© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4025-4033 doi:10.1242/jcs.192096

RESEARCH ARTICLE RNA-binding HuR regulates nuclear import of protein Wei Zhang1, Amanda C. Vreeland1 and Noa Noy1,2,*

ABSTRACT Donato et al., 2007; Dong et al., 1999; Manor et al., 2003; Schug The RNA-binding protein HuR binds to elements rich in adenylate and et al., 2007; Sessler and Noy, 2005). Surprisingly, we found that, in uridylate (AU-rich elements) in target mRNAs and stabilizes them the absence of RA, CRABP2 binds to HuR and dramatically increases against degradation. The complete spectrum of whose its RNA-binding affinity. Consequently, CRABP2 enhances the expression is regulated by HuR and are the basis for the broad stability of various HuR target transcripts and upregulates their levels range of cellular functions of the protein is incompletely understood. (Vreeland et al., 2014b). Binding of RA triggers dissociation of We show that HuR controls the expression of multiple components of CRABP2 from HuR and activates a nuclear localization signal (NLS), the nuclear import machinery. Consequently, HuR is crucial for the allowing it to mobilize to the nucleus where it delivers RA to RAR. nuclear import of cellular retinoic acid-binding protein 2 (CRABP2), Our observations showed that the well-established anti-carcinogenic which delivers RA to the nuclear retinoic acid receptor (RAR) and activity of CRABP2 is mediated both by its ability to activate RAR, whose mobilization to the nucleus is mediated by a ‘classical-like’ leading to induction of anti-proliferative RAR target genes (Budhu nuclear localization signal (NLS). HuR is also required for heregulin- et al., 2001; Donato and Noy, 2005; Donato et al., 2007; Manor et al., induced nuclear translocation of the NFκB subunit p65, which 2003), and by cooperation with HuR, resulting in stabilization of anti- contains both classical and non-canonical NLSs. HuR thus oncogenic HuR target mRNAs (Vreeland et al., 2014a,b). regulates the transcriptional activities of both RAR and NFκB. The Interestingly, we found that reducing the expression of HuR not observations reveal that HuR plays a central role in regulating nuclear only abolished the ability of CRABP2 to upregulate HuR target import of . transcripts but also inhibited the transcriptional activity of RAR (Vreeland et al., 2014a). The additional observation that reducing the KEY WORDS: HuR, RNA stability, Nuclear import, Retinoic acid, expression of HuR blocked nuclear translocation of CRABP2 Cellular retinoic acid-binding protein, NFκB suggested that HuR is involved in some aspects of nuclear import (Vreeland et al., 2014a). INTRODUCTION Proteins that localize to the nucleus contain nuclear targeting One of the best-characterized RNA-binding proteins in animals is sequences that link them to carrier proteins of the β- HuR (encoded for by Elavl1), a ubiquitously-expressed member (-β) superfamily. Linkage can be accomplished by direct of the embryonic lethal abnormal vision (ELAV)/Hu family of binding to an importin-β or through mediation by adaptor proteins RNA-binding proteins. In the nucleus, HuR is involved in various such as importin-α (karyopherin-α). The best-characterized nuclear functions, including RNA splicing and nuclear export. In the targeting signal is the classical NLS recognized by an importin-α that, extranuclear compartment, HuR is associated with polysomes and in turn, links the NLS-containing protein to importin-β1(encodedfor binds elements rich in adenylate and uridylate (AU-rich elements; by Kpnb1) (Gorlich et al., 1995). Classical NLSs are usually ARE) in 3′UTR regions of target transcripts and protects them identifiable in the primary sequence of a protein as a series of basic against degradation (Brennan and Steitz, 2001; Chen et al., 2002; residues (Chook and Blobel, 2001; Hodel et al., 2001; Kalderon et al., Hinman and Lou, 2008; Lebedeva et al., 2011; Lopez de Silanes 1984; Robbins et al., 1991). We previously showed that CRABP2 et al., 2004; Mukherjee et al., 2011; Myer et al., 1997). By contains an unusual variation of a classical NLS. Instead of existing in stabilizing target mRNAs, HuR is involved in key biological the primary sequence, this NLS is assembled in the three-dimensional processes including cell-cycle progression, apoptosis, immune structure of the protein in response to RA binding. Consequently, function, inflammation and oncogenic activities (Abdelmohsen and holo-CRABP2 is recognized by importin-α1 (encoded for by Gorospe, 2010; Ghosh et al., 2009; Gupta et al., 2012; Hinman and Kpna2), enabling its ligand-induced nuclear translocation (Sessler Lou, 2008; Lebedeva et al., 2011). and Noy, 2005). Other importin-β proteins, such as transportin We recently discovered that transcript stabilization by HuR is aided 1/importin-β2 (encoded for by Tnpo1), directly bind cargoes that by cellular retinoic acid-binding protein 2 (CRABP2) (Vreeland et al., contain a broad range of ‘non-classical’ NLSs characterized by a C- 2014b). The best-understood function of CRABP2 is its ability to terminal sequence comprised of Arg separated from a Pro-Tyr (PY) shuttle retinoic acid (RA) from the cytosol to the nucleus, where it is motif by two to five residues (Christie et al., 2015). An additional key directly delivered to the nuclear retinoic acid receptor (RAR) and component of the nuclear import machinery is the small GTPase . thereby promotes transcriptional activity (Budhu and Noy, 2002; The activation state of Ran is determined by Ran guanine nucleotide exchange factor (RanGEF), which loads the protein with GTP, and Ran GTPase-activating protein (RanGAP), which facilitates the 1Department of Cellular & Molecular Medicine, Lerner Research Institute, hydrolysis of Ran-bound GTP to GDP (Bischoff et al., 1994; Cleveland Clinic Foundation, Cleveland, OH 44195, USA. 2Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Bischoff and Ponstingl, 1991; Gorlich et al., 1996). GTP hydrolysis by Ran is also stimulated by the Ran-binding protein RanBP1 *Author for correspondence ([email protected]) (Bischoff et al., 1995). Nuclear RanGEF and cytosolic RanGAP/ N.N., 0000-0002-2404-1443 RanBP1 thus form a RanGDP/GTP gradient that allows cargo-loaded to release their cargo in the nucleus and to dissociate from

