Cells and cell lines.

Human 293T cells and HeLa cells were maintained under standard conditions. CEM-SS cells stably expressing NTAP-3G were generated by standard retrovirus-mediated transduction (40) using pCMS28/NTAP-A3G, or untagged A3G as a control, and selection with 0.5 μg/ml puromycin. Expression of the NTAP-A3G fusion protein was confirmed by immunoblotting. Peripheral blood mononuclear cells (PBMCs) were obtained by venipuncture, isolated using a Ficoll gradient, stimulated with 5 μg/ml phytohemagglutinin-L, and maintained in medium supplemented with interleukin-2 (105).

Virus infectivity.

HIV-1 GFP reporter viruses were prepared using 60-mm-diameter monolayers of 293T cells by cotransfection of ∂CMVp8.91, pMDG VSV-G, and pHR′SINcPPT-SGW (5 μg, 3 μg, and 5 μg, respectively) together with 1 μg of control vector (pcDNA3.1, pNTAP, or pCTAP) or an expression vector for A3G (pA3G, pNTAP-A3G, or pCTAP-A3G). Virus-containing supernatants were harvested at ~48 h posttransfection and filtered, and p24^Gag concentrations were measured by enzyme-linked immunosorbent assay. Sixteen-millimeter-diameter HeLa cell monolayers were challenged with normalized viral stocks, and relative infectivity was determined at ~24 h by flow cytometry (FACSCalibur) by measuring the number of GFP-positive cells. Values are presented as percentages of infectivity relative to the value obtained for virus produced in the presence of pcDNA3.1.

Expression and purification of tagged A3G-containing complexes.

A3G-containing complexes were isolated using the InterPlay mammalian TAP system according to the manufacturer's instructions (Stratagene). Briefly, two 150-mm-diameter cultures of 70% confluent 293T cells were transfected with 2 μg of pNTAP-A3G or pCTAP-A3G (or pNTAP or pA3G as controls) and harvested at ~40 h; for purification from the CEM-SS line stably expressing NTAP-A3G, 2 × 10⁸ cells were used. Following harvest, cells were washed three times in phosphate-buffered saline (PBS) and lysed in lysis buffer by three successive rounds of freezing in dry ice and thawing. Cell extracts were centrifuged for 10 min at 16,000 × g at 4°C, the supernatants passed over streptavidin resin, and eluted complexes bound to calmodulin coated-beads. Following elution from the calmodulin column, proteins were precipitated with methanol and chloroform (121) and resuspended in gel-loading buffer. The samples were resolved on 4 to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, which were then fixed in 50% (vol/vol) methanol, 10% (vol/vol) acetic acid, and 10% fixative enhancer (Bio-Rad) and stained using a Silver Stain Plus kit (Bio-Rad). In experiments that also contained Vif, the 293T cells were cotransfected with 4 μg of pcVIF and 2 μg of pNTAP-A3G and treated with 10 μM MG132 (a proteasome inhibitor) for 18 h prior to lysis. In some experiments, cell lysates were treated with 10 μg/ml RNase A (Sigma) for 60 min at room temperature before purification of the NTAP-A3G-containing complexes.

Mass spectrometry.

In-gel reduction, alkylation, and trypsin digestion were performed using standard procedures prior to subsequent analysis by mass spectrometry, as described previously (126). Peptides were extracted from the gel pieces by a series of acetonitrile and aqueous washes, lyophilized, and resuspended in 50 mM ammonium bicarbonate before analysis by liquid chromatography-tandem MS. Reversed-phase chromatography separations of peptides were performed using 75-μm C18 PepMap columns and an Ultimate liquid chromatography system (Dionex, United Kingdom). Peptides were then ionized by electrospray ionization using a Z-spray source fitted to a Q-Tof micro instrument (Waters Corp.), and the tandem MS analyses were conducted using collision energy profiles that were chosen based on the m/z ratio and the charge state of the peptide. The mass spectral data were processed into peak lists (containing the precursor ion m/z and charge state and the m/z and intensity of the fragment ions) and searched against the SwissProt and NCBI nonredundant databases using Mascot software (Matrix Science, United Kingdom). All results were manually verified.

Coimmunoprecipitation and immunoblotting.

