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Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the invivo stability of ARE-containing mRNAs

1998; Springer Nature; Volume: 17; Issue: 12 Linguagem: Inglês

10.1093/emboj/17.12.3448

1460-2075

Xiucai Fan,

RNA modifications and cancer

Article15 June 1998free access Overexpression of HuR, a nuclear–cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs Xinhao Cynthia Fan Xinhao Cynthia Fan Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT, 06536 USA Search for more papers by this author Joan A. Steitz Corresponding Author Joan A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT, 06536 USA Search for more papers by this author Xinhao Cynthia Fan Xinhao Cynthia Fan Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT, 06536 USA Search for more papers by this author Joan A. Steitz Corresponding Author Joan A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT, 06536 USA Search for more papers by this author Author Information Xinhao Cynthia Fan1 and Joan A. Steitz 1 1Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT, 06536 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3448-3460https://doi.org/10.1093/emboj/17.12.3448 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3′ untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of β-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti-HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa–L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment. Introduction Selective mRNA turnover is an important mechanism of eukaryotic gene regulation. The expression of proto-oncogenes, lymphokines and cytokines is usually transient, requiring rapid mRNA removal through destabilization following the cessation of transcription. AU-rich elements (AREs) located in their 3′ untranslated regions (3′ UTRs) comprise a major class of cis-elements that target these mRNAs for rapid degradation (Caput et al., 1986; Shaw and Kamen, 1986; for reviews, see Belasco and Brawerman, 1993; Chen and Shyu, 1995). Loss of this negative regulatory control conferred by AREs has been shown to be associated with transforming phenotypes (Miller et al., 1984; Meijlink et al., 1985; Lee,W. et al., 1988). Besides mRNAs, small nuclear RNAs (snRNAs) can also be targeted by AREs for rapid degradation, presumably through similar decay pathway(s) (Fan et al., 1997). ARE-mediated decay may also be differentially regulated. For instance, in a monocyte tumor cell line, c-fos mRNA is degraded constitutively, whereas the GM-CSF mRNA is stable (Schuler and Cole, 1988); in a human T cell line upon co-stimulation with anti-CD28 antibody, GM-CSF and interleukin-3 (IL-3) mRNAs have been reported to be selectively stabilized (Lindsten et al., 1989). It is not known, however, whether the decay pathway(s) are somehow inhibited or stabilizing pathway(s) are activated to override the decay pathways under such circumstances. Most AREs contain multiple copies of the sequence AUUUA (Caput et al., 1986; Shaw and Kamen, 1986; for reviews, see Belasco and Brawerman, 1993; Chen and Shyu, 1995). During ARE-mediated decay, shortening of the poly(A) tail precedes the degradation of the mRNA body (Shyu et al., 1991; Chen et al., 1995). Based on their sequence features, deadenylation and degradation kinetics, AUUUA-containing AREs have been classified into two groups (Shyu et al., 1991; Chen et al., 1995; Xu et al., 1997). Class I AREs, found mainly in proto-oncogene mRNAs (such as c-fos), contain one to three copies of dispersed AUUUA motifs in a U-rich region and mediate distributive synchronous poly(A) shortening followed by rapid degradation of the mRNA body. In contrast, class II AREs, found mostly in cytokine mRNAs (such as GM-CSF), contain multiple copies of clustered AUUUA motifs and direct asynchronous deadenylation, suggesting a processive nucleolytic digestion of the poly(A) tail followed by mRNA decay (Shyu et al., 1991; Chen et al., 1995; Xu et al., 1997). To define the cellular degradation machinery