Received 6 May 2016; Accepted 2 September 2016 Ran in the cytosol (reviewed in Christie et al., 2015). Journal of Cell Science

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Here, we show that HuR regulates the expression of several endoplasmic reticulum (ER) marker calnexin, both in the absence components of the nuclear import machinery and thus controls the and presence of CGP-74514A (Fig. 1A), demonstrating that nuclear localization of both CRABP2, which contains a classical extranuclear HuR is associated with the ER. NLS, and the NFκB subunit p65, which contains both a classical We previously reported that, in the absence of RA, CRABP2 NLS and a non-canonical NLS. We also show that, although localizes at the ER (Majumdar et al., 2011). Considering that importin-β1 and RanBP1 are necessary for nuclear import of CRABP2 is highly soluble, this localization is probably mediated by CRABP2, they are dispensable for the cross-cytosol trafficking that protein–protein interactions and not by interactions with the ER allows the protein to accumulate at the nuclear membrane in membrane. Together, the observations that HuR is associated with response to RA. the ER and can bind CRABP2 (Vreeland et al., 2014b) suggest that the ER localization of CRABP2 is mediated by HuR. To examine RESULTS this possibility, a cell line derived from tumors that arose in a HuR mediates the endoplasmic reticulum localization of CRABP2-null MMTV-neu mouse model of breast cancer (M2−/− apo-CRABP2 cells) (Schug et al., 2008) was used. M2−/− lines that stably express HuR shuttles between the nucleus and the cytosol but is short hairpin RNA (shRNA) targeting luciferase (shCtrl) or shRNA predominantly nuclear in non-dividing cells (Fig. 1A). The cyto– targeting HuR (shHuR) were generated (Fig. 1B). Cells were nuclear shuttling of this protein is regulated by cyclin-dependent depleted of retinoids by culturing in charcoal-treated medium and kinase 1 (Cdk1), and inhibition of the kinase results in retention of were transfected with a vector encoding Flag-tagged CRABP2. HuR in the cytosol (Kim et al., 2008). Indeed, a larger fraction of Parental cells and cells with reduced expression of HuR were co- HuR was found in the extranuclear compartment of cells treated immunostained for Flag–CRABP2 and the ER marker calnexin with the Cdk1 inhibitor CGP-74514A than in control cells (Fig. 1C). As expected, CRABP2 co-localized with calnexin in (Fig. 2A). Cytosolic HuR extensively co-localized with the parental cells, reflecting its ER association. Notably, the co-

Fig. 1. HuR mediates the association of CRABP2 with endoplasmic reticulum. (A) M2−/− cells were untreated (−) or treated with CGP74514A (2 µM, 2 h) (+CGP). The ER marker calnexin and HuR were visualized by immunostaining. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. (B) Immunoblot demonstrating expression of HuR in M2−/− cells stably expressing shRNAs targeting luciferase (shCtrl) or HuR (shHuR). (C) M2−/− cells stably expressing shCtrl or shHuR were transiently transfected with a vector encoding Flag–CRABP2. Flag–CRABP2 (red) and ER marker calnexin (green) were immunostained and visualized using confocal microscopy. DAPI was used to visualize nuclei. Journal of Cell Science

