituents of these structures. We pro- pose a model wherein mRNA released from polysomes during stress is routed to SGs for triage, sorting, and mRNP remodeling, after which certain transcripts are selectively exported to asso- ciated PBs for degradation. Results SGs and PBs are induced by different stimuli Previous studies have shown that the composition of SGs var- ies with the stimulus used to elicit their assembly; e.g., heat shock–induced SGs contain HSP27, whereas arsenite-induced SGs do not (Kedersha et al., 1999), and SGs containing G3BP (Ras-GSP SH3 domain–binding protein) have been described as lacking TIA-1 (Tourriere et al., 2003). Therefore, we used a number of SG-inducing stimuli to survey SG and PB composi- tion. U2OS cells and HeLa cells were treated with arsenite (ox- idative stress), clotrimazole (mitochondrial stress), or heat shock, and were stained for SG markers eIF4E (Fig. 1) and eIF3 and PB marker DCP1a. As shown in Fig. 1 (A and E), some unstressed cells contain DCP1a-positive PBs (yellow ar- rows), whereas others do not. Remarkably, eIF4E appears Figure 2. Distribution of proteins between G3BP-induced SGs and PBs. present in PBs together with DCP1a. Arsenite treatment (Fig. SGs were induced in DU145 cells by the transfection of GFP-G3BP and 1, B and F) induces both SGs (Fig. 1, white arrowheads) 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; PBs in all cells, and the great majority of the PBs appear clus- (C) eIF4G and eIF4E; (D) eIF3b and FLAG-eIF4E; (E) PABP-1 and DCP1a; tered around SGs in both U2OS (Fig. 1 B) and HeLa (Fig. 1 F) and (F) FAST and eIF4E. Yellow arrows indicate representative PBs; white cells. In contrast, cells treated with the mitochondrial poison arrowheads indicate SGs in the merged views. clotrimazole (Fig. 1, C and G) or heat shock (Fig. 1, D and H) display SGs but do not show an increase in PBs, nor do PBs ap- cally irregular in shape and are frequently juxtaposed with PBs pear associated with SGs. We conclude that SGs and PBs are (Fig. 2, yellow arrows). GFP-G3BP transfectants were counter- coordinately induced by arsenite, but that other stress stimuli stained for the PB marker DCP1a and the SG marker TIA-1 induce SGs in cells lacking PBs. (Fig. 2 A). DCP1a is found in PBs but is largely excluded from Shared versus unique protein the SG, as shown by TIA-1 staining. This indicates that GFP- components of SGs and PBs G3BP and TIA-1 are present in SGs but are excluded from PBs, whereas DCP1a is present in PBs but not in SGs. Similar The presence of eIF4E in PBs was unexpected. Therefore, we analysis indicates that another PB component, XRN1 (Fig. 2 sought to confirm this result and determine whether other pre- B), is present in both PBs and G3BP-induced SGs. Consistent viously described SG components might also be present in with the data shown in Fig. 1, eIF4E (Fig. 2 C) is found in both PBs. We used DU145 cells, which had been previously used to SGs and PBs, whereas eIF4G is found in SGs but not in eIF4- analyze SG components (Kedersha et al., 2002), and induced positive PBs. Two approaches confirm that the eIF4E signal in SGs by the transient transfection of GFP-G3BP, an SG compo- PBs is not caused by antibody cross-reactivity with some PB nent whose expression induces the assembly of very large SGs protein: (1) a different eIF4E antibody gives identical results readily amenable to microscopic analysis (Tourriere et al., (unpublished data); and (2) transfected FLAG-tagged eIF4E re- 2003). GFP-G3BP (Fig. 2) induces the formation of large SGs veals the same PB–SG distribution when detected using anti- (1–5 m in diameter; Fig. 2, white arrowheads) that are typi- STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 873 eIF2 (Fig. 3 D), and endogenous G3BP (Fig. 3 E) are only in SGs, whereas GW182 (Fig. 3 F) and FAST (Fig. 2 F) predomi- nate in PBs. We conclude that G3BP, eIF4G, eIF3, phospho- eIF2, and PABP-1 are restricted to SGs, whereas DCP1a and 2 (unpublished data) are confined to PBs. GW182 autoantibody staining suggests that it is present in both PBs and SGs (Fig. 3 F, green); however, anti-GW182 is not monospecific by Western blot analysis, and a GFP-tagged construct encoding most of GW182 (aa 313–1709) is only found in PBs (Yang et al., 2004). Thus, GW182 localizes to PBs, whereas its association with SGs remains inconclusive. Of considerable interest is the finding that XRN1, FAST, and eIF4E are present in both PBs and SGs. The dual SG–PB localization of each of these pro- teins was confirmed by using tagged constructs in transient transfection assays (Fig. 2 D and see Fig. 8, B–D). FAST inter- acts with TIA-1 and antagonizes the translational silencing of TIA-1 (Li et al., 2004b). In unstressed COS7 cells, most FAST is nuclear and is associated with mitochondria (Li et al., 2004a). Its presence in PBs and its relocalization to SGs may reflect its function as a translational regulator of TIA proteins. PBs are present in AA cells that cannot phosphorylate eIF2 or assemble SGs Little is known about the signaling pathways and specific mo- lecular events that govern PB assembly, although their size and number increase when 5–3 mRNA decay is blocked (Sheth and Parker, 2003) and vary throughout the cell cycle (Yang et al., 2004). SG assembly requires the phosphoryla- tion of eIF2 (Kedersha et al., 1999) and is mediated by the aggregation of one of several RNA-binding proteins, includ- ing TIA proteins (Gilks et al., 2004), Fragile X Mental Retar- dation protein (Mazroui et al., 2002), G3BP (Tourriere et al., 2003), and the survival of motor neurons protein (Hua and Figure 3. Distribution of proteins between arsenite-induced SGs and PBs. Zhou, 2004). We therefore asked whether