MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles Line S

Research Article

MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles Line S. Reinert,1 Bo Shi,3 Suvobroto Nandi,1 Krystyna Mazan-Mamczarz,2 Michele Vitolo,1 Kurtis E. Bachman,1 Huili He,1 and Ronald B. Gartenhaus1

1University of Maryland, Marlene and Stewart Greenebaum Cancer Center; 2Laboratory of Cellular and Molecular Biology, National Institute on Aging, Intramural Research Program, NIH, Baltimore, Maryland; and 3Division of Rheumatology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois

Abstract In the past several years, it has become increasingly evident MCT-1 is an oncogene that was initially identified in a human that mRNA translation is also an important control point, and T cell lymphoma and has been shown to induce cell this is particularly the case in embryonic development and proliferation as well as activate survival-related pathways. during the regulation of cell growth and differentiation (11, 12). MCT-1 contains the PUA domain, a recently described RNA- The efficiency of expression of key proteins involved in cell binding domain that is found in several tRNA and rRNA growth regulation, proliferation, or cell death may be controlled modification enzymes. Here, we established that MCT-1 at the translational level by changes in the activity of protein interacts with the cap complex through its PUA components of the protein synthesis machinery. Experimentally, domain and recruits the density-regulated protein (DENR/ aberrant expression of translation factors has been shown to induce the malignant transformation of cells. The eIF4E protein DRP), containing the SUI1 translation initiation domain. binds to the cap structure m7GpppN (where N is any Through the use of microarray analysis on polysome- ¶ associated mRNAs, we showed that up-regulation of MCT-1 nucleotide), which is found at the 5 -terminus of all cellular was able to modulate the translation profiles of BCL2L2, eukaryotic mRNAs (except those in organelles) (13), and over- expression of eIF4E has been shown to result in the TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite in vitro in vivo equivalent levels of mRNAs in the cytoplasm. Our data transformation of cells both and (14, 15). establish a role for MCT-1 in translational regulation, and Typically, mRNAs coding for proteins positively involved in support a linkage between translational control and onco- growth regulation are poorly translated in resting cells and it is genesis. (Cancer Res 2006; 66(18): 8994-9001) the translation of those mRNAs that is specifically induced after growth stimulation of cells (16, 17). Here, we present the novel effects of MCT1 interacting with the density-regulated protein Introduction (DENR/DRP), which contains a SUI1 domain found in the The oncogene MCT-1 is amplified in certain human T cell translation initiation factor, eIF1 (18, 19). We showed that MCT1 lymphomas and has been mapped to chromosome Xq22-24 (1). is a cap-binding protein recruiting DENR and subsequently This region is amplified in a subset of primary B cell non– altering the mRNA translational profile. The data presented Hodgkin lymphomas, suggesting that increased copy number of a supports a role for the MCT1 oncogene in translation and gene(s) located in this region confers a growth advantage to suggests a mechanism for its role in transformation. certain primary human lymphomas (2–4). Lymphoma cell lines overexpressing MCT-1 exhibited increased growth rates and Materials and Methods displayed increased protection against serum starvation–induced Plasmid constructions and cell culture transfection. The cDNA of apoptosis when compared with matched controls (1, 5, 6). full-length MCT-1 (543 nucleotides) was amplified from total RNA isolated Recently, we showed that MCT-1 impairs cell cycle checkpoint from normal lymphocytes and cloned into pcDNA3.1/V5-Histidine tag control after exposure to DNA-damaging agents and transforms vector (Invitrogen, Carlsbad, CA). This was represented as V5-MCT human mammary epithelial cells (7). Furthermore, MCT-1 may plasmids in the text. Similar cloning was also done for MCT-1 deletion also play a role in the pathogenesis of human breast cancer by constructs: DPUA (1-351 nucleotides) and PUA (245-543 nucleotides). Full- inhibiting apoptosis and promoting angiogenesis (8). The length MCT-1 was subcloned into a retroviral vector pLXSN (Clontech, molecular mechanism(s) underlying the broad activity of MCT-1 Mountain View, CA) and was used for the stable transfection of Raji and Farage B cell non–Hodgkin lymphoma cell lines. MCT-1 and its deletion is currently unknown. MCT1 contains a PUA domain, a recently constructs in pcDNA3.1 were used for in vitro translations and for transient described RNA-binding domain that is found in several tRNA and transfection of 293HEK cell lines (human embryonic kidney cell lines), rRNA modification enzymes (9, 10), suggesting that MCT1 might NIH3T3, Raji and Farage cells (American Type Culture Collection, have an RNA-binding function. Manassas, VA) with LipofectAMINE 2000 (Invitrogen) or Nucleofector Kit V (Amaxa, Inc., Ko¨ln, Germany). MCT-1 and deletion constructs were also cloned into pGEX-5X-1 vector (Invitrogen) and transformed into Escherichia coli BL21 (DE3) strains (Invitrogen) to obtain glutathione S-transferase (GST)–tagged fusion proteins. The open reading frame from Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). the human DENR sequence (GenBank accession no. AF038554) was Requests for reprints: Ronald B. Gartenhaus, University of Maryland, Marlene amplified and cloned into pCMV-myc (Clontech) and pLXSN vector and Stewart Greenebaum Cancer Center, 9-011 BRB, 655 West Baltimore Street, (Clontech) which was transfected in Raji cell lines by using Nucleofector Baltimore, MD 21201. Phone: 410-328-3691; Fax: 410-328-6559; E-mail: rgartenhaus@ (Amaxa). DENR was also cloned into the pET28c His-tagged expression som.umaryland.edu. E. I2006 American Association for Cancer Research. vector (Novagen, Madison, WI) to purify His-tagged DENR proteins using doi:10.1158/0008-5472.CAN-06-1999 coli BL21 (DE3) strain (Invitrogen).