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Fig. 2. HuR is required for RA-induced nuclear translocation of CRABP2. (A) M2−/− cells stably expressing shCtrl or shHuR were cultured in delipidated medium and transfected with vector encoding Flag–CRABP2. Flag–CRABP2 was immunostained in untreated cells and in cells treated with RA for 30 min, and visualized using confocal microscopy. DAPI was used to visualize nuclei. (B) Immunoblot demonstrating expression of HuR in MCF-7 cells stably expressing shCtrl or shHuR. (C) MCF-7 cells expressing shCtrl or shHuR were cultured in delipidated medium and treated with vehicle or RA for 30 min. Endogenous CRABP2 was immunostained and visualized using confocal microscopy. DAPI was used to visualize nuclei. (D) M2−/− cells stably expressing shHuR were transfected with a vector encoding Flag–CRABP2 and treated with vehicle or RA. Flag–CRABP2 (red) and nuclear membrane marker lamin B (green) were immunostained and visualized using confocal microscopy. DAPI was used to visualize nuclei. localization was markedly reduced in cells with decreased contrast, in MCF-7 cells with reduced expression of HuR, treatment expression of HuR (Fig. 1C), suggesting that CRABP2 is with RA resulted in accumulation of CRABP2 around the nucleus associated with the ER through interaction with HuR. but, again, failed to affect complete nuclear import (Fig. 2C). Fluorescence microscopy showed that, in the presence of RA, HuR is required for RA-induced nuclear import of CRABP2 CRABP2 displays extensive co-localization with the nuclear Upon binding RA, CRABP2 dissociates from the ER and mobilizes membrane marker lamin B1 (Fig. 2D). Hence, HuR is not to the nucleus, a process that is complete within 30 min of RA necessary for mobilization of holo-CRABP2 to the nuclear treatment (Majumdar et al., 2011; Sessler and Noy, 2005) (Fig. 2A). membrane but is crucial for enabling translocation of the protein Strikingly, in M2−/− cells with stable reduced expression of HuR, across this membrane into the nucleus. RA induced a discernible shift in the subcellular localization of CRABP2, but this shift did not culminate in nuclear import. Instead, HuR controls the nuclear import of CRABP2 in part by CRABP2 accumulated around the nucleus and did not enter this regulating the expression of importin-β1 compartment (Fig. 2A). Similar responses were observed in M2−/− Binding of RA to CRABP2 induces structural rearrangements that cells in which HuR expression was transiently reduced using two activate an NLS recognized by importin-α1 (Sessler and Noy, 2005). different shRNAs (Fig. S1), confirming that the effect reflects a The possibility that HuR regulates the nuclear import of CRABP2 reduction in HuR expression and not off-target effects of the by controlling importin-α1 expression was therefore examined. shRNA. MCF-7 mammary carcinoma cells, which express a high However, data showed that decreasing the expression of HuR did not level of CRABP2, were used to examine the involvement of HuR in affect the expression of importin-α1 in either M2−/− (Fig. 3A) or the nuclear import of endogenous protein. MCF-7 cell lines that MCF-7 cells (Fig. 3B). Nuclear import of importin-α1 and its cargo is stably expressed either shCtrl or shHuR were generated (Fig. 2B). known to rely on an importin-β such as importin-β1 (Chook and Cells were treated with RA, immunostained for CRABP2, and Blobel, 2001; Gorlich et al., 1995; Hodel et al., 2001; Kalderon et al., imaged. Endogenous CRABP2 in parental MCF-7 cells underwent 1984; Robbins et al., 1991). Examination of importin-β1(encodedfor nuclear translocation in response to RA, which was similar to its by the Kpnb1 ) expression revealed that decreasing the −/− behavior in M2 cells that ectopically overexpress CRABP2. By expression of HuR reduced the levels of both importin-β1protein Journal of Cell Science

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Fig. 3. HuR stabilizes mRNA for importin-β1(Kpnb1) but not for importin-α1. (A,B) Immunoblots of importin-α1inM2−/− (A) or MCF-7 (B) cells stably expressing shCtrl or shHuR. (C,D) Immunoblots of importin-β1inM2−/− (C) or MCF-7 (D) cells stably expressing shCtrl or shHuR. (E) Levels of mRNA for importin- β1(Kpnb1)inM2−/− cells stably expressing shCtrl or shHuR, as measured by Q-PCR and normalized to 18s (mean±s.d.; n=3). *P<0.05, paired Student’s t-test. (F) M2−/− cells stably expressing shCtrl or shHuR were treated with actinomycin D (2.5 µg/ml). Levels of Kpnb1 mRNA at various time points following treatment were measured by Q-PCR. Data (mean±s.e.m.; n=3) were normalized to corresponding values at time zero. (G) HuR was immunoprecipitated from M2−/− cells. Kpnb1 mRNA that co-precipitated with HuR was detected by semi-quantitative PCR.