PBs are present in SGs were induced in DU145 cells by arsenite treatment, and cells were mutant AA cells, in which the normal eIF2 allele has been 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, replaced with a nonphosphorylatable mutant (S51A eIF2) phospho-eIF2, and eIF3b; (E) eIF4E, G3BP, and eIF3b; and (F) GW182, allele by homozygous replacement (Scheuner et al., 2001). As FAST, and TIA-1. Yellow arrows indicate representative PBs; white arrow- shown in Fig. 4 A, treatment of wild-type SS cells with arse- heads indicate SGs in the merged views. nite results in robust SG assembly (white arrowheads), as as- sessed using three independent SG markers (eIF3b; G3BP; FLAG (Fig. 2 D, blue). We conclude that eIF4E is present in and TIA related [TIAR]). In contrast, no SG assembly is seen both PBs and SGs. In contrast, eIF3b (Fig. 2 D) and PABP-1 with any of these SG markers in arsenite-treated AA mouse (Fig. 2 E) are restricted to SGs. The TIA-1–interacting protein cells (Fig. 4 A, right). Likewise, SGs are not induced in AA FAST (Fig. 2 F) exhibits a pattern similar to XRN1; i.e., it is cells by any other treatments, including heat shock, puromy- predominantly associated with PBs and is weakly associated cin treatment, or transfection with G3BP (unpublished data). with SGs. Only the enforced expression of the phosphomimetic form of To confirm that the SGs induced by G3BP overexpres- eIF2 generates SGs in AA cells (supplemental Fig. 1 in sion are compositionally similar to SGs induced by stress, we McEwen et al., 2005), demonstrating their competence to as- exposed DU145 cells to oxidative stress using arsenite and semble SGs given this essential trigger. stained for endogenous SG and PB markers (Fig. 3). Although Staining arsenite-stressed control SS cells and mutant arsenite-induced SGs are smaller than those induced by GFP- AA cells for PB marker proteins GW182 and DCP1a (Fig. 4) G3BP overexpression, the results are generally comparable. As reveals that both cell lines display numerous PBs (Fig. 4 B, shown in Fig. 3 A, DCP1a is confined to PBs (yellow arrow), yellow arrows). In contrast, SGs (Fig. 4, white arrowheads) eIF3b is confined to SGs (white arrowhead), and eIF4E is are induced in SS cells, as shown by TIA-1 staining, but are present in both structures. PABP-1 and TIA-1 are restricted to absent in AA cells treated similarly. To verify that these ap- SGs, whereas XRN1 (Fig. 3 B) predominates in PBs, but a mi- parent PBs in both SS and AA cells behave normally, we con- nor amount is detectable in SGs. eIF4G (Fig. 3 C), phospho- firmed that they were abolished upon treatment of the cells 874 JCB • VOLUME 169 • NUMBER 6 • 2005 Figure 4. Role of eIF2 phosphorylation and Lsm4 expression in SG and PB formation. (A) Arsenite-treated wild-type (SS) and eIF2 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 trans- fected 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. Physical juxtaposition and transient with emetine or cycloheximide (unpublished data). We con- interactions between SGs and PBs clude that PBs, unlike SGs, do not require the phosphorylation We were struck by the observation that arsenite-induced SGs of eIF2 for their assembly. appear juxtaposed with PBs and contain eIF4E but no other PBs are induced by arsenite initiation factors (e.g., Figs. 1 and 3). Therefore, we investi- gated the kinetics of SG–PB assembly by using combinations As arsenite induces both PBs and SGs (Fig. 1), we asked of stress-inducing conditions. Fig. 5 shows HeLa cells sub- whether the knockdown of PBs would affect SG assembly in jected to different stresses and triple-stained for eIF3b (SG- response to arsenite. Several small interference RNAs (si- specific marker), FAST (PBs), and eIF4E (found in both SGs RNAs) were used to knockdown different PB components and PBs). Untreated cells (Fig. 5 A) display few PBs (Fig. 5, (unpublished data), but only siRNA against Lsm4 was mini- yellow arrows), which appear as yellow dots because of the mally effective in preventing PB assembly in response to merge of green (eIF4E) and red (FAST) signals. The treatment arsenite. DU145 and HT1080 cells were transfected with of cells with arsenite for 30 min (Fig. 5 B) resulted in a dra- control or Lsm4 siRNA, untreated or treated with arsenite, matic increase in the number of PBs coordinate with robust fixed and stained for PBs, scored microscopically, and SG assembly (Fig. 5, white arrowheads); remarkably, virtually counted. As shown in Fig. 4 C, RT-PCR reveals that efficient all PBs were found adjacent to SGs, as shown in Fig. 1. SG knockdown Lsm4 mRNA is obtained, which reduces PBs and PB formation appear synchronously in response to shorter (Fig. 4, D [dark gray bars] and E). However, upon arsenite arsenite treatments. treatment, the percentage of cells with PBs increases mark- Disassembly of both SGs and PBs is enforced by eme- edly despite knockdown for Lsm4. In HT1080 cells, Lsm4 tine and cycloheximide, which are drugs that inhibit transla- knockdown is able to reduce the percentage of PB-positive tional elongation and block the disassembly of polysomes, cells to 5% of control levels in the absence of stress (Fig. 4, D thereby preventing the translocation of mRNA into SGs and [right bars] and E). Arsenite treatment induces PBs in 75% PBs (Kedersha et al., 2000; Sheth and Parker, 2003; Cougot et of these cells, whereas 95% display SGs. As heat shock and al., 2004a). As the size of both PBs and SGs should be propor- clotrimazole also induce SGs in cells lacking PBs, the data tional to the amount of mRNA within each, we determined indicate that assembly of SGs and PBs is regulated by dis- whether adding emetine to arsenite-treated cells would cause