Cancer Res 2006; 66: (18). September 15, 2006 8994 www.aacrjournals.org

Screening of human HeLa cell cDNA library by the yeast two-hybrid Depletion of eIF4E from reticulocyte lysate. The V5-control, V5- system. A yeast two-hybrid screen was done using the MATCHMAKER MCT-1, V5-DPUA, and V5-PUAs plasmids were transcribed and translated system III (Clontech) and was used according to the manufacturer’s according to the manufacturer in a TNT Quick coupled transcription/ instructions. The bait construct pGBK-T7-HA-MCT-1 encoding the full- translation system (Promega). Half of this reaction (50 AL) was used for length MCT-1 protein (181 amino acids) fused in frame to the GAL4 DNA- overnight depletion of eIF4E by using 4 Ag of eIF4E antibody (Santa Cruz binding domain, was constructed by inserting the PCR-generated fragment Biotechnology), 20 AL of protein A/G agarose beads and protease into the SalIand the BamHIsites of pGBKT7 vector. The human HeLa cell inhibitor. Prior to depletion, 4 AL of the extract, 7 AL of the supernatant cDNA library, which was fused with the GAL4 activation domain in the which was depleted for eIF4E, and 10% of the eluate from the beads, i.e., pACT2 vector, was obtained from Clontech. The MATa strain H109, which the depleted eIF4E, was loaded on a SDS gel followed by Western blot was stably transformed with bait construct MCT-1, was mixed with yeast with EIF4E antibody to control the depletion level. The cap-binding assay strain Y187 that was pretransfected by human HeLa cell cDNA library was done as described above but also with an equal amount of the (Clontech). The binding specificities of the isolated prey clones were extract after depletion of eIF4E. checked after cotransformations by growth on high-stringency selective Polysome isolation. 293HEK cells were transiently transfected with yeast media. Plasmid DNA from prey yeast clones was extracted using yeast vector, V5-MCT-1, or pCMV-c-Myc-DENR plasmids using Lipofectamine plasmid isolation kit (Clontech) and sequenced. (Invitrogen) according to the manufacturer’s protocol. Thirty-six hours MCT-1 and DENRcoimmunoprecipitation. Raji cell lines were later, they were collected and washed with PBS containing 100 Ag/mL of transiently transfected with the following plasmids: pcDNA empty vector, cycloheximide. The cells were lysed in a buffer containing 0.3 mol/L of KCl, V5-MCT-1, V5-DPUA, V5-PUA, and pCMV-myc-DENR. Cell pellets were lysed 5 mmol/L of MgCl2, 10 mmol/L of Tris (pH 7.4), 0.5% NP-40, 5 mmol/L of and reciprocal coimmunoprecipitation experiments with either c-Myc DTT, proteinase K inhibitors, RNase inhibitors, and 1 mg of heparin per monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or mL at 4jC. Centrifugation was done to pellet nuclei and unlysed cells. The anti-V5 monoclonal antibody (Invitrogen) were done using protein A beads. supernatant was centrifuged through a 10% to 50% sucrose gradient, as Eluted and denatured samples were loaded on 15% acrylamide SDS-PAGE. described in ref. (23). Equal volumes of the fractions were loaded on a 15% Western blot was carried out using standard methods. Coimmunoprecipita- SDS gel and blotted with antibody against eIF2a, eIF4E, rS6p, h-actin, V5 tion with endogenous MCT-1 and transfected pCMV-myc-DENR in 293HEKs for detecting MCT-1, and c-Myc for detecting DENR (Santa Cruz was done similarly, but used MCT-1 antibody (Sigma, Research Genetics, Inc., Biotechnology). Similar experiments were carried out on Farage and St. Louis, MO) and c-Myc monoclonal antibody (Santa Cruz Biotechnology). Daudi B cell lines that were stably transfected with vector or V5-MCT-1 GST pull-down assay. Ten micrograms of the E. coli expressed and plasmid. purified His-DENR, GST, GST-MCT-1, GST-DPUA, and GST-PUA were used Microarray and data analysis. 293HEK cells were transfected with for GST pull-down assays as previously described (20). The glutathione- V5-MCT and V5-vector plasmids, and the polysomes were separated as agarose beads were washed four times and eluted in 2Â SDS buffer. The described above. Polysome-associated RNA from vector and MCT-1- eluted proteins were loaded on 15% acrylamide SDS-PAGE and immuno- transfected cells were purified by RNeasy mini kit (Qiagen, Valencia, CA) blotted with GST or His antibody. and pooled separately. Five micrograms of polysome-associated RNA were Immunostaining. 