Thirty-five-millimeter-diameter cultures of 293T cells were transiently transfected with 1.5 μg of the plasmid encoding the protein to be immunoprecipitated (pA3G-HA, pmyc-YB-1, or the empty vector control) and 3 μg of vector expressing the protein of interest by use of polyethylenimine (Polysciences, Inc.) at a ratio of 3 μg to 1 μg DNA. At 24 h posttransfection, cells were lysed in immunoprecipitation (IP) buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1 mM EDTA), and complete protease inhibitor cocktail (Roche) for 30 min at 4°C and then cleared by centrifugation for 15 min at 18,000 × g at 4°C. For coimmunoprecipitation assays, protein A or G agarose (Invitrogen) was preincubated with an anti-HA (mouse monoclonal; 12CA5) or anti-myc (mouse monoclonal; 9E10) antibody for 1 h at 4°C and then mixed with cell lysate for 4 h at 4°C on a rotator. Thereafter, the agarose beads were washed three times with cold IP buffer and bound complexes either eluted in gel loading buffer or treated with RNase. The latter procedure was performed by resuspending washed beads in IP buffer, dividing them into two equal volumes, adding 10 μg/ml RNase mixture (DNase free; Roche) to one, and incubating both at room temperature for 20 min. After two washes in cold IP buffer, proteins were eluted in gel-loading buffer. Samples were analyzed by SDS-PAGE using 10% gels for proteins with relative molecular masses of less than 60 kDa and 7% gels for proteins with relative molecular masses of greater than 60 kDa. Resolved proteins were detected by immunoblotting using anti-HA (mouse monoclonal HA.11; Covance Inc.), anti-myc (rabbit polyclonal ab9106; Abcam), anti-A3G (rabbit polyclonal [92]), anti-GFP (mouse monoclonal clones 7.1 and 13.1; Roche), anti-VSV-G (rabbit polyclonal, PRB-192P; Covance) or anti-LRP130 (mouse monoclonal; 9C9 [83]) as primary antibodies. Bound antibodies were visualized using appropriate horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Pierce).

Immunofluorescence.

Unmodified PBMCs, H9 T cells, or CEM-SS T cells stably expressing NTAP-A3G (2 × 10⁴ to 4 × 10⁴) were fixed to glass slides by centrifugation for 5 min at 800 rpm using a Shandon Cytospin 4 instrument (Thermo Electron Corporation) and were air dried for 10 min at room temperature. Adherent HeLa cell monolayers were seeded onto glass coverslips (Cover Glass, 22 mm; BDH) in 35-mm-diameter plates and were transfected with 1 μg of pA3G by use of FuGENE 6 (Roche). Cells were washed three times in PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min. After being washed with PBS, cells were incubated with 50 mM ammonium chloride, permeabilized with 0.5% Triton X-100 for 5 min at room temperature, and incubated for 30 min with blocking solution (4% goat serum in PBS) at room temperature. Staining was performed by incubating the cells for 60 min with an A3G-specific antibody raised in rabbits (92) at a 1:100 dilution, washing them with 10% fetal calf serum in PBS, and incubating them for 45 min with an Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G (Molecular Probes) at a 1:1,000 dilution. To visualize the nuclei, cells were additionally stained for 5 min with 4′,6′-diamidino-2-phenylindole (DAPI) dilactate (Molecular Probes) at a 1:25,000 dilution. Samples were mounted using Mowiol (Calbiochem), and images were collected with a confocal laser scanning microscope (DM IRE2; Leica) and processed with the LCS (version 2.02) software (Leica) and Adobe Photoshop (version 6.0). Images are Z-series compilations of 6 to 10 images in a stack.

Identification of APOBEC3G-associated proteins.

It was conceivable that overexpression of TAP-tagged A3G in 293T cells may have led to the assembly of nonphysiological complexes. To try to address this point, we used a retrovirus vector to generate a stable cell line expressing NTAP-A3G; to avoid possible competition between endogenous A3G and the ectopic NTAP-A3G for incorporation into complexes, we used the CEM-SS cell line, as it naturally expresses very low levels of A3G (13, 101). Immunoblot analysis indicated that these CEM-SS/NTAP-A3G cells express only ~4-fold more A3G than H9 T cells, a line that naturally restricts vif-deficient HIV-1 replication (data not shown) (105). Using the same procedure as for 293T cells, NTAP-A3G-containing complexes were purified from these T cells and the isolated proteins separated by SDS-PAGE (Fig. ​(Fig.1C1C shows a representative gel). Most of the bands that were visible following silver staining appear to match those noted in the 293T experiments. Indeed, many were present at higher quantities relative to NTAP-A3G, particularly band 4. CEM-SS cells stably expressing untagged A3G served as a negative control for the specificity of purification (data not shown). Based on these findings, the CEM-SS/NTAP-A3G cell line became our preferred source for the isolation of A3G-associated proteins.