tinct signaling pathways. STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 875 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 M for 30 min) induces both SGs (white arrowhead) and PBs (yellow arrow). (C) Cells were treated with arsenite for 60 min, and 20 g/ml emetine was added dur- ing the last 30 min. (D–F) Cells were subjected to heat shock (44C) for 15 min (C), 30 min (D), or 60 min (E). Yellow arrows indicate rep- resentative 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. the disassembly of SGs before PBs, or vice versa. Emetine ad- sembly. Cells that were exposed to heat shock (44C) for 15 dition to arsenite-treated cells followed by an additional 30- and 30 min (Fig. 5, D and E) displayed SGs in cells lacking min incubation (Fig. 5 C) resulted in partial SG disassembly PBs. Continued heat shock treatment for 1 h resulted in the (Fig. 5, white arrowheads) without affecting PBs. A longer disappearance of SGs and the appearance of PBs (Fig. 5 F). emetine treatment (1–2 h) completely dispersed SGs without Thus, heat shock appears to trigger a coordinate sequence of affecting PBs (unpublished data), but the treatment of cells for events: an early and transient induction of SGs followed by a 1 h with emetine in the absence of arsenite disassembled all late induction of PBs. Remarkably, eIF4E distribution appears the PBs (unpublished data). This indicates that emetine treat- to shift between the two compartments under these conditions, ment disassembles SGs before disassembling PBs and that suggesting that some eIF4E-bound mRNA may move from eIF4E is still present in PBs upon emetine-enforced SG disas- SGs to PBs during heat shock. 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 rep- resentative PBs; white arrowheads indicate SGs in the merged views. 876 JCB • VOLUME 169 • NUMBER 6 • 2005 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 37C 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. A single class of mRNA transcripts is with DCP1a and TIA-1 in Fig. 6 B and with XRN1 and eIF4E in present in both SGs and PBs Fig. 6 C. We conclude that a single class of mRNA localizes to SGs are thought to be sites of mRNA sorting rather than decay both SGs and PBs. (Kedersha and Anderson, 2002). As PBs are putative sites of SG–PB interactions in real time using 5–3 mRNA degradation (Sheth and Parker, 2003; Cougot et al., time-lapse microscopy 2004a), the juxtaposition of the two structures during arsenite treatment (Fig. 1) and their sequential assembly/disassembly dur- To investigate the physical interaction of PBs with SGs over ing heat shock (Fig. 5) suggests that mRNAs destined for decay time, we obtained a series of red (RFP) or green (GFP or YFP)- are sorted in SGs and are subsequently transported into PBs. If so, tagged proteins and transiently expressed various combinations a single class of mRNA transcripts should be detected in both in COS7 cells, which were subsequently viewed live using a SGs and PBs at different stages of its processing. To test this heated stage and inverted confocal microscope. The extremely prediction, we expressed a -globin mRNA containing the motile nature of PBs (Yang et al., 2004) required that each MS2-binding site in its 3 untranslated region (pEF-7B- frame be made from a volume-rendered image derived from MS2bs) together with a fusion protein comprised of GFP, MS2 10 z-axis sections (see Materials and methods) so as to visual- coat protein, and a nuclear localization signal (Fig. 6 A, GFP- ize all of the PBs within each cell. Cells that were cotransfected MS2-NLS). Transfection of GFP-MS2 alone or with a globin with RFP-DCP1a (PB marker) and GFP–TIA-1 (SG marker) reporter lacking the MS2-binding site resulted in a signal ex- display spontaneous SGs in 30–70% of the transfectants, and clusively localized to the nucleus (Rook et al., 2000; unpublished these SGs frequently associate with one or more PBs. When fol- data). When GFP-MS2 is cotransfected with the globin reporter lowed over time (Fig. 7 A and Video 1, available at http:// containing the MS2-binding site, nuclear export of the tethered www.jcb.org/cgi/content/full/jcb.200502088/DC1), some “at- GFP signal is observed in 2–10% of transfected cells; only in tached” PBs (Fig. 7 A, white arrowheads) remain stably bound cells expressing high amounts of globin-MS2 is the tethered GFP to SGs. Other PBs (Fig. 7 A, yellow arrows) appear intermit- exported from the nucleus. Cytoplasmic GFP signal is found in tently attached to SGs or move freely in the cytoplasm without SGs and PBs, as shown in Fig. 6 (B and C). The RNA-tethered interacting with SGs. “Free” or unbound PBs exhibit greater signal is equally distributed between PBs (Fig. 6, yellow arrows) motility than SG-associated PBs, even when both types of PBs and SGs (Fig. 6, white arrowheads), as shown by colocalization are present in the same cell. SGs exhibit fission, fusion, and STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 877 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. occasional dispersal, which are properties consistent with on- sought to verify the ability of TTP to induce SG–PB fusion by going sorting and export of their contents. Similar results are using GFP-G3BP to induce SGs and test whether the coexpres- obtained when GFP-G3BP is used to induce and detect SG–PB sion of nonfluorescent TTP would alter the interaction of interactions. The expression of FAST-YFP (Fig. 7, green) with SGs with PBs. As shown in Video 4 (available at http:// RFP-DCP1a (Fig. 7, red) resulted in the incorporation of FAST www.jcb.org/cgi/content/full/jcb.200502088/DC1) and Fig. 7 C, into SGs in some cells but into PBs in other cells (Video 2, avail- the coordinate