3T3 cells were transiently transfected with myc-DENR used to generate cDNA and thereafter generate cRNA probes labeled with and V5-MCT-1 or mock-transfected as controls in two-chambered slides Biotin-16-UTP (Roche Molecular Biochemicals, Basel, Switzerland) using (Nunc, Roskilde, Denmark). Forty-eight hours after transfection, the cells the AmpoLabeling-LPR kit (SuperArray, Frederick, MD). The cRNA probe were washed in PBS, fixed with a 1:1 mixture of ice-cold methanol and was hybridized to GEArray-human apoptosis and cell cycle gene arrays acetone, and blocked with 3% bovine serum albumin for 2 hours. The cells (SuperArray) and the hybridization signal was detected with a chemi- were then washed thrice with washing buffer (PBS containing 0.2% Tween luminescence detection kit (SuperArray). The relative mRNA level of each 20) and incubated overnight with 1:200 mouse monoclonal anti-V5 gene was analyzed using GEArray Analyzer software. From the quantified antibody and 1:200 rabbit polyclonal anti-myc antibody at room signals, we calculated the log2 ratio and then normalized it with a temperature. The cells were then washed thrice with washing buffer and quantile normalization method (24). After the normalization, we cal- incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa culated the mean log-fold change in MCT-1 samples compared with the Fluor 568 at a concentration of 1:200 each. The cells were then washed vector control samples. The fold changes were calculated by 2(log2 ratio). again, as described, and mounted using Vectashield mounting medium The assays were independently carried out in triplicate. (Vector Labs, Burlingame, CA) and coverslips were placed on them. Real-time RT-PCR analysis. Total RNA was isolated from V5-MCT-1 Photomicrographs were taken using a Leica DM4000B immunofluorescence and V5 control vector expressing 293HEK cells and using the RNeasy microscope (64Â magnification) with Improvision software. system (Qiagen). Single-stranded cDNA was generated using first-strand In vitro translation of luciferase. pCMV-luciferase was translated cDNA synthesis kit (Amersham) following the manufacturer’s directions. in vitro as previously described (21), with minor modifications. The assay Control template reactions were prepared in parallel without the addition was done in the presence of luciferase 7mGpppG RNA and [35S]methionine of reverse transcriptase. Real-time RT-PCR was then done as previously and 0, 0.1, or 1 Ag of purified GST-tagged MCT-1 proteins at 30jC for 30 described (25). Each reaction was carried out in triplicate; in addition, minutes. The translated proteins were separated on a 15% SDS gel and three independent reactions were run simultaneously. Glyceraldehyde-3- autoradiographed. phosphate dehydrogenase (GAPDH) cDNA served as the internal control Cap-binding assay. The cap-binding assay was done as previously template. The change in mRNA level was calculated by: DDcycle threshold described (22), with minor modifications. Nontagged and linearized (Ct) = (DCt MCT-1 expressing sample) À (DCt vector expressing sample), MCT-1 plasmid was in vitro–translated using [35S]methionine, 20 ALof where DCt = (Ct for the target gene) À (Ct for GAPDH gene). ANOVAs this rabbit reticulocyte lysate (Promega, Madison, WI) with 300 ALof (P values) were calculated by using a null hypothesis H_0 that all the three buffer D [50 mmol/L HEPES (pH 7.4), 40 mmol/L NaCl, 2 mmol/L EDTA, experiments had the same mean as reviewed in ref. (26). We let Â_1, Â_2, 0.1% Triton X-100] and 30 AL of a 50% slurry of 7-methyl-GTP-sepharose and Â_3 denote the means of the three independent DDCt, respectively. (Amersham, Buckingham, United Kingdom) and incubated for 1 hour at If P values were >0.05, we could not reject the null hypothesis H_0 4jC. After washing the resin four times with buffer D, bound proteins (i.e., all three DDCt means are equal). were eluted with 2Â SDS sample buffer, resolved by 15% SDS-PAGE, and Western blots. We did Western blots to quantify the protein levels autoradiographed. Competition reaction was carried out as described of several mRNAs that were actively recruited to the polysomes. The above but with the indicated amount of Cap analogue (m7GpppG; protein levels of MCT-1, Mre11, cyclin D1, E2F1, Bcl-w, Dp-1, and GAPDH Ambion) or GTP (Ambion). The cap-binding assay was also done in the were examined from either stably transfected Farage or Daudi cell lines, presence of 1 Ag of purified His-DENR. The eluted His-DENR protein was or transiently transfected 293HEK whole cell lysates that were obtained visualized by performing Western blot with anti-His antibody. at three different time points: 24, 36, and 48 hours after transfection www.aacrjournals.org 8995 Cancer Res 2006; 66: (18). September 15, 2006