The eight most prominent bands, as indicated (1 to 8) in Fig. ​Fig.1C,1C, were excised, subjected to tryptic digestion, and analyzed by MS to identify the constituent proteins; the lower-abundance bands were not analyzed initially but will be the subject of future work. The Mascot score (94) was used to justify the accuracy of protein identification, with a score of greater than 74 indicating a significant match. Table ​Table11 lists the proteins present in each gel slice in descending order according to the numbers of identified peptides for each protein. Twenty-two matches corresponding to discrete proteins were identified, with A3G being a major component of bands 6 and 7 (the latter likely corresponding to a degraded A3G product). Strikingly, many of the proteins identified in this analysis are known cellular RNA-binding proteins: poly(A)-binding protein, cytoplasmic 1 (PABPC1), and various hnRNP proteins are commonly found in RNP complexes (85); HuR is an AU-rich element-binding protein that affects stability and translation regulation of mRNAs (19, 42); the 130-kDa leucine-rich PPR motif-containing protein (LRP130) has been shown to be present in RNP complexes associated with nuclear and mitochondrial RNAs (83, 84); the 60-kDa ribonucleoprotein autoantigen SS-A/Ro (Ro60) and the Sjogren syndrome antigen B (SS-B/La) proteins are core components of Ro RNP complexes (128); and Mov10 was recently described as a component of an Argonaute protein-associated RNP complex (82, 113). In addition, a number of cytoskeletal and mitochondrial proteins were also found. Because A3G's association with many factors is RNase sensitive (Fig. ​(Fig.1B)1B) (25), thus defining these complexes as RNP complexes, we focused our immediate attention on the RNA-binding proteins we had identified.

RNA-dependent interactions with APOBEC3G.

The interactions of several proteins identified in NTAP-A3G complexes were further examined by coimmunoprecipitation using 293T cells cotransfected with an expression vector for HA-tagged A3G and one of several vectors each expressing an epitope-tagged version of the test protein of interest (Fig. ​(Fig.2).2). Following immunoprecipitation with an HA-specific antibody, samples were divided, and one half were treated with RNase. The maintenance or loss of the interaction with A3G was then evaluated by immunoblotting using antibodies specific for the epitopes present in the test proteins (upper panels).

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FIG. 2.

Coimmunoprecipitation of cellular proteins with A3G. Transfected 293T cells coexpressing A3G-HA (or empty vector as indicated) and tagged versions of cellular proteins (except LRP130) identified in the NTAP-A3G RNP complexes or Vif were lysed and immunoprecipitated with anti-HA or anti-myc antibodies and protein A or G linked to agarose. When indicated, the immunoprecipitates were treated with an RNase mixture (w/o, without RNase treatment). Samples were analyzed by immunoblotting using the indicated primary antibodies. In the La analyses, a background band of mobility slightly faster than that of tagged La was consistently seen for immunoprecipitated samples. For YB-1, the coimmunoprecipitation was reversed and A3G was detected on the blot. Bands indicated by asterisks correspond to immunoglobulin heavy or light chains.

Most of the proteins we tested, e.g., Mov10, Ro60, La, HuR, PABPC1, and SRP68, were specifically coimmunoprecipitated with A3G but were not recovered from the empty vector control samples (Fig. ​(Fig.2).2). In the case of YB-1, the experiment was performed in reverse because of the occlusion of YB-1-myc by the antibody heavy chains during immunoblotting. The cell lysates were therefore immunoprecipitated using a myc-specific antibody, and the association of A3G was then monitored by immunoblotting using an A3G-specific antibody (Fig. ​(Fig.2).2). Importantly, none of these particular proteins were detected in A3G-containing complexes after RNase treatment (Fig. ​(Fig.2),2), indicating that their presence in such complexes is dependent on RNA. The most likely explanation is that these proteins do not contact A3G directly but that they are associated with A3G-containing complexes through indirect interactions that are bridged by RNA. Similar experiments conducted with hnRNP U and LRP130 failed to confirm their presence in A3G complexes (Fig. ​(Fig.2).2). Whether this reflects a genuine lack of association or fortuitous copurification during TAP tag purification remains to be determined. However, it is perhaps worth noting that hnRNP U was also identified by mass spectrometry in similar affinity purification procedures of Staufen-containing RNP complexes, but its presence could not be confirmed by immunoblotting (17, 119). As a positive control for the detection of direct protein-protein interactions in these analyses, we confirmed that Vif could be coimmunoprecipitated with A3G in this experimental format and that binding was maintained following RNase treatment (Fig. ​(Fig.2).2). This observation was consistent with our earlier studies (Fig. ​(Fig.1B).1B). In sum, these coimmunoprecipitation experiments confirmed that many of the proteins identified in A3G complexes by TAP tag purification are indeed found in association with A3G, though the interactions are generally reliant upon RNA: it therefore appears reasonable to describe such complexes as A3G RNP complexes.