expression of GFP-G3BP, RFP-DCP1a, and able at http://www.jcb.org/cgi/content/full/jcb.200502088/DC1). myc-tagged vector does not alter the relationship between These data are consistent with the distribution of endogenous GFP-G3BP SGs and PBs; both free and interacting structures FAST, as shown in Figs. 1 and 2, and suggest that FAST (like are observed. However, cells expressing myc-tagged TTP with eIF4E) may be present in both structures. Unfortunately, YFP- GFP-G3BP and RFP-DCP1a (Fig. 7 D and Video 5) display a eIF4E constructs fail to recapitulate the localization of endoge- nearly complete recruitment of PBs to SGs. Similar results nous or FLAG-tagged eIF4E, so we are unable to examine its were seen using GFP–TIA-1 as the SG inducer/marker, as the distribution between SGs and PBs in real time. coexpression of GFP–TIA-1 with TTP promotes interactions We reasoned that SG–PB interaction may be influenced between SGs and PBs (unpublished data). by the amount of mRNA being transported from the SG into We then asked whether FAST, XRN1, eIF4E, or the TTP- the PB and hypothesized that the expression of TTP, an SG- related protein BRF1 would promote interactions between GFP- associated protein that promotes mRNA decay (Stoecklin et al., G3BP SGs and PBs. Fig. 8 depicts GFP-G3BP and RFP-DCP1a 2004), might increase SG–PB interactions by increasing the cotransfected with one of the following: empty vector (Fig. 8 A), amount of mRNA routed from SGs into PBs. As shown in Fig. FAST-myc (Fig. 8 B), FLAG-XRN1 (Fig. 8 C), FLAG-eIF4E 7 B and Video 3 (available at http://www.jcb.org/cgi/content/ (Fig. 8 D), TTP-myc (Fig. 8 E), or FLAG-BRF1 (Fig. 8 F). Only full/jcb.200502088/DC1), the expression of YFP-TTP with TTP (Fig. 8 E) and its close homologue BRF1 (Fig. 8 F) are RFP-DCP1a results in the quantitative and stable association of found to induce SG–PB fusion. Remarkably, BRF1 promotes the PBs with SGs. Remarkably, the YFP-TTP SGs appear to en- complete engulfment of large PBs by SGs, whereas in TTP capsulate single or multiple PBs. Although fusion events be- transfectants, smaller, more numerous PBs are embedded in a tween these conglomerate SG–PB structures were observed, single SG. Although not affecting the SG–PB relationship, fission events were rare. The data indicate that both the number eIF4E overexpression appears to reduce the size of PBs but in- and duration of SG–PB interactions is stabilized by the expres- creases their number (Fig. 8 D, red), whereas XRN1 expression sion of TTP. As YFP-TTP is also diffusely present in the cyto- results in fewer, larger PBs (Fig. 8 C, red). Altogether, the data plasm, making the borders of the SG difficult to determine, we indicate that the expression of different SG–PB components 878 JCB • VOLUME 169 • NUMBER 6 • 2005 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. affects their size and interaction. Most important, the expression selected SG (Fig. 9 A, arrow) was obtained before bleaching of TTP and BRF1, which functionally accelerate mRNA decay (Fig. 9, pink tracing) and was subsequently bleached at the po- in these cells, also promote the interaction of SGs with PBs. sition indicated by the vertical yellow line. The dark blue line indicates the scan intensity taken immediately after bleaching. Dynamics of different SG–PB A scan taken 30 s later (Fig. 9, aqua tracing) reveals the com- components in real time using FRAP plete recovery of GFP–TIA-1 fluorescence. The FRAP be- The TTP- and BRF1-induced fusion of SGs and PBs could either havior of GFP–PABP-1, shown in Fig. 9 B, also recapitulates be direct (i.e., a physical linkage between SG–PB structural previous findings (Kedersha et al., 2000). GFP–PABP-1 exhibits components) or indirect (i.e., by shunting more substrate slower and less complete recovery than does GFP–TIA-1 be- mRNA destined for decay through SGs into PBs). To analyze cause only 60% of SG-associated GFP–PABP-1 fluorescence the dynamic nature of SG and PB components within these recovers after 30 s. This suggests that TIA-1 and PABP-1 are structures, we used FRAP using GFP and/or YFP-tagged ver- not quantitatively present in the same mRNP complexes. sions of different SG–PB-associated proteins. Previous studies TTP overexpression generates spontaneous SGs, and ar- (Kedersha et al., 2000) demonstrated that GFP–TIA-1 rapidly senite treatment induces TTP to leave SGs (Stoecklin et al., moves in and out of SGs. In these experiments, 90% recovery 2004) but not PBs (unpublished data), which is an effect depen- of the bleached signal occurred within 10 s. We used this sys- dent on TTP phosphorylation that mediates its binding to 14-3-3 tem to analyze the FRAP kinetics of representative members of (Stoecklin et al., 2004). YFP-TTP SG bleaching (Fig. 9 C) is the “SG-only proteins” GFP–PABP-1 and GFP-G3BP, the followed by rapid and complete recovery; this result was consis- “SG–PB shared proteins” YFP-TTP and FAST-YFP, and the tently obtained in 10 cells and was the same when either large “PB-only proteins” GFP-GW182 and YFP-DCP1a. As shown (probable SGs) or small foci (probable PBs) were bleached. in Fig. 9 A, GFP–TIA-1 forms large, distinct SGs in response to Cells coexpressing RFP-DCP1a were used to verify the rapid arsenite treatment. A linear scan of the region containing a kinetics of YFP-TTP that was unambiguously localized to STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 879 PBs (Fig. S1, available at http://www.jcb.org/cgi/content/full/ of poly(A) RNA at PBs, supporting the contention that these jcb.200502088.DC1). Thus, YFP-TTP rapidly moves in and out foci are sites of mRNA degradation (Cougot et al., 2004a). of both PBs and SGs, suggesting that TTP constitutes a