responsive to ARE signals, much effort has been devoted to identifying ARE-binding proteins (Malter, 1989; Bohjanen et al., 1991, 1992; Brewer, 1991; Malter and Hong, 1991; Vakalopoulou et al., 1991; Myer et al., 1992, 1997; Hamilton et al., 1993, 1997; Zhang et al., 1993; Katz et al., 1994; Nakagawa et al., 1995; Wennborg et al., 1995). We have focused on a protein of 32 kDa apparent molecular weight, first identified by UV-crosslinking to the c-fos ARE in HeLa cell extracts (Vakalopoulou et al., 1991). This protein also binds to the AU-rich regions of Herpesvirus saimiri U snRNAs (HSURs) 1, 2 and 5, which are highly expressed in virally transformed marmoset T cells (Lee,S. et al., 1988; Lee and Steitz, 1990; Myer et al., 1992). Repeated copies of AUUUA at the 5′ end of HSUR 1 have been shown to target this and other snRNAs for rapid degradation upon transient transfection into several mammalian cell lines (Fan et al., 1997). Characterization of the purified 32 kDa protein demonstrated that it is identical to HuR (also called HuA; Myer et al., 1997), a ubiquitously expressed member of the Hu family of proteins (Ma et al., 1996). Three other Hu proteins, namely Hel-N1 (or HuB), HuC and HuD, have been previously identified as target antigens in paraneoplastic encephalomyelitis sensory neuronopathy (the Hu syndrome), which is characterized by diverse neuronal degeneration associated with small cell lung cancer (SCLC) (Dalmau et al., 1991; Szabo et al., 1991; Levine et al., 1993). It is believed that patients develop autoantibodies against Hu antigens abnormally expressed in SCLC, which then attack the central nervous system, leading to neuronal dysfunction and finally death (for reviews, see Posner, 1995; Darnell, 1996). All four Hu proteins contain three highly conserved RNA binding domains belonging to the RRM (RNA recognition motif; also called RBD) superfamily (Burd and Dreyfuss, 1994), but their auxiliary regions (the sequence N-terminal to RRM 1 and the hinge region between RRMs 2 and 3) differ. All Hu proteins exhibit high affinity for AU-rich RNA sequences (Levine et al., 1993; Gao et al., 1994; Abe et al., 1996; Ma et al., 1996, 1997; Jain et al., 1997; Myer et al., 1997). Deletion studies conducted on HuC, HuD and HuR have suggested that RRMs 1 and 2 are responsible for ARE-binding, whereas RRM 3 binds simultaneously to the poly(A) tail of an mRNA (Abe et al., 1996; Ma et al., 1997). Hu proteins are highly conserved. Their RRMs exhibit strong homology to those of the Drosophila RNA binding proteins Elav (embryonic lethal, abnormal vision) and sxl (sex-lethal). Elav was originally cloned in a screen designed to identify Drosophila nervous system defects and was found to be essential for normal neural development (Campos et al., 1985; Robinow et al., 1988). Homologs of each of the four human Hu proteins have been identified in other vertebrates, such as Xenopus (Good, 1995), zebrafish (Good, 1995), chicken (Wakamatsu and Weston, 1997) and mouse (Okano and Darnell, 1997). They have been classified into four groups called Elav-like ribonucleoproteins A–D (elr A–D, or Hu A–D, respectively), based on both sequence similarities in the auxiliary regions and the tissue specificity of their mRNA expression (Good, 1995; Okano and Darnell, 1997). Elr B, C and D are neuron-specific, while elr A is expressed ubiquitously at the mRNA level (Good, 1995; Okano and Darnell, 1997). Human HuR (Hu A) protein is the most closely related to Drosophila Elav and sxl among the four human Hu proteins and has been considered ancestral to the other three (Okano and Darnell, 1997). It is over 99% identical to mouse HuA at the amino acid level (Ma et al., 1996), 98.2% to chicken (Wakamatsu and Weston, 1997) and over 90% to the Xenopus protein (Good, 1995; Ma et al., 1996). HuR has been implicated in ARE-mediated rapid mRNA destabilization because of a direct correlation between its in vitro affinity for a variety of ARE sequences and the ability of these sequences to direct in vivo degradation