APOBEC3G localizes to discrete cytoplasmic foci.

Having begun to localize A3G biochemically in terms of proximity to, and interactions with, other cellular proteins, we next wished to examine localization using cell-imaging approaches, in particular confocal microscopy. Here, the literature is inconsistent, with some differing observations having been made; for instance, early results using transfected cells suggested that A3G is dispersed throughout the cytosol with no particular pattern of localization (2, 74, 76), whereas some more recent work has suggested that accumulation in cytoplasmic microdomains can occur (122). We therefore analyzed the subcellular localization of endogenous A3G in untransfected PBMCs and H9 cells by indirect immunofluorescence using an A3G-specific antibody (Fig. 3A and B, respectively). In both cell types, A3G was distributed throughout the cytoplasm, with a pronounced localization to discrete cytoplasmic foci. These varied in relative size and number from one cell to another and were not observed in parental CEM-SS cells, indicating that this staining pattern reflects the localization of the endogenous A3G protein (data not shown). Similar intense foci of A3G were also observed for the CEM-SS/NTAP-A3G line (Fig. ​(Fig.3C),3C), indicating that the tagging of A3G does not perturb subcellular localization, as well as for HeLa cells transfected with pA3G (Fig. ​(Fig.3D).3D). Finally, various fusions of A3G to fluorescent proteins, such as CFP or YFP, also localized to these cytoplasmic foci (data not shown; also see below). Importantly, appending these tags to A3G did not disrupt the ability of A3G to inhibit HIV-1 infectivity (data not shown).

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FIG. 3.

A3G is localized in the cytoplasm and is enriched in discrete cytoplasmic foci. PBMCs (A), H9 cells (B), CEM-SS cells stably expressing the NTAP-A3G protein (C), or HeLa cells transfected with pA3G (D) were fixed and probed with rabbit anti-A3G antibodies. The cells were then stained with a rabbit-specific secondary antibody directly conjugated to Alexa Fluor 594 dye. Cell nuclei were stained with DAPI dilactate, and the samples examined by confocal microscopy. Scale bars = 10 μm.

APOBEC3G localizes to P bodies.

Cytoplasmic RNP complexes can cluster or accumulate in discrete microdomains within the cytoplasm. In particular, P bodies and SGs are two types of nonmembraned RNP granules where translationally silenced mRNA is stored and/or degraded in mammalian cells (5, 7, 18, 27, 30, 38, 57, 58, 103, 111). P bodies are sites of accumulation for components of the mRNA-decapping and 5′-to-3′ mRNA decay machinery (30, 50, 71, 103, 117), the nonsense-mediated decay pathway (41, 104, 115), and RNA-induced silencing complexes (RISC) (68, 82, 96, 100), whereas SGs are sites where mRNAs coding for “housekeeping” proteins are transiently stored in a translationally silenced state during cellular stress, thereby facilitating the preferential translation of proteins important for the stress response (4, 6, 57). Although the precise roles of P bodies versus SGs have yet to be fully defined—and they may well overlap to some extent—mRNA localization to P bodies is generally associated with the terminal stages of RNA degradation pathways. Provocatively, Mov10, one of the proteins identified in A3G RNP complexes (Table ​(Table11 and Fig. ​Fig.2),2), is an established constituent of P bodies (82, 113), whereas certain other A3G-associated proteins such as PABPC1 and HuR can each accumulate in SGs (42, 59). Taken together with A3G's pronounced localization to cytoplasmic foci, these findings prompted us to investigate the possible targeting of A3G to P bodies and/or SGs.