transient Recently, dual immunofluorescence using antibodies tether between mRNA and the decay machinery. against the SG marker TIA-1 and the PB marker DCP1a SG-associated GFP-G3BP also displays rapid, complete re- clearly showed that SGs and GW bodies/PBs are distinct and covery (Fig. 9 D) that is unaltered by arsenite treatment (unpub- independent cytoplasmic structures (Cougot et al., 2004a), but lished data). FAST-YFP induces spontaneous SGs in 30–70% of the relationship between them has not been addressed. Our re- transfectants and is localized to PBs in most of the remaining sults confirm that SGs and PBs are compositionally and mor- transfectants. As shown in Fig. 9 E, its FRAP kinetics are very phologically distinct entities, each of which can be assembled slow, and recovery is minimal in all cells that were tested (n  in the absence of the other and are compositionally distinct. 20) and unaltered by arsenite (unpublished data). The slow kinet- However, there are strong spatial and functional links be- ics of FAST and its presence in both SGs and PBs suggests that it tween SGs and PBs. First, oxidative stress induces the assem- may play a scaffolding role in organizing SGs and PBs. bly of both SGs and PBs and promotes interactions between The overexpression of YFP-DCP1a induces very large them. Second, time-lapse microscopy reveals that a subset of PBs in many cells and more normal-sized PBs in others. PBs PBs is stably tethered to SGs, whereas another subset is inde- normally exhibit size variation depending on metabolic state pendent and highly mobile within the cytoplasm. Third, sev- (Sheth and Parker, 2003) and cell cycle (Yang et al., 2004). eral proteins (i.e., FAST, XRN1, eIF4E, and TTP) and mRNAs Fig. 9 F shows photobleaching of a medium-sized YFP-DCP1a (i.e., globin-MS2 reporter) are found in both SGs and PBs. PB, which exhibits kinetics similar to those of GFP–PABP-1. Fourth, SGs and PBs are induced to fuse by the overexpres- However, YFP-DCP1a FRAP kinetics is variable: the ex- sion of TTP or BRF1, which are RNA-binding proteins that change rate is rapid in small PBs but is slower in larger PBs. promote mRNA decay and are components of both SGs and GFP-GW182 exhibits very slow FRAP recovery (Fig. 9 G), PBs. Finally, pharmacologic inhibitors of translational elon- similar to that of FAST. The transit time of PABP-1 and gation promote the disassembly of both structures, suggesting DCP1a is intermediate between that of TIA-TTP-G3BP and that both PBs and SGs are assembled from translationally GW182-FAST, suggesting that PABP-1 and DCP1a either competent mRNA. shuttle in and out of SGs and PBs independently of the RNA The SG–PB fusion induced by TTP and BRF1 suggests substrates or are removed from the transcripts during mRNP that these proteins regulate the dynamic interactions between remodeling that occurs in tandem with their movement. SGs and PBs. Both TTP and BRF1 promote the degradation of mRNAs bearing ARE in their 3 untranslated regions. TTP has been proposed to direct these transcripts to exosomes, which Discussion are degradative machines that promote 3–5 exonucleolytic Stress-induced phosphorylation of eIF2 results in stalled degradation of deadenylated transcripts (Chen et al., 2001). translational initiation such that actively translating ribosomes The ability of TTP to promote interactions between PBs and “run off” their transcripts, resulting in polysome disassembly SGs suggests that this class of destabilizing factor might also concurrent with SG assembly (Anderson and Kedersha, 2002; promote 5–3 mRNA degradation at the SG, which is consis- Kedersha and Anderson, 2002). SG assembly is regulated by tent with recent data suggesting that TTP and BRF1 comprise one or more RNA-binding proteins, including TIA-1 (Gilks et molecular links between ARE-containing mRNAs and mRNA al., 2004), G3BP (Tourriere et al., 2003), Fragile X Mental Re- decay enzymes present in PBs (Lykke-Andersen and Wagner, tardation protein (Mazroui et al., 2002), survival of motor neu- 2005). Our data indicate that this molecular link has morpho- rons protein (Hua and Zhou, 2004), and/or TTP (Stoecklin et logical as well as functional consequences. It is important to al., 2004). Another cytoplasmic mRNP domain termed the note that the SGs observed in the real-time experiments are in- GW body was first visualized by using a patient-derived au- duced by the overexpression of either TTP or G3BP. As such, toantisera reactive with GW182, a 182-kD RNA-binding pro- they may not have the same composition and function as arse- tein (Eystathioy et al., 2002). GW bodies contain RNA, but nite or heat-induced SGs. Nevertheless, the TTP- and BRF-1– unlike SGs, GW bodies are prominent in actively growing un- induced stabilization of PB–SG interactions reveals a unique stressed cells (Eystathioy et al., 2002). Convergent studies mechanism whereby this class of protein might regulate from several laboratories have shown that GW bodies contain mRNA metabolism. proteins involved in mRNA degradation, including the decap- The presence of eIF4E in PBs is somewhat surprising, as ping enzymes DCP1a and 2, a heptamer of Lsm proteins re- it binds to the seven-methyl guanosine cap and is thought to quired for mRNA decapping, and the exonuclease XRN1 protect the integrity of the cap (Ramirez et al., 2002; Liu et al., (Eystathioy et al., 2002, 2003; Ingelfinger et al., 2002; Cougot 2004). In S. cerevisiae, eIF4G and eIF4E are removed from et al., 2004a,b; Yang et al., 2004). In Saccharomyces cerevisiae, mRNA before the recruitment of DCP1 and decapping the accumulation of nondegradable mRNAs at compositionally (Tharun and Parker, 2001). Our data show that eIF4G, PABP, similar