(Fig. 3C,D; Fig. S2A,B) and Kpnb1 mRNA (Fig. 3E). The possibility In the nucleus, CRABP2 delivers RA to the nuclear receptor RAR, that HuR regulates importin-β1 expression by stabilizing its mRNA thereby promoting its transcriptional activity. Transcriptional was then evaluated. M2−/− cells expressing shCtrl or shHuR were activation assays were carried out to examine whether treated with transcription inhibitor actinomycin D (2.5 µg/ml), and downregulation of HuR interferes with the activation of RAR and the rate of degradation of Kpnb1 mRNA was monitored. The half-life whether importin-β1 can rescue this inhibition. M2−/− cells stably of Kpnb1 mRNA was found to be 16.5±0.75 h and 10.2±1.12 h in expressing shCtrl or shHuR were transfected with empty vector (ev) or shCtrl- and shHuR-expressing cells, respectively (Fig. 3F), indicating vector encoding CRABP2 in the presence or absence of co- that this transcript is indeed stabilized by HuR. The observation that transfection of importin-β1 (Fig. S3A). Cells were also transfected downregulation of HuR did not affect the stability of Gapdh mRNA with a luciferase reporter driven by an RAR response element (RARE) (Fig. S2C) attested to the specificity of the response. Moreover, and a vector encoding β-galactosidase, which served as transfection ribonucleoprotein immunoprecipitation (RIP) assays showed that control. Cells were treated with RA (1 μM, overnight) and luciferase Kpnb1 mRNA co-precipitates with HuR protein (Fig. 3G). The activity measured and normalized to the activity of β-galactosidase observations thus demonstrate that HuR modulates the level of (Fig. 5C; Fig. S3B). Ectopic expression of CRABP2 or importin-β1 importin-β1 by binding and stabilizing its RNA. alone enhanced the activation of RAR, and expression of both further The importin-β1 inhibitor importazole (Soderholm et al., 2011) promoted transactivation in parental cells. The transcriptional activity was used to evaluate whether HuR controls the nuclear import of of RAR was significantly lower in shHuR-expressing cells and was CRABP2 by governing the expression of importin-β1. M2−/− cells partially restored upon ectopic expression of importin-β1and ectopically expressing Flag–CRABP2, were treated with vehicle or CRABP2. The effects of the direct endogenous RAR target gene importazole (30 µM, 1 h) prior to treatment with RA. Similar to the Cyp26a1 on the expression level were then examined. Cells were effect of downregulation of HuR, importazole efficiently blocked treated with RA (4 h) and then Cyp26a1 mRNA was measured by the RA-induced nuclear translocation of CRABP2 (Fig. 4). The quantitative real-time PCR (Q-PCR) (Fig. 5D). Similar to the effects effect of restoring the level of importin-β1 in shHuR-expressing observed in transactivation assays, ectopic expression of CRABP2 or cells was then examined. Ectopic overexpression of importin-β1in importin-β1 alone increased Cyp26a1 expression, and expression of these cells (Fig. 5A) enabled RA-induced nuclear translocation of both proteins further increased Cyp26a1 expression. Cyp26a1 CRABP2. However, completion of import was delayed from 30 min expression was significantly lower in cells with reduced expression in parental cells (Fig. 2A) to 60 min (Fig. 5B). Hence, ectopically of HuR and, in contrast to control cells, ectopic expression of CRABP2 expressed importin-β1 rescued the nuclear import of CRABP2 but alone had no effect on Cyp26a1 mRNA in these cells. Overexpression did not fully restore the kinetics of the process. of importin-β1 partially restored Cyp26a1 expression levels and Journal of Cell Science