We first examined A3G localization to P bodies (Fig. ​(Fig.4).4). Here, proliferating HeLa cell monolayers were cotransfected with expression vectors for tagged versions of A3G and various P-body components that carried either YFP, CFP, red fluorescent protein (RFP), or a myc epitope tag as required. The P-body proteins analyzed included Mov10, a putative RNA helicase (belonging to the dead-like helicase superfamily) that can associate with the Ago1 and Ago2 proteins (82, 113), both of which also accumulate in P bodies and are components of the RISC complex (39, 67, 81, 106); Ago1 and Ago2 themselves; the dead box helicase rck/p54, which facilitates P-body formation and acts as a translational repressor (26, 27); Dcp1a, the mRNA-decapping enzyme involved in the 5′-to-3′ degradation of mRNA (30, 71); and GW182, a protein that has been ascribed roles in RNA silencing and P-body integrity (53, 68). After 24 to 36 h, the cells were fixed, stained as necessary, and examined using confocal microscopy. Figure 4A to F show representative fields for each sample, with the single-channel analyses and merged images displayed. The results were essentially indistinguishable among these six samples, with A3G and all P-body markers displaying diffuse cytosolic staining and prominent localization to cytoplasmic microdomains. Most importantly, the majority of the A3G foci colocalized with P bodies, indicating that A3G can indeed accumulate in these RNP-rich regions of the cytoplasm. It was also clear, however, that not all areas of intense A3G accumulation coincided with specific P-body proteins (this is particularly obvious in the merged panel E), suggesting either that P bodies may be heterogeneous in their composition or that A3G can also localize to other cytoplasmic microdomains.

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FIG. 4.

A3G colocalizes with P bodies in the cytoplasm. HeLa cells coexpressing YFP-A3G, CFP-A3G, or myc-A3G (left panels) and the indicated P-body markers myc-Mov10 (A), myc-Ago1 (B), myc-Ago2 (C), RFP-rck/p54 (D), mRFP-Dcp1a (E), GFP-GW182 (F), and myc-YB-1 (G) (middle panels) were fixed and stained with the 9E10 antibody and a mouse-specific secondary antibody conjugated to Alexa Fluor 594 dye. Samples were viewed by confocal microscopy, and merged views are displayed on the right. Scale bars = 10 μm.

We also examined the possible colocalization of A3G with a number of the other proteins identified in NTAP-A3G complexes (Table ​(Table1).1). Again, unstressed cultures of HeLa cells were cotransfected with combinations of tagged expression vectors and subcellular localization was assessed using double-label confocal microscopy. To summarize a large number of experiments, we did not observe significant colocalization between A3G and LRP130, hnRNP U, La, Ro60, SRP68, PABPC1, or HuR (data not shown; also refer to Fig. ​Fig.5E).5E). Indeed, in some cases, such as those of hnRNP U and La, the test protein localized to the nucleus, consistent with published results (51, 77). Whether the capacity of these proteins to copurify with A3G-containing RNPs reflects artifactual associations taking place during the purification procedure or the physiologic binding of small fractions of protein naturally present in the cytoplasm remains to be determined. The exception in these findings was YB-1, which displayed a cytoplasmic staining but also clearly colocalized with A3G in cytoplasmic microdomains (Fig. ​(Fig.4G);4G); the accumulation of YB-1 in P bodies was confirmed in costaining experiments with Dcp1a (data not shown). A number of activities have been ascribed to YB-1, including its presence in translationally inactive RNP complexes, where it competes for initiation factors (35-37, 90). Given the current understanding of P-body function(s), it is perhaps not surprising that YB-1 can accumulate in them.

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FIG. 5.

A3G localizes to stress granules after heat shock. HeLa cells coexpressing CFP-A3G, YFP-A3G, or myc-A3G (left panels) and the indicated SG markers myc-PABPC1 (A), myc-HuR (B), YFP-TIA-1 (C), or SRP68-GFP (D) were incubated at 44°C for 30 min to induce SG formation. (E) Patterns of localization of myc-PABPC1, myc-HuR, and YFP-TIA-1 under normal cell growth conditions. Fixed cells were stained with the 9E10 antibody (A, B, D, and the first two panels of E), processed as for Fig. ​Fig.44 and viewed by confocal microscopy. Merged views are displayed to the right. Scale bars = 10 μm.

Localization of APOBEC3G to stress granules.