cytoplasmic foci (PBs) implicated these phylogeneti- and eIF3 are present in SGs but not in PBs, suggesting that cally conserved foci in the process of mRNA degradation eIF4G is removed from the cap before its transit into PBs, (Sheth and Parker, 2003). In mammalian cells, the interference whereas eIF4E remains bound to the cap. Because the rate at RNA–mediated knockdown of XRN1 enhances the accumulation which eIF4E dissociates from capped mRNA is accelerated in 880 JCB • VOLUME 169 • NUMBER 6 • 2005 the absence of eIF4G (Haghighat and Sonenberg, 1997), In the model shown in Fig. 10, we posit that SGs contain capped mRNA may be liberated within the PB, allowing transcripts routed from disassembling polysomes in accord DCP1a/2-mediated decapping. with the absolute requirement for eIF2 phosphorylation in SG We have proposed that SGs are sites of mRNA triage in assembly. This idea is in agreement with the studies of Thomas which individual transcripts are sorted for storage, reinitia- et al. (2005), who demonstrated that newly synthesized mRNAs tion, or degradation (Anderson and Kedersha, 2002; Kedersha are not present in SGs. Error-containing transcripts selected for and Anderson, 2002). This model predicts that those mRNAs nonsense-mediated decay during the pioneer round of transla- targeted for decay will be exported from the SG to sites of tion (before polysome assembly) may contribute to free PBs, as mRNA decay such as PBs. The aforementioned interactions nonsense-mediated decay occurs via decapping and 5–3 de- between SGs and PBs may allow mRNA to move from the cay (Maquat, 2002; Neu-Yilik et al., 2004) and is inhibited by SG to the PB. Two lines of evidence suggest the direction of cycloheximide. SGs induced by stress are likely to contain a this process. First, arsenite induces the formation of juxta- mixture of transcripts, but SGs induced by the overexpression posed SGs and PBs, and subsequent emetine treatment forces of different RNA-binding proteins (e.g., TIA, G3BP, and TTP) the disassembly of SGs before the disassembly of PBs. Sec- are likely to differ in their mRNA composition. For example, ond, heat shock induces SG formation before PB formation. TIA-induced SGs are likely enriched for TIA-bound transcripts Initially, eIF4E is concentrated at SGs in cells lacking PBs, that are targeted for translational silencing, whereas TTP- but in the continued presence of heat, SGs are disassembled, induced SGs may be enriched for TTP-bound transcripts that and PBs containing eIF4E are concomitantly assembled. are targeted for decay. Thus, TTP-induced SG–PB fusion occurs These results imply (but do not mandate) that eIF4E is first because TTP-induced SGs are assembled from mRNAs se- incorporated into SGs and later translocates into PBs. As lected by TTP binding for rapid decay. Given the very rapid eIF3, eIF4G, PABP-1, small ribosomal subunits, and G3BP flux of TTP within SGs and PBs assessed by photobleaching, are found in SGs but not in PBs, these proteins must be re- it is unlikely that TTP itself constitutes a stable component of moved from mRNA before its export from the SG. Because either compartment. It is more likely that TTP serves to de- eIF4G and PABP-1 are directly involved in mRNA circular- liver its mRNA cargo to PBs by interacting with stable com- ization, it is probable that mRNAs exported from SGs into ponents of these particles (Lykke-Andersen and Wagner, PBs are decircularized before translocation, which is concur- 2005). However, FAST has the properties of a putative scaf- rent with their deadenylation (the activation step for mRNA fold protein that might stabilize SG–PB interactions; it dis- decay by both mRNA decay pathways). Finally, as eIF4E and plays a very slow exchange rate, as measured by photobleach- TTP are components of both SGs and PBs, these RNA-bind- ing, lacks known RNA binding motifs, nucleates both SGs ing proteins may remain with mRNA as it moves from the SG and PBs upon overexpression, and interacts with TIA-1. Pos- to the PB. sibly, TTP or TTP-associated proteins promote SG–PB fusion Figure 10. Hypothetical model of the rela- tionship between SGs and PBs. Proteins found exclusively in SGs are shown in yellow; pro- teins found in both SGs and PBs are depicted in green; and proteins restricted to PBs are shown in blue type. STRESS GRANULES AND PROCESSING BODIES ARE LINKED • KEDERSHA ET AL. 881 For pcDNA3-Flag-BRF1, the human BRF1 cDNA was excised as a by interacting either directly or indirectly with FAST to re- BamHI/blunt–XbaI fragment from bsdHis-BRF1 (Stoecklin et al., 2002) model the SG–PB scaffold. and inserted into the BamHI/blunt–XbaI sites of pcDNA3-Flag-Bak (a gift The data presented in this study establish that SGs and from T. Chittenden, ImmunoGen, Inc., Cambridge, MA). For pcDNA3- Flag-eIF4E, eIF4E was amplified from plasmid pcDNA3-eIF4E (a gift from PBs are discrete cytoplasmic structures that share some protein D. Dixon, University of South Carolina, Columbia, SC) using primers 5- and mRNA components as well as some functional properties. TTTGAATTCGCGACTGTCGAACCG-3 and 5-TGTTCTAGATTAAACAA- Both structures are induced by stress but are regulated by dis- CAAACCTATTTTTAG-3, digested with EcoRI and XbaI, and ligated into the EcoR–XbaI sites of pCDNA3-Flag. GFP-G3BP was a gift from J. Tazi tinct signaling events, and each can exist without the other. PBs (Centre National de la Recherche Scientifique, Montpellier, France). and SGs exhibit a high degree of motility when independent pGFP-GWaa313-1709 (Eystathioy et al., 2002) and FLAG-FAST (Li et al., but appear less motile when they are tethered together, and 2004b) were described previously. For pEF-FAST-myc, FAST was ampli- fied