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importin-α5 (encoded for by Kpna1) (Fagerlund et al., 2008) and a non-classical NLS recognized by (Liang et al., 2013). Downregulation of HuR decreased the levels of both Kpna1 and Tnpo1 mRNAs (Fig. 7A), and RIP assays showed that both transcripts can be co-precipitated with HuR (Fig. 7B). The half-life of Tnpo1 mRNA decreased from 6.6±0.51 to 4.16±0.26 h in response to downregulation of HuR (Fig. 7C), whereas the half-life of Kpna1 mRNA was 25.67±4.51 and 9.63±1.89 h in parental and shHuR-expressing cells, respectively. Hence, HuR regulates the expression of both genes by stabilizing their transcripts. NFкB subunit p65 translocates to the nucleus after activation by various signals, including the growth factor heregulin (Kannan- Thulasiraman et al., 2010). We showed that heregulin (0.1 µg/ml, 30 min) indeed induced nuclear translocation of p65 (Fig. 7D). Heregulin-induced nuclear translocation of p65 was completely abolished in cells with decreased expression of HuR, and overexpression of importin-α5 partially restored the import (Fig. 7D). In the nucleus, p65 regulates the transcription of various proteins, including the fatty acid-binding protein FABP5 (Kannan- Thulasiraman et al., 2010). The involvement of HuR in regulating the transcriptional activity of p65 was examined by transactivation assays utilizing a luciferase reporter driven by an 800-bp sequence of the Fig. 4. The importin-β inhibitor importazole blocks nuclear import of proximal promoter of FABP5. The data showed that although −/− – CRABP2. M2 cells were transfected with a vector encoding Flag CRABP2. heregulin-β1 activated the reporter in parental cells, decreasing the Cells were pretreated with importazole (30 µM) for 1 h prior to treatment with RA. Flag–CRABP2 (red) was immunostained and visualized using confocal expression of HuR completely abolished the response (Fig. 7E). microscopy. DAPI was used to visualize nuclei. DISCUSSION recovered the effect of CRABP2. Taken together, the data indicate that The ability of HuR to stabilize target transcripts allows this RNA- importin-β1 is necessary for the nuclear import of CRABP2 and for binding protein to control biological processes ranging from cell transcriptional activation by RAR, and support the notion that HuR growth and death to inflammatory responses (Abdelmohsen and regulates these processes by controlling the stability of Kpnb1 mRNA Gorospe, 2010; Ghosh et al., 2009; Hinman and Lou, 2008). and thus the level of expression of importin-β1 protein. However, the complete spectrum of genes whose expression is regulated by HuR and are thus the basis for the broad range of cellular HuR regulates the expression of multiple components of the functions of this protein remains incompletely understood. We show nuclear import machinery here that HuR plays a central role in regulating nuclear protein import. The observation that importin-β1 only partially rescued the nuclear The data reveal that HuR controls the expression of multiple import of CRABP2 suggests that additional factors involved in components of the nuclear import machinery including RANBP1, nuclear import are regulated by HuR. importin-β1/KPNB1, transportin 1 and importin-α5/KPNA1. In Decreasing HuR levels decreased the level of mRNA for Ranbp1 agreement, it was previously reported that HuR binds to mRNA for (Fig. 6A). The half-life of Ranbp1 mRNA was found to be 8.25± both KPNB1 and TNPO1 (Mukherjee et al., 2011). At least in the 1.16 h and 4.88±1.10 h in cells expressing shCtrl and shHuR, cases of KPNA1, KPNB1 and TNPO1, HuR functions in this respectively (Fig. 6B). Furthermore, RIP assays demonstrated that capacity by enhancing the stability of their transcripts. Consequently, Ranbp1 mRNA co-precipitates with HuR protein (Fig. 6C). Hence, downregulation of HuR severely impaired the nuclear import of HuR regulates expression of RanBP1 by binding and stabilizing its CRABP2, a protein that harbors a ‘classical-like’ NLS (Fig. 2), and mRNA. The ability of Ranbp1 to rescue the impaired nuclear import that of NFκB subunit p65, which reaches the nucleus using both of CRABP2 brought about by downregulation of HuR was then classical and non-canonical NLSs (Fig. 7). In both cases, ectopic examined. Ectopic expression of RANBP1 enabled RA-induced supplementation of factors that are lost in the absence of HuR either nuclear translocation of CRABP2, although completion of the partially or almost completely ‘rescued’ nuclear translocation. The process was delayed from 30 min following treatment with RA in control of nuclear import by HuR must have important consequences parental cells (Fig. 2A) to 60 min (Fig. 6D). Interestingly, for nuclear functions beyond the regulation of nuclear translocation of coexpression of RANBP1 together with importin-β1 restored CRABP2, as addressed here. Indeed, decreasing the expression of the time course, allowing CRABP2 to complete its nuclear HuR not only interfered with the ability of CRABP2 to promote the translocation within 30 min of RA treatment (Fig. 6D). transcriptional activity of its cognate nuclear receptor RAR (Fig. 5C, CRABP2 contains a ‘classical-like’ NLS and is mobilized to the D) but also significantly depressed the activity of the receptor in the nucleus by the importin-α1/importin-β1 pathway. However, nuclear absence of CRABP2 (Fig. 5C,D). Because HuR appears to be crucial import of proteins can also be directly mediated by an importin-β for nuclear import mediated by different classes of NLSs, its (Lee et al., 2006; Palmeri and Malim, 1999). For example, the depletion must interfere with the nuclear localization of many importin-β transportin 1 (encoded for by Tnpo1) can directly bind proteins, including those necessary for transcriptional activity in and mobilize to nucleus cargo proteins containing non-canonical general. The role of HuR in regulating the nuclear import of such NLS (Fridell et al., 1997). Moreover, some nuclear proteins contain proteins (e.g. transcription factors, transcriptional co-regulators and both classical and non-canonical NLSs. One such protein is the components of the general transcription machinery) remains to be