(n = 3). Western blot for total proteins from Jurkat cell lines that expressed and all of them were found to encode for the same protein known pSIREN-RetrpQ vector containing random (NT) small interfering RNA as density-regulated protein (DENR/DRP) or smooth muscle cell– (siRNA) scrambled oligo or siRNA against MCT-1 (NT 503-523; ref. 7) were associated protein 3 (SMAP-3) (27). The search for a conserved transfected by using Nucleofector (Amaxa). Controls were nontransfected domain database revealed that DENR contained a SUI1 domain Jurkat cells. The cells were harvested 72 hours posttransfection and 40 Agof similar to the archetypal translation initiation factor eIF1 (Fig. 1A). total cell extract was loaded on 15% SDS gel followed by Western blot. Quantitative analysis was carried out using Alpha Innotech quantitation The SUI1 domain in eIF1 has a secondary structure fold cor- software. responding to that of a number of ribosomal proteins and RNA- binding domains (28). In order to confirm the yeast two-hybrid data, we examined the in vivo interaction of these proteins using Results stably transfected lymphoid cell lines. The two proteins were

MCT-1 physically interacts with DENR. The NH2 terminus of shown to physically interact in reciprocal coimmunoprecipitation MCT-1 contains a domain with significant homology to the assays in vivo in transfected Raji cells (Fig. 1B) and in 293HEK protein-protein binding domain found in cyclin H (1, 13). To when only DENR was overexpressed (Fig. 1C). Using a GST pull- identify which proteins physically interact with MCT-1, we used a down assay, we attained similar results (Fig. 1D). Subcellular yeast two-hybrid approach. A total of 16 positive clones were found localization of MCT-1 and DENR proteins was examined using (data not shown), the plasmids from these clones were sequenced immunohistochemistry. Both MCT-1 and DENR were shown to