using primers 5-CCACCATGGAATAGCCACCATGAGGAGGC- their interaction is promoted by the mRNA-destabilizing pro- CGCGGGGGGAA-3 and 5-ATAAGAATGCGGCCGCGCCCCCT- tein TTP. The Janus-like juxtapositioning of SGs and PBs is TCAGGCCCCCAGCG-3, digested with NcoI and NotI, and ligated into reminiscent of the relationship between the nuclear gemini of the NcoI–NotI sites of pEF-myc. To make FAST-YFP, the coding region of FAST was amplified using primers 5-TGTGAGATCTAGTAGGAGGC- coiled bodies and Cajal bodies (Dundr et al., 2004), a case in CGCGGGGG-3 and 5-CCGAAGCTTGCCCCCTTCAGGCCC-3, di- which the morphology of linked compartments arises from or- gested with BglII and HindIII, and ligated into pEF-YFP-N1 vector (CLON- dered, compartmentalized stages in nuclear small nuclear RNP TECH Laboratories) that was digested with the same enzymes. biogenesis. The dynamic relation between SGs and PBs reiter- siRNA transfection ates the importance of compartmentalization in regulating the Du145 and HT1080 cells were transfected with 1.25 l/ml of Lipo- fate of cytoplasmic mRNA. fectamine 2000 and 100 nM siRNA duplexes for 48 h. Subsequently, cells were reseeded and, after 8 h, were transfected again with siRNA for another 40–44 h. siRNAs were designed using published recommenda- Materials and methods tions (Reynolds et al., 2004) and were purchased from Ambion. The fol- lowing target sequences (sense strand) were chosen: control siRNA (D0), Cell lines 5-GCAUUCACUUGGAUAGUAA-3; and Lsm4 siRNA (L4), 5-ACA- COS7, HeLa, and DU145 cells were obtained from the American Type ACUGGAUGAACAUUAA-3. Culture Collection, and U2OS cells were obtained from J. Blenis (Harvard Medical School, Boston, MA). HT1080 cells were obtained from C. Mo- RT-PCR roni (University of Basel, Basel, Switzerland). Cells were maintained in HT1080 cells were transfected with siRNA D0 or L4. Total cytoplasmic DME containing 10% FBS at 7.0% CO RNA was extracted, and 5 g RNA was used for reverse transcription us- ing oligo-dT and MMLV-RT (Promega). cDNA was purified with the Antibodies Qiaquick PCR purification kit (QIAGEN), and one tenth was used per PCR Antibodies against eIF4E (monoclonal and rabbit polyclonal), eIF4G, reaction using Taq polymerase (2.5 U/50 l) and solution Q (QIAGEN). eIF3b, myc, TIA-1, FXR1, and TIAR were obtained from Santa Cruz Bio- Annealing was performed at 56C using primers 5-CCTTGTCACTGCT- -GAGACTGTGGAGCGGAATC-3 for the amplifica- GAAGACG-3 and 5 technology, Inc. Phospho-specific anti-eIF2 was obtained from StressGen tion of Lsm4 and 5-GGTGGTCGGAAAGCTATC-3 and 5-GAGCTTCT- Biotechnologies. Human autoantisera against GW182 was an index se- TATAGACACCAG-3 for the amplification of ribosomal protein S7 as a rum from a 48-yr-old female with mixed motor and sensory neuropathy, control. Parallel reactions were performed using 15, 20, 25, 30, and 35 which was obtained from Advanced Diagnostics Laboratory. Antibodies PCR cycles, and the products were resolved by 1.5% agarose gel electro- against DCP1a and XRN-1 were previously described (Lykke-Andersen phoresis and were stained with ethidium bromide. and Wagner, 2005). Antisera against FAST (anti–FAST-N) were de- scribed previously (Li et al., 2004a). Monoclonal anti-myc was a gift from L. Klickstein (Brigham and Woman’s Hospital, Boston, MA). Anti-HA was Fluorescence microscopy obtained from Covance Research Products. Anti–PABP-1 was a gift from Cells were stained and processed for fluorescence microscopy as previ- G. Dreyfuss (University of Pennsylvania, Philadelphia, PA). Anti-G3BP was ously described (Gilks et al., 2004). Conventional fluorescence micros- a gift from I. Gallouzi (McGill University, Montreal, Canada). Anti- copy was performed using a microscope (model Eclipse E800; Nikon) Dcp2was a gift of B. Seraphin (Centre de Genetique Moleculaire, Gif-sur- with epifluorescence optics and a digital camera (model CCD-SPOT RT; Yvette, France) and M. Kildejian (Rutgers University, Piscataway, NJ). Diagnostic Instruments). The images were compiled using Adobe Photo- shop software (v7.0). Plasmids Plasmids encoding FLAG-DCP1a, FLAG-XRN1, and FLAG-DCP2 were pre- Confocal microscopy viously described (Lykke-Andersen and Wagner, 2005). To make pEYFP- Cells transfected with combinations of GFP- and RFP-tagged vectors were DCP1a, the human DCP1a cDNA was amplified from plasmid pcDNA3- viewed live at 37C using an inverted microscope (model TE2000-U; Ni- Flag-DCP1a by using primers 5-GTGCTCGAGCTGAGGCGCTGAGT-3 kon) equipped with a 60 oil objective Cfi planapo lens (NA 1.40; Ni- and 5-GTGGAATTCTCATAGGTTGTGGTTG-3 and was ligated as an kon) and a confocal system (model C-1; Nikon). Each image was volume XhoI–EcoRI fragment into the Xho–EcoRI sites of pEYFP-C1 (CLONTECH rendered from 10 Z-stacks of 0.85-m thickness using EZ-C1 software (Ni- Laboratories). To make mRFP-DCP1a, monomeric RFP (provided by R.Y. kon). Timed series were acquired at a rate of 1 min per frame; each frame Tsien, Howard Hughes Medical Institute, University of California, San Di- represents a volume-rendered image. Videos are shown in the supplemen- ego, La Jolla, CA; Campbell et al., 2002) was amplified using primers tal material; frames taken 10 min apart are shown in Fig. 7. Videos were 5-ATTCATACCGGTCCACCATGGCCTCCTCCG-3 and 5-TAAATTCTC- made using Adobe Image Ready software (v7.0) to animate the volume- GAGAGGCGCCGGTGGAG-3 and was ligated as an AgeI–XhoI frag- rendered TIF images exported from the EZ-C1 software (Nikon). ment into the AgeI–XhoI sites of pEYFP-DCP1a, thereby replacing YFP. GFP-MS2-NLS was a gift from K. Kosik (University of California, Santa FRAP photobleaching analysis Barbara, Santa Barbara, CA) and was previously described (Rook et al., Fluorescently tagged