NFкB subunit p65, which contains a classical NLS recognized by explored. Journal of Cell Science

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Fig. 5. Importin-β1 partially rescues the nuclear import of holo-CRABP2 in cells with reduced expression of HuR. (A) Immunoblot demonstrating expression of importin-β1inM2−/− cells stably expressing shHuR transfected with an empty vector (ev) or vector encoding importin-β1 (impβ1). (B) Cells were transfected with a vector encoding Flag–CRABP2 and treated with vehicle or RA for the denoted times. Flag–CRABP2 (red) was immunostained and visualized using confocal microscopy. DAPI was used to visualize nuclei. (C) Transactivation assays in denoted cells transfected with an empty vector or vector encoding CRABP2 or importin-β1 or both. Protein levels were assessed by immunoblot (Fig. S3A). Cells were treated with RA and the luciferase activity measured and normalized to β-galactosidase. (D) Expression of the RAR target gene Cyp26a1 in denoted cells transfected with empty vector, vector encoding CRABP2 or importin-β1 or both. Cells were treated with RA for 4 h and CYP26a expression measured by Q-PCR. Graphs show mean±s.e.m. (n=3). *P<0.05, paired Student’s t-test, compared with cells expressing shCtrl and transfected with empty vector.

We previously reported that, in the absence of RA, CRABP2 is MATERIALS AND METHODS not uniformly distributed in the cytosol but, instead, localizes at the Cells − − ER (Majumdar et al., 2011). We show here that this localization is We generated the M2 / cell line from tumors that arose in MMTV-neu/ disrupted in cells with reduced levels of HuR (Fig. 1B), indicating CRABP2-null mice (Schug et al., 2008). MCF7 cells were recently that CRABP2 associates with the ER through its interactions with purchased from ATCC. Cell lines were recently tested for contamination. ’ ’ HuR. Notably, although the nuclear translocation of CRABP2 was Cells were maintained in Dulbecco s modified Eagle s medium (DMEM) containing 4.5 g/l glucose, 4.5 g/l L-glutamine, 10% fetal bovine serum completely blocked in cells with reduced expression of HuR, RA ’ (FBS; Life Technologies), 100 IU/ml penicillin and 100 µg/ml induced a discernible shift in the protein s subcellular localization. streptomycin. Cell transfections were carried out using PolyFect (Qiagen). RA binding not only triggered dissociation of CRABP2 from the ER, reflecting dissociation of the CRABP2–HuR complex (Fig. 1B), but also induced accumulation of the protein at the Reagents nuclear membrane (Fig. 2D). The observation that modulation of Retinoic acid (RA) was purchased from Calbiochem. Actinomycin D was α from Sigma-Aldrich. Antibodies against HuR (sc-5261; 1:1000 WB), HuR expression did not affect the level of importin- 1 (Fig. 3A,B) β-actin (sc-57778; 1:1000 WB), karyopherin-β1 (importin-β1) (sc-1863; raises the possibility that HuR mediates the movement of its cargo 1:1000 WB) and lamin B (sc-6217; 1:300 IF) were from Santa Cruz towards the nucleus, even in the absence of a partner importin-β. Biotechnology. Antibody against KPNA2 (importin-α1) (AB6036; 1:1000 How CRABP2 travels to the nuclear surface under conditions that WB) was from Abcam. Antibody against CRABP2 (1:1000 WB) was a gift

do not allow it to translocate into the nucleus remains to be clarified. from Cecile Rochette-Egly (IGBMC, Strasbourg, France). Journal of Cell Science