Figure 1. MCT-1 interacts with the SUI1 domain containing DENR protein. A, DENR was pulled down in a yeast two-hybrid screen by using MCT-1 as a bait protein. The PUA domain of MCT-1 is a predicted RNA-binding domain. DENR contains the SUI1 domain, which is found in the translation factor eIF1. B, MCT-1 coimmunoprecipitates with DENR in transiently transfected Raji cell lines. Lanes 1, 2, 7 and 8, pcDNA vector; lanes 3 and 4, pCMV-Myc-DENR; lanes 9 and 10, pcDNA-MCT-1-V5; lanes 5, 6, 11, and 12, pcDNA-MCT-1-V5 and pCMV-Myc-DENR cotransfected. Immunoprecipitation was carried out with both c-Myc monoclonal antibody and anti-V5 monoclonal antibody and Western blot was carried out using the indicated antibodies. C, coimmunoprecipitation assay in 293HEK cells with transfected DENR and endogenous MCT-1. D, GST pull-down assay. GST and GST-MCT-1 proteins together with His-DENR or His-DENR alone are incubated with glutathione Sepharose beads. Western blot of immobilized GST and GST-MCT-1 (bottom); the interacting His-DENR (top). E, MCT-1 and DENR both colocalized in the cytoplasm whereas 4¶,6-diamidino-2-phenylindole (Dapi) staining identified the nucleus.

Cancer Res 2006; 66: (18). September 15, 2006 8996 www.aacrjournals.org

Figure 2. MCT-1 binds specifically to the cap and requires the PUA domain. A, a schematic presentation of MCT-1 and deletion constructs. The full-length MCT-1 (543 nucleotides) is 181 amino acids long, DPUA (1-351 nucleotides) is 117 amino acids long, and PUA (245-543 nucleotides) is 100 amino acids long. B, [35S]methionine-labeled full-length and deletion MCT-1 proteins were in vitro translated using rabbit reticulocyte lysate and incubated with 7-methyl-GTP-sepharose and the bound proteins were eluted and resolved by 15% SDS-PAGE and autoradiographed. Right, 5% of the input MCT-1 proteins. The same cap-binding assay as above was carried out in the presence of the indicated amount of (m7GpppG) cap analogue competitor (C) or GTP competitor (D). E, the cap-binding assay was also done in the presence of purified His-DENR. The eluted His-DENR protein was visualized by performing Western blot with anti-His antibody. colocalize predominantly in the cytoplasm of 3T3 cells (Fig. 1E). 3¶-untranslated region RNA fragments (Supplementary Fig. S1). Very little background staining was found in the merge slide of To identify if DENR binds directly to the cap complex, MCT-1 and mock-transfected control 3T3 cells (data not shown). DPUA proteins were incubated separately with m7GTP-sepharose The PUA domain of MCT-1 is required for Cap complex– beads in the presence of His-DENR protein. DENR only came binding. The above data pointed to MCT-1 and DENR forming a down with the cap in the presence of full-length MCT-1. The cap functional complex involved in translation initiation. The PUA complex incubated with the DPUA deletion mutant was not able domain present in MCT-1 at the COOH-terminal end is a to bring down the DENR protein (Fig. 2E). predicted RNA-binding domain (Fig. 2A). Here, we show that MCT-1 interacts with the translation machinery. Next, we MCT-1 is able to bind a cap complex, which is present at the asked if MCT-1 binding to the cap complex requires eIF4E. In order 5¶-ends of mRNA. Equal amounts of in vitro–translated MCT-1 to address this question, we expressed MCT-1 in reticulocyte lysates proteins were incubated with m7GTP-sepharose beads and the and then depleted eIF4E (Fig. 3A). Reticulocyte lysates containing bound proteins were eluted, run on SDS gel and autoradio- equal amounts of MCT-1 proteins, before and after the depletion of graphed (Fig. 2B). Full-length MCT-1 and the PUA domain both eIF4E were then used for the cap binding assay (Fig. 3B). Both full- exhibited strong binding to the m7GTP-sepharose, whereas the length MCT-1 and PUA-containing mutant lose their cap-binding binding of DPUA was very modest. This indicated that the PUA function in eIF4E-depleted extracts, consistent with eIF4E being domain of MCT-1 was required for the interaction with m7GTP- required for their interaction with the cap complex. sepharose. To confirm the specificity of the cap binding by Because MCT-1 interacts with the cap complex, we asked MCT-1, a competition assay with sepharose beads alone or whether the MCT-1/DENR complex interacts with the translation together with a soluble cap analogue was carried out and the machinery by analyzing polysomal fractions isolated through a binding was successfully competed out in a dose-dependent sucrose gradient. This separates the unbound mRNA from manner (Fig. 2C). The free GTP does not compete the cap translationally active mRNAs bound to polysomes of increasing complex-MCT-1 binding, consistent with MCT-1 binding specifi- size. Low-passage 293HEK cells (human embryonic kidney cell cally to GTP in the m7GTP-sepharose beads (Fig. 2D). Further- lines) that readily achieve highly efficient transient transfections more, MCT-1 does not bind to other RNA structures including (>80% transfection rate) were used for this assay. Cytosolic extracts uncapped 5¶-untranslated region, open reading frame, or from transiently transfected 293HEK cells expressing empty vector www.aacrjournals.org 8997 Cancer Res 2006; 66: (18). September 15, 2006