constructs of SG and PB proteins were tested to de- 2000). For pEF-7B-MS2bs, the T7-tagged rabbit -globin gene containing termine whether they exhibited localization that was compatible with their a sixfold repeat of the MS2bs was excised from plasmid pcDNA3-7B- endogenous or (in the case of TTP) FLAG-tagged counterparts; those fail- MS2bs as a HindIII/blunt–XbaI fragment and ligated into the NcoI/blunt– ing to meet this criterion were not used. COS7 cells were transiently trans- XbaI sites of pEF/myc/cyto (Invitrogen). The plasmid pcDNA3-TTP-mycHis fected with the indicated constructs using SuperFect (QIAGEN), were re- was made as described previously (Stoecklin et al., 2004). For YFP-TTP, plated onto glass coverslips after 10 h of transfection, and were analyzed murine TTP cDNA was amplified from plasmid pcDNA3-TTP-mycHis by us- 38–46 h posttransfection. Transfectants were viewed using a 60 oil ob- ing primers 5-TATCAAGCTTATGAATTCCGTTCC-3 and 5-TCAGATC- jective (NA 1.40) on an interactive laser cytometer (model Ultima; Merid- CTCTTCTGAGATG-3 digested with HindIII and XbaI and inserted into the ian Instruments). Appropriate cells were located, and images were taken HindIII–XbaI sites of pEYFP-C1 (CLONTECH Laboratories). using a two-dimensional scanning mode before bleaching. Selected SGs 882 JCB • VOLUME 169 • NUMBER 6 • 2005 or PBs (Fig. 9, arrows) were photobleached for 1 s at 0.5 mW of power yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3 to 5 exonucleases of the exosome using a beam radius of 0.7 m and an excitation wavelength of 488 nm. complex. EMBO J. 17:1497–1506. Fluorescence emission was detected at 530 15 nm. The results shown Jacobson, A. 2004. Regulation of mRNA decay: decapping goes solo. Mol. Cell. were representative of three independent transfections in which a total of 15:1–2. 10 different cells were analyzed. In some cases (see Fig. S1), two-color scans were obtained by simultaneously exciting both fluorophores at 488 Kedersha, N., and P. Anderson. 2002. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. Biochem. Soc. Trans. nm and separating the two emissions using a 575-nm dichroic filter and 30:963–969. the appropriate emission filters (green emission 530 15 nm; red emis- sion 630 nm). Kedersha, N.L., M. Gupta, W. Li, I. Miller, and P. Anderson. 1999. RNA-bind- ing proteins TIA-1 and TIAR link the phosphorylation of eIF-2 to the assembly of mammalian stress granules. J. Cell Biol. 147:1431–1441. Online supplemental material Fig. S1 shows photobleaching of PB-localized YFP-TTP. Video 1 shows the Kedersha, N., M.R. Cho, W. Li, P.W. Yacono, S. Chen, N. Gilks, D.E. Go- lan, and P. Anderson. 2000. Dynamic shuttling of TIA-1 accompanies dynamics of GFP–TIA-1 SGs and PBs; Video 2 shows the dynamics of the recruitment of mRNA to mammalian stress granules. J. Cell Biol. FAST-YFP and RFP-DCP1a PBs; Video 3 shows YFP-TTP and RFP-DCP1 PBs; 151:1257–1268. and Video 4 shows GFP-G3BP SGs and RFP-DCP1 PBs, all in real time. Kedersha, N.L., S. Chen, N. Gilks, W. Li, I.J. Miller, J. Stahl, and P. Anderson. Video 5 shows that TTP coexpression promotes fusion between GFP-G3BP 2002. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-defi- SGs and RFP-DCP1 PBs. Online supplemental material is available at http: cient preinitiation complexes are core constituents of mammalian stress //www.jcb.org/cgi/content/full/jcb.200502088.DC1. granules. Mol. Biol. Cell. 13:195–210. We thank R. Tsien for the mRFP construct, K. Kosik for GFP-MS2-NLS, J. Tazi Krishnamoorthy, T., G.D. Pavitt, F. Zhang, T.E. Dever, and A.G. Hinnebusch. 2001. Tight binding of the phosphorylated alpha subunit of initiation fac- for GFP-G3BP, M. Kiledjian and B. Serephin for antibodies, and H. Gilbert tor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide ex- and M. Gurish for help with the confocal microscopy. We thank W. Li change factor eIF2B is required for inhibition of translation initiation. (Brigham and Women’s Hospital, Boston, MA) for the FAST-myc construct and Mol. Cell. Biol. 21:5018–5030. N. Gilks and members of the Anderson lab for lively discussions. Laroia, G., R. Cuesta, G. Brewer, and R.J. Schneider. 1999. Control of mRNA de- This work was supported by the National Institutes of Health grants cay by heat shock-ubiquitin-proteasome pathway. Science. 284:499–502. AI50167, AR051472, and AI33600 (to P. Anderson); DK42394 and Li, W., N. Kedersha, S. Chen, N. Gilks, G. Lee, and P. Anderson. 2004a. FAST HL52173 (to R.J. Kaufman); GM 066811 (to J. Lykke-Andersen); HL32854 is a BCL-X(L)-associated mitochondrial protein. Biochem. Biophys. Res. and HL070819 (to D.E. Golan) and the Canadian Institutes for Health Re- Commun. 318:95–102. search Grant MOP-38034 (to M.J. Fitzler). M.J. Fitzler holds the Arthritis Soci- Li, W., M. Simarro, N. Kedersha, and P. Anderson. 2004b. FAST is a survival ety Chair at the University of Calgary. J. Lykke-Andersen is a Pew Scholar. protein that senses mitochondrial stress and modulates TIA-1-regulated changes in protein expression. Mol. Cell. Biol. 24:10718–10732. Submitted: 14 February 2005 Liu, S.W., X. Jiao, H. Liu, M. Gu, C.D. Lima, and M. Kiledjian. 2004. Functional Accepted: 16 May 2005 analysis of mRNA scavenger decapping enzymes. RNA. 10:1412–1422. Long, R.M., and M.T. McNally. 2003. mRNA decay: x (XRN1) marks the spot. Mol. Cell. 11:1126–1128. References Lykke-Andersen, J., and E. Wagner. 2005. 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GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation. J. Cell Sci. 117: 5567–5578. 884 JCB • VOLUME 169 • NUMBER 6 • 2005

Journal

The Journal of Cell Biology – Pubmed Central

Published: Jun 20, 2005

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