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Fig. 6. Stabilization of Ranbp1 mRNA by HuR contributes to its ability to control the nuclear import of CRABP2. (A) Levels of Ranbp1 mRNA in M2−/− cells stably expressing shCtrl or shHuR, as measured by Q-PCR and normalized to 18s. Levels of mRNAs in shHuR- expressing cells normalized to shCtrl-expressing cells are shown (mean±s.d.; n=3). *P<0.05, paired Student’s t-test. (B) Cells were treated with actinomycin D (2.5 µg/ml) and then levels of Ranbp1 mRNA at various time points were measured by Q-PCR. Data (mean±s.e.m.; n=3) were normalized to corresponding values at time zero. (C) HuR was immunoprecipitated from M2−/− cells. Ranbp1 mRNA that co-precipitated with HuR was detected by semi- quantitative PCR. (D) shHuR- expressing M2−/− cells were transfected with a vector encoding Flag–CRABP2 in concert with a vector encoding RanBP1 or RanBP1 and importin-β1. Cells were treated with RA and Flag–CRABP2 (red), immunostained and visualized using confocal fluorescence microscopy. DAPI was used to visualize nuclei.

Lentiviral shRNA production harboring a luciferase reporter driven by a RAR response element (RARE- pLKO.1 vectors harboring shRNAs for HuR (Elavl1, TRCN0000112085; Luc) and co-transfected with an expression vector for β-galactosidase, Elavl1 TRCN0000112087; Elavl1 TRCN0000112088; Mouse) (ELAVL1 serving as transfection efficiency control. Cells were also co-transfected TRCN0000017275; Human) were from Open Biosystems; pLKO.1 vector with pSG5 vector encoding CRABP2 or pCMV-Sport6 encoding harboring luciferase shRNA (SHC007) or non-targeting shRNA (SHC002) importin-β1. At 24 h post-transfection, cells were treated with 1 μMRA was from Sigma-Aldrich. Using pCMV packaging vector and pMD2.G or vehicle overnight. Luciferase activity was measured and corrected for envelope vector, lentiviruses were produced in HEK293T cells. Target cells transfection efficiency by the activity of β-galactosidase, as previously were infected using standard protocols and selected with 5 µg/ml and 1 µg/ml described (Budhu et al., 2001). puromycin for M2−/− cells and MCF-7 cells, respectively. Confocal fluorescence microscopy Real-time quantitative PCR Cells, cultured in DMEM containing 10% charcoal-treated FBS, were Q-PCR was performed using a StepOnePlus real-time PCR system with transfected with pCMV-3Tag-1 vector encoding Flag–CRABP2. Cells were the following TaqMan probes: Elavl1, Mm00516012_m1; Kpna1, treated with RA or heregulin-β1 as described in the text, fixed with 4% Mm00434700_m1; Kpnb1, Mm00434318_m1; Tnpo1, Mm00839059_g1; paraformaldehyde in phosphate-buffered saline (PBS), and permeabilized Tnpo2, Mm00520392_m1; Ranbp1, Mm00650862_m1; Cyp26a1, with 0.2% Triton X-100. Endogenous CRABP2 in MCF-7 cells and Flag- Mm00514486_m1; and 18s rRNA (4352930E, Applied Biosystems). tagged CRABP2 in M2−/− cells were visualized by immunostaining. Levels of mRNAs were normalized to 18s rRNA using the threshold cycle Antibodies: CRABP2 (Santa Cruz sc-10065; 1:300 IF), Flag (Sigma- (ΔΔCT) method (Applied Biosystems technical bulletin no. 2). Aldrich F1804; 1:250 IF), calnexin (Sigma-Aldrich C4731; 1:300 IF), Alexa Fluor®-conjugated secondary antibodies (Invitrogen A11008, Immunoblotting A11059, A11005; 1:1000 IF). Myc-DDK-pCMV-Entry vector encoding Cells were lysed in RIPA buffer containing 150 mM NaCl, 10 mM Tris pH mKPNA1 and Myc-His A-pCDNA3.1(+) vector encoding mRanBP1 were 7.2, 0.1% SDS, 1% Triton X-100, 1% deoxycholate and Halt protease used. Cells were imaged using a PerkinElmer UltraVox spinning disk inhibitor cocktail (Thermo Scientific). Protein concentration was determined confocal microscope. Analysis of images was carried out using Fiji ImageJ using the Bradford protein assay. Cell lysates (50 µg protein) were resolved and Office PowerPoint. by SDS-PAGE and immunoblotted using appropriate antibodies. Ribonucleoprotein immunoprecipitation Transactivation assays Ribonucleoprotein immunoprecipitations were performed as previously Some 5×104 cells were plated in six-well plates in DMEM supplemented described (Keene and Tenenbaum, 2002; Vreeland et al., 2014b). Semi- with 10% charcoal-treated FBS. Cells were transfected with vectors quantitative PCR was performed using the following primers: Kpnb1:forward Journal of Cell Science