of a reporter mRNA (Myer et al., 1997). To ascertain HuR's involvement in mRNA decay in vivo, we have assayed the decay rates of ARE-containing mRNAs in mouse L929 cells overexpressing HuR and have observed stabilization of mRNAs containing AREs of both class I and II (c-fos and GM-CSF). We have also established the subcellular localization of HuR in HeLa and mouse L cells as nuclear. Yet, both the endogenous and epitope-tagged HuR actively shuttle between nucleus and cytoplasm. We discuss models for HuR function in mammalian cells. Results Expression of HuR in cultured mammalian cell lines We raised rabbit polyclonal antibodies against His-tagged HuR recombinant protein (kindly prepared by Dr V.Myer) and affinity-purified them through a Ni2+-agarose column coupled to His-tagged recombinant mouse HuR (mHuA) protein (see Materials and methods; the pET-mHuA plasmid was kindly provided by Dr R.Darnell). The rabbit antiserum and the affinity-purified antibody were analyzed by Western blot using HeLa whole-cell extract (Figure 1A, lanes 1 and 2). To verify that the single band detected in Figure 1A, lane 2 (indicated by an arrow) is indeed HuR, the purified antibody was depleted by passing the solution through an affinity column that was pre-loaded with His-mHuA at a 5-fold excess relative to the antibody. Use of this depleted antibody preparation resulted in a complete loss of signal (Figure 1A, lane 3) in a strip loaded with the same amount of HeLa cell lysate as in lanes 1 and 2 and run on the same gel. Figure 1.Western blot analysis of HuR expression. (A) Specificity of the anti-HuR polyclonal antibody. Rabbit polyclonal antibody was raised against His-tagged recombinant HuR protein, affinity-purified through a Ni2+-agarose column coupled to recombinant His-mHuA protein. HeLa total cell extract was fractionated (50 μg protein/lane) on 12.5% SDS–PAGE and blotted with crude anti-HuR rabbit antiserum at 1:2000 dilution (lane 1), or with affinity-purified anti-HuR polyclonal antibody at 0.7 μg/ml (also 1:2000, lane 2). The affinity-purified anti-HuR was depleted by passage through an antigen affinity column pre-loaded with a 5-fold excess (molar ratio) mHuA relative to the antibody, and blotted at the same (1:2000) dilution (lane 3). The HuR signal is indicated by an arrow in lane 2. (B) Expression of HuR in cultured cell lines. Total cell lysates from 1670, HEK 293, NIH 3T3, L929, HeLa and COS were prepared by sonication, fractionated at 200 μg/lane on 12.5% SDS–PAGE and immunoblotted using the affinity-purified anti-HuR antibody at 0.7 μg/ml (lanes 1–7, respectively). The blots were stripped and re-probed with anti-tubulin monoclonal antibody (Calbiochem) at 1 μg/ml as an internal control. L929 cells transiently transfected with pCDNA3-HuR and pCDNA3-HuR-C-Flag were analyzed in the same way (lanes 8 and 9, respectively). This blot was also stripped and re-blotted with anti-Flag (Sigma) at 1 μg/ml dilution (lanes 5–9). The signals were analyzed using the NIH Image Program. HuR levels in 1670, HEK 293, NIH 3T3, HeLa, COS and L929+HuR were found to be ∼6-, 7-, 0.6-, 5-, 5- and 3-fold, respectively, relative to that in L929 cells. The overexpressed HuR-C-Flag signal (lane 9) was ∼2-fold relative to the endogenous HuR. The secondary bands in lanes 2 and 6 are likely to be gel artifacts, rather than degradation products, as they do not appear in repeated Western blots of the same extracts. Download figure Download PowerPoint Since HuR expression has only been reported to occur at the RNA level in non-neuronal mammalian tissues (Ma et al., 1996; Okano and Darnell, 1997), we examined HuR protein expression in various cell lines through Western analysis. Total cell lysates from 1670 (marmoset T cells), HEK 293 (human embryonic kidney cells), NIH 3T3 (mouse fibroblasts), L929 (mouse connective tissue cells), HeLa (human epithelial cells) and COS (monkey kidney fibroblasts) were prepared by sonication and immunoblotted using the affinity-purified anti-HuR antibody. As shown in