Figure 3. eIF4E is required for MCT-1 interaction with the cap complex. The eIF4E was immunodepleted from the reticulocyte lysate containing the MCT-1 and MCT-1 deletion proteins. A, Western blot with eIF4E antibody of the lysate before and after eIF4E depletion as well as the depleted eIF4E. B, the cap binding assay was done as in Fig. 2B, but also with the eIF4E-depleted extract (left). Right, 5% of the input MCT-1 and deletion proteins. C, MCT-1 and DENR sediment with the translation initiation complex. Transiently transfected 293HEK cells were used for polysome preparation as described. Fractions 1 to 3 are typically completely devoid of ribosome components and contain multifactor complex. Representative polysome distribution profile (top). Representative Western blot analysis with antibodies against eIF2a, eIF4E, Myc-DENR, V5-MCT-1, h-actin, and rS6p (bottom). or V5-MCT-1 and pCMV-Myc-DENR were applied to a linear 10% full-length MCT-1 enhanced translation in vitro and that the PUA to 50% sucrose gradient. The ribosomal units were monitored at an RNA-binding domain was critical for cap binding, but not absorbance of 260 nm and the profile is shown in Fig. 3C (top). sufficient to function as a translation enhancer. This indicated In order to examine if MCT-1 and DENR interact with ribosomes, that MCT-1 required both PUA to bind to the cap RNA, and DPUA we did Western blot analyses of the polysome fractions from cells to possibly interact with other proteins to enhance translation. expressing both V5-MCT-1 and Myc-DENR. The eIF4E and eIF2 MCT-1 was previously shown to play a role in cell cycle components of the translation initiation complex are found in the progression, apoptosis, and DNA damage response (5–8, 34). In RNP sedimentation fraction and in 40S fractions as previously order to identify if the increased translation of genes participating reported (29–31). Our data showed that MCT-1 sediments in the in these processes was caused by the up-regulation of MCT-1, we same fractions as eIF4E and the eIF2a under our assay conditions isolated polysomal RNA fractions as previously described from (Fig. 3C, bottom). The DENR protein was detected in similar 293HEK cells transiently transfected with V5-MCT-1 or vector fractions as both MCT-1 and eIF2a. Both h-actin and a component control plasmids. The mRNAs that were actively recruited to the of ribosomal subunit, rpS6, were fractionated with monosomes and polysomes were analyzed on a targeted microarray containing 267 polysomes as expected (refs. 32, 33; Fig. 3C). We obtained nearly genes involved in apoptosis and cell cycle (SuperArray). For the identical profiles and Western blots employing Farage and Daudi B quantified signals from three independent experiments, we cell lines (data not shown), that were stably transfected with either calculated the log2 ratio, and then normalized it with a quantile MCT-1 or empty vector plasmids. normalization method to obtain the same background intensity MCT-1 is involved in translational regulation. Having (24). After the normalization, we calculated the mean fold change established that MCT-1 sediments predominantly with the RNP V5-MCT-1 polysomal samples compared with the vector polysomal and 40S sedimentation fraction, we asked whether MCT-1 was able samples. We identified 67 genes (25%) that were z2-fold increased to enhance translation. We showed that MCT-1 enhances the in V5-MCT-1 polysomal fractions compared with the vector translation of the luciferase reporter gene when GST-MCT-1 polysomal fractions (Fig. 4B; Supplementary Table 1). protein was added in excess to the rabbit reticulocyte lysate In order to verify that protein levels were increased in accord translation system in a dose-dependent manner (Fig. 4A). This with the enhanced recruitment to the polysome of mRNAs, we function was lost, when GST alone, GST-DPUA, or only GST-PUA carried out Western blot analyses of selected proteins. To confirm protein was substituted for GST-MCT-1. This established that the that the Western blot data actually reflected an increase in the