4031 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4025-4033 doi:10.1242/jcs.192096

Fig. 7. Stabilization of Tnpo1 and Kpna1 mRNAs by HuR contributes to its ability to control the nuclear import of p65. (A) Levels of mRNA for Tnpo1/ importin-β1, Tnpo2 and Kpna1/importin-α5inM2−/− cells stably expressing shCtrl or shHuR, as measured by Q-PCR and normalized to 18s. Levels of mRNAs in shHuR-expressing cells normalized to cells expressing shCtrl are shown (mean±s.d.; n=3). *P<0.05, paired Student’s t-test, compared to cells expressing shCtrl. (B) HuR was immunoprecipitated from M2−/− cells. Tnpo1 and Kpna1 mRNAs that co-precipitated with HuR were detected by semi-quantitative PCR. (C) Cells were treated with actinomycin D (2.5 µg/ml) and levels of Tnpo1 (left) and Kpna1 (right) mRNAs at various time points were measured by Q-PCR. Data were normalized to corresponding values at time zero (mean±s.e.m.; n=3). (D) M2−/− cells stably expressing shCtrl or shHuR and shHuR-expressing cells transfected with a vector encoding importin-α5 were treated with vehicle or heregulin-β1 (0.1 µg/ml) for 30 min. p65 was immunostained and visualized. DAPI was used to visualize nuclei. (E) Transactivation assays in denoted cells transfected with a vector harboring a luciferase reporter driven by 800 bp of the proximal promoter of FABP5. Cells were serum starved and treated with vehicle or heregulin-β1 (HRG; 50 ng/ml, overnight). Luciferase activity was measured and normalized to β-galactosidase (means±s.d.; n=3). *P<0.05, paired Student’s t-test, compared to vehicle-treated cells.

TGGCAAACCCAGGAAACAGT, reverse GCCCAACGTCTGCAAAACA Funding T; Kpna1: forward CGAGCGTCTCCCTCTTCGTA, reverse CCTGGT- This work was supported by National Institutes of Health [grant numbers DK060684 GCCATCTCCATGTT; Ranbp1: forward GCAGATGAGTCCAACCAC- and CA166955 to N.N.]. Deposited in PMC for release after 12 months. GA, reverse GCTTGACATCTCCAGTGCCT; Tnpo1: forward GCTCGT- Supplementary information CCCTTACCTTGCTT, reverse TGTGGCAACAGACGAGAGAC. Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.192096.supplemental Statistical analysis Statistical significance of difference was analyzed by a paired Student’s t References -test. Analyses were performed using SPSS 16.0 software. Abdelmohsen, K. and Gorospe, M. (2010). Posttranscriptional regulation of cancer traits by HuR. Wiley Interdiscip. Rev. RNA 1, 214-229. Acknowledgements Bischoff, F. R. and Ponstingl, H. (1991). Catalysis of guanine nucleotide exchange We thank Cecile Rochette-Egly (IGBMC, Strasbourg) for providing CRABP2 on Ran by the mitotic regulator RCC1. Nature 354, 80-82. antibodies. The work utilized a confocal microscope that was acquired with National Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A. and Ponstingl, H. Institutes of Health shared instrument grant (SIG) [grant number 1S10OD019972-01]. (1994). RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc. Natl. Acad. Sci. USA 91, 2587-2591. Bischoff, F. R., Krebber, H., Smirnova, E., Dong, W. and Ponstingl, H. (1995). Competing interests Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP The authors declare no competing or financial interests. binding protein RanBP1. EMBO J. 14, 705-715. Brennan, C. M. and Steitz, J. A. (2001). HuR and mRNA stability. Cell. Mol. Life Sci. Author contributions 58, 266-277. W.Z. designed and carried out experiments and contributed to the writing of the Budhu, A. S. and Noy, N. (2002). Direct channeling of retinoic acid between cellular manuscript, A.C.V. designed and carried out experiments and contributed to the retinoic acid-binding protein II and retinoic acid receptor sensitizes mammary writing, N.N. conceived and supervised the project, designed experiments and wrote carcinoma cells to retinoic acid-induced growth arrest. Mol. Cell. Biol. 22, the manuscript. 2632-2641. Journal of Cell Science

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