Figure 1B, HuR protein levels are high in 1670, HEK 293, HeLa and COS cells (Figure 1B, lanes 1, 2, 5 and 6, respectively) and lower, but still detectable in NIH 3T3 and L929 cells (lane 3 for 3T3 cells; lanes 4 and 7 for L cells). The blots were stripped and re-probed with anti-tubulin monoclonal antibody as an internal loading control. We conclude that HuR protein is expressed in a variety of mammalian cell lines. We next investigated whether transient transfection of a human HuR cDNA [pCDNA3-HuR, driven by the human cytomegalovirus (CMV) immediate early gene promoter] into mouse L929 cells could provide overexpression of HuR. As analyzed by Western blot, a protein of the same size as endogenous HuR (∼36 kDa) is detected in transfected cell lysates (Figure 1B, lane 8); the level of the HuR band is increased ∼3-fold compared with non-transfected cells (Figure 1B, lane 7). Since the transient transfection efficiency of mouse L cells using DEAE–dextran is ∼30% (Fan et al., 1997), the overexpression of HuR is therefore estimated to be ∼8-fold relative to the endogenous level in those L cells that have been transfected, similar to the level of HuR protein expression in HEK 293 cells (Figure 1B, lane 2). A C-terminal Flag-tagged HuR, expressed from the same CMV promoter (pCDNA3-HuR-C-Flag), was also transiently transfected into L cells and immunoblotted with anti-HuR. A protein of ∼37 kDa, consistent with the addition of the Flag epitope, is detected in these transfected cells (Figure 1B, lane 9) at approximately the same level as in the non-tagged HuR transfectants (Figure 1B, lane 8). The blot was stripped and probed with anti-Flag monoclonal antibody, yielding a band at 37 kDa only in extracts from cells transfected with HuR-C-Flag (Figure 1B, lane 9). These results further verify the specificity of the affinity-purified anti-HuR polyclonal antibody. Nuclear localization of HuR While HuR was originally isolated from HeLa cell nuclear extract (Myer et al., 1997), a fraction of the protein had also been reported to be cytoplasmic (Vakalopoulou et al., 1991). We investigated the subcellular localization of HuR by indirect immunofluorescence. HeLa cells grown on coverslips were fixed with 3% paraformaldehyde for 20 min, permeabilized with 0.5% Triton for 15 min, and stained using the affinity-purified anti-HuR polyclonal antibody. The cells were co-stained with either the Y12 monoclonal antibody that recognizes Sm snRNPs (which exhibit nuclear localization with nucleolar exclusion) (Lerner et al., 1981) or with the Y10B monoclonal antibody, which is immunoreactive with ribosomal RNA (rRNA; showing cytoplasmic and nucleolar staining) (Lerner et al., 1981). The secondary antibody for anti-HuR, goat-anti-rabbit IgG, was coupled to Texas red, and that for the monoclonal antibodies, goat-anti-mouse IgG, to FITC. Normal rabbit IgG and anti-HuR pre-cleared by exposure to the mHuA antigen affinity column were also applied at the same dilution as negative controls. As shown in Figure 2, staining of HuR in HeLa cells with the affinity-purified anti-HuR antibody reveals a nearly exclusive nuclear localization, with nucleolar exclusion (Figure 2B and F). Normal rabbit IgG and the antigen-depleted anti-HuR antibody yield no immunofluorescence signals (Figure 2Q and N, respectively), while Y12 (Figure 2C, J, O and R) and Y10B (Figure 2G) stain their respective antigens as expected. HuR's nucleoplasmic distribution is confirmed by the superimposed images (Figure 2D and H): it displays an overlapping pattern with Sm snRNPs, as shown by the orange color (Figure 2D), and is complementary to the rRNA staining (Figure 2H). A second fixation/permeabilization method was also used; the cells were fixed with 2% formaldehyde for 30 min followed by 3 min at −20°C in acetone. HuR exhibited the same nucleoplasmic staining (data not shown) as in Figure 2, suggesting that the fixation procedures do not alter the intracellular distribution of HuR. Figure 2.Nucleoplasmic localization of endogenous and transfected