As discussed above, SGs are another category of RNP-rich cytoplasmic microdomains involved in cellular mRNA metabolism. They can assemble in response to cellular insult (59), contain many RNA-binding proteins (for a review, see reference 4), and possibly serve as a storage compartment for mRNAs whose translation is concomitantly suppressed (5). Because some components of A3G RNP complexes, such as PABPC1 and HuR, are known to accumulate in SGs under stress conditions (42, 59), we next asked whether A3G can also localize to SGs. To address this, we used the same double-label microscopy strategy described above, except that the transfected HeLa cells were subjected to heat shock at 44°C for 30 min prior to fixation and examination (Fig. ​(Fig.5).5). Previous work had indicated that this protocol induces the formation of SGs but not P bodies (58).

As seen in the panels displaying A3G staining alone, heat shock triggered dramatic changes in A3G localization such that much of the protein now accumulated in large cytoplasmic granules of irregular morphology. As expected, these areas of intense staining coincided with the localization of PABPC1 (Fig. ​(Fig.5A),5A), HuR (Fig. ​(Fig.5B),5B), and a previously defined SG marker, TIA-1 (Fig. ​(Fig.5C).5C). As discussed above, the striking colocalization of A3G with PABPC1 or HuR could not be observed in the absence of cellular stress (refer to Fig. ​Fig.5E5E for illustrations of PABPC1, HuR, and TIA-1 localization before heat shock), even though biochemical studies had indicated their presence in the same RNP complexes (Table ​(Table11 and Fig. ​Fig.2).2). Accordingly, we suspect that these complexes may contribute to the diffuse cytosolic A3G staining that is seen under unstressed conditions (Fig. ​(Fig.33 and ​and4)4) and that likely represents association with polysomes.

We also tested a number of other proteins found in A3G RNP complexes for their ability to colocalize with A3G in SGs following heat shock. Most proteins did not display this phenotype (data not shown); the exception was SRP68 (Fig. ​(Fig.5D).5D). It is unclear whether other components of the signal recognition particle and the 7SL RNA are also targeted to SGs, and further study will be required to address this question.

Redistribution of APOBEC3G and P-body components to stress granules.

Since A3G can accumulate in P bodies or SGs depending upon the physiologic status of the cell, we next asked whether any other P-body proteins redistribute to SGs under stress conditions. HeLa cells were triply transfected with pCFP-A3G, pEYFP-TIA-1 (to label SGs), and an expression vector for a myc- or mRFP-tagged P-body marker. Cells were later subjected to heat shock and examined by confocal microscopy as described above (Fig. ​(Fig.6).6). Most P-body proteins that were examined relocalized to SGs together with A3G: results are shown for Mov10 (Fig. ​(Fig.6A),6A), Ago2 (Fig. ​(Fig.6B),6B), and YB-1 (Fig. ​(Fig.6C),6C), but Ago1 and rck/p54 behaved similarly (data not shown; also see reference 125). The exceptions to this pattern were the decapping enzyme Dcp1a, which remained confined to foci of intense staining that were clearly distinct from SGs, and GW182, which remained predominantly associated with P bodies (Fig. ​(Fig.6D;6D; also data not shown and reference 58). Thus, a subset of P-body proteins continues to colocalize with A3G under changing cellular environments.

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FIG. 6.

Some P-body proteins colocalize to stress granules with A3G after heat shock. HeLa cells coexpressing CFP-A3G, YFP-TIA-1 (to define SGs), and the P-body markers myc-Mov10 (A), myc-Ago2 (B), myc-YB1 (C), and mRFP-Dcp1a (D) were stressed and analyzed as for Fig. ​Fig.5.5. Merged views are displayed to the right. Scale bars = 10 μm.

This work was supported by the United Kingdom Medical Research Council. S.G.-M. is a Fellow of the European Molecular Biology Organization, and M.H.M. is an Elizabeth Glaser Scientist supported by the Elizabeth Glaser Pediatric AIDS Foundation.

We thank Monica Agromayor, Paul Anderson, Edward Chan, Greg Hannon, Vicky Katsanou, Nancy Kedersha, Megerditch Kiledjian, Dimitris Kontoyiannis, Tim Ley, Jidong Liu, Zhi Hong Lu, Jens Lykke-Andersen, Juan Martin-Serrano, Gunter Meister, Thoru Pederson, Serafín Piñol-Roma, Ger Pruijn, Thomas Tuschl, and Dominique Weil for reagents and invaluable advice.