Cancer Res 2006; 66: (18). September 15, 2006 8998 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. The MCT-1 Oncogene Modulates Translation translation of these genes and was not due to differences in the kinase activity, elevated levels of MCT-1 protein in primary diffuse transcription level, we did real-time PCR on the total mRNA from large B cell lymphoma, and overriding cell cycle checkpoints. control vector and V5-MCT expressing 293HEK cells. The real-time The molecular mechanism(s) underlying the broad activity of PCR was done in triplicate for each sample from the three MCT-1 is currently unknown. Using a yeast two-hybrid approach, independent experiments (Fig. 5A). The differences in cycle MCT-1 was shown to interact with the density-regulated protein threshold (Ct) from the V5 vector and the V5-MCT-1 samples, (DENR/DRP), a protein containing the SUI1 domain. This com- which were normalized to GAPDH, were compared. The total plex was shown to interact with the cap complex and to alter mRNA levels were virtually identical in all target genes examined the mRNA translational profile of human embryonic kidney cell despite the differences in protein levels consistent with differences lines. in translation. The MCT-1 mRNA levels were f5-fold increased in The SUI1 domain present in DENR is homologous to the the V5-MCT-1 sample compared with vector (Fig. 5A). prototypical one found in eIF1, containing a secondary structure Total cell lysates either from stably transfected Farage B cell fold corresponding to that of a number of ribosomal proteins lymphoma cells expressing empty vector or PLXSN-V5-MCT-1 or and RNA-binding domains (28). The function of SUI1 in eIF1, is from 293HEK cells were isolated at 24, 36, and 48 hours post- to scan along the mRNA to locate the initiation codon and to transfection with either MCT-1 or empty vector control plasmids. discriminate between cognate and noncognate initiation codons Western blotting was carried out using antibodies to Mre11, Bcl-w, (35). MCT-1 contains a PUA domain, which is a recently cyclin D1, E2F1, Dp-1, GAPDH, and MCT-1 (Fig. 5B and C). After described RNA binding domain and is found in several RNA standardizing to control GAPDH protein levels, there was a modification enzymes. Previous studies with proteins containing consistent increase observed in the protein levels of cyclin D1 a PUA domain suggested that they are involved in tRNA and protein by 2.8-fold, Mre11 protein levels by 2-fold, Bcl-w protein rRNA modifications as exemplified by the yeast pseudouridine by 3-fold, the transcription factors E2F1 by 4-fold, and Dp-1 by synthases (9, 36). We have provided experimental evidence that 2-fold in the MCT-1 transiently transfected cells compared with MCT-1 is a cap complex–binding protein interacting with vector controls. We also showed that MCT-1 increased Mre11 m7GTP through the PUA domain and that this interaction protein levels by 5-fold and Dp-1 by 2-fold in Farage cells (Fig. 5C). requires the presence of eIF4E. In light of our data demonstrat- These differences were most apparent at 48 hours posttransfection ingthatbothMCT-1andDENRsedimentintheRNP in 293HEK cells (Fig. 5B) and were reproducible in at least three sedimentation fraction and in 40S fractions, as was the case independent experiments. To further support the effect of elevated for eIF2, it is plausible to hypothesize that MCT-1 by binding to MCT-1 protein levels on translation, we employed a knock-down the cap structure brings the SUI1 domain containing DENR in approach in Jurkat lymphoma cells that exhibit high levels of close proximity with the translation initiation complex. We endogenous MCT-1 (6). The cyclin D1 and Dp-1 