HuR in HeLa and L929 cells. HeLa (A–H, M–O), mouse L929 cells (I and J, P–R) and L929 cells transfected with pCDNA3-HuR-C-Flag (K and L) were fixed with 3% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 and stained with either the affinity-purified anti-HuR polyclonal antibody (B, F, I and K), affinity-purified anti-HuR depleted by exposure to mHuA antigen (N), or normal rabbit IgG (Q). Cells were co-stained with either the Y12 monoclonal antibody that recognizes Sm snRNPs in the nucleoplasm (C, J, O and R), Y10B monoclonal antibody against rRNA in the nucleoli and cytoplasm (G), or anti-Flag monoclonal antibody (Sigma) (L). The transfected L929 cell overexpressing HuR-C-Flag is indicated by an arrow (K and L). The secondary antibody for anti-HuR, goat-anti-rabbit IgG (Jackson ImmunoResearch Laboratory), was coupled to Texas red, and that for the monoclonal antibodies, goat-anti-mouse IgG, to FITC. Superimposed red and green images are shown in (D) (for B and C) and (H) (for F and G). (A) and (E) show HeLa cells in phase-contrast while (M) and (P) are Nomarski images. Bars, 10 μm. Download figure Download PowerPoint Mouse L929 cells untransfected or transfected with pCDNA3-HuR-C-Flag were also subjected to immunofluorescent staining. As shown in Figure 2, endogenous HuR is localized in the L cell nucleus (Figure 2I), but the signal is weaker than in HeLa cells, consistent with the results of Western analysis (Figure 1B). Overexpressed HuR-C-Flag in transfected L cells (indicated by arrows in Figure 2K and L) is likewise predominantly nucleoplasmic (Figure 2K). Double staining with both affinity-purified anti-HuR polyclonal (Figure 2K) and anti-Flag monoclonal antibodies (Figure 2L) confirms the co-localization of the endogenous and the Flag-tagged HuR proteins. Overexpression of HuR stabilizes ARE-containing mRNAs The correlative data that previously implicated HuR in the ARE-mediated mRNA decay pathway (Myer et al., 1997) could not distinguish whether HuR functions as a stabilizer or a destabilizer. Overexpression and correct nuclear localization in transfected cells provided an opportunity to dissect HuR's role further by examining the in vivo decay of ARE-containing reporter mRNAs co-transfected with HuR. We chose the β-globin reporter gene with a serum inducible c-fos promoter and a c-fos ARE [pB-ARE(fos)], utilized in many transient transfection studies of mRNA stability (Shyu et al., 1989; Schiavi et al., 1994; Zubiaga et al., 1995; Fan et al., 1997; Myer et al., 1997). The 51-nucleotide class I c-fos ARE (Shyu et al., 1989) was shown previously to have a high affinity for HuR (Myer et al., 1992). Plasmids pCDNA3-HuR and pB-ARE(fos) (Myer et al., 1997) were co-transfected into mouse L cells along with a control plasmid pEF-BOS-CAT, which constitutively expresses the CAT mRNA (Zubiaga et al., 1995; Myer et al., 1997). After 24 h of serum starvation, cells were collected at 0, 1.5, 3.25, 4.5 and 5.5 h following serum induction; total RNA was isolated and analyzed by RNase T1 protection (Figure 3A). The data, averaged from several experiments and standardized to the CAT mRNA internal control, are plotted in Figure 3B. The maximum signal in each case was considered 100%. Plasmid pCDNA3 alone, without the HuR insert, was assayed as a negative control. Figure 3.Overexpression of HuR stabilizes β-globin reporter mRNAs containing both class I (c-fos) and II (GM-CSF) AREs. (A) Transient transfection analyses. The β-globin reporter [pB-ARE(fos), pB-ARE(GM-CSF) or pBBB] and CAT (pEF-BOS-CAT) plasmids were transiently co-transfected into mouse L929 cells along with pCDNA3 plasmids overexpressing HuR, HuR-C-Flag or hnRNP A0, as indicated. After serum starvation, β-globin transcription was stimulated by serum addition and cells were harvested at the time intervals indicated. Total RNA was isolated for T1 RNase protection analyses using both a β-globin and a CAT antisense probe. (B) Time-course of β-globin mRNA decay. The β-globin signals in the RNase protection assays (as in Figure 3A) were quantitated on a Molecular Dynamics PhosphorImager, standardized to the CAT internal control (the lower band of the doublet was quantitated as the major T1 digestion product) and plotted. 100% was arbitrarily assigned to the time point with the highest signal. The results are the average of duplicate experiments (except for pCDNA3-HuR, which is the average of three repeats); the variability at each time point was within 5% relative to the maximal signal. Download figure Download PowerPoint As is evident from Figure 3A and B, in the negative control cells (which are co-transfected with the pCDNA3 vector alone), the pB-ARE(fos) mRNA level decays to 40% at 5.75 h (half-life = 2.5 h) after induction in cells overexpressing HuR. Conversely, in cells transfected with vector alone or with hnRNP A0, pB-ARE(GM-CSF) mRNA levels decayed to <20% (half-lives = 1.3 or 1.2 h, respectively). We conclude that overexpression of HuR can stabilize mRNAs containing AREs of both class I and II. One explanation for the observed stabilization is that excess HuR traps ARE-containing mRNAs in the nucleus, keeping them away from the degradation machinery. We eliminated this possibility by cloning the AU-rich sequence into the 3′ UTR of a luciferase reporter gene, and carrying out luciferase activity assays. In eight independent transient transfection experiments, we found the luciferase activity to be 2.0 ± 0.4-fold higher in cells overexpressing HuR than in control cells transfected with vector alone. Enhanced translation of the ARE-containing reporter mRNA indicates that the major effect of overexpressing HuR cannot be to sequester bound mRNAs in the nucleus. HuR shuttles between nucleus and cytoplasm We have shown that HuR is a nuclear protein and that its overexpression interferes with mRNA decay, which is believed to occur in the cytoplasm. This raised the possibility that HuR may shuttle between nucleus and cytoplasm, binding initially to an ARE-containing mRNA in the nuclear compartment, but then providing protection during its cytoplasmic life. We utilized the ability of HeLa and mouse L929 cells to form heterokaryons (Pinol-Roma and Dreyfuss, 1992) to test whether HuR in fact shuttles between nucleus and cytoplasm. Since the C-terminal Flag-tagged HuR exhibits the same subcellular localization (Figure 2) and mRNA stabilization activity (Figure 3) as the non-tagged HuR, we transfected HeLa cells with HuR-C-Flag and then fused them with mouse L cells. If HuR shuttles, the tagged protein visualized by immunostaining using anti-Flag antibodies should be transported to the mouse nuclei, even in the absence of protein synthesis. A myc-epitope-tagged hnRNP A1, which had been shown previously to shuttle (Pinol-Roma and Dreyfuss, 1992; Michael et al., 1995; Siomi and Dreyfuss, 1995), was transfected in parallel as a positive control. The non-shuttling hnRNP C1 (also myc-tagged) provided a negative control (Pinol-Roma and Dreyfuss, 1992; Nakielny and Dreyfuss, 1996). As shown in Figure 4, at 4 h after fusion in the presence of the protein synthesis inhibitor cycloheximide, HuR-C-Flag appears in both the HeLa and L cell nuclei of the heterokaryons (Figure 4A). [The HeLa and mouse nuclei can be distinguished by co-staining with Hoechst dye (Moser et al., 1975), which produces bright dots in mouse cell nuclei, indicated by arrows in Figure 4B, E and H)]. HuR's behavior therefore mimics that of the known shuttling protein hnRNP A1-myc, which is likewise detected in both HeLa and L nuclei (Figure 4D). Conversely, the non-shuttling protein hnRNP C1-myc is confined to the HeLa nucleus in the heterokaryon (panel G), confirming that the amount of cycloheximide used is sufficient to shut down protein synthesis. Figure 4.HuR shuttles between the nucleus and cytoplasm. HeLa cells were transiently transfected with plasmid pCDNA3-HuR-C-Flag, pCDNA3-hnRNP A1-myc (Pinol-Roma and Dreyfuss, 1992), or pCDNA3-hnRNP C1-myc (Pinol-Roma and Dreyfuss, 1992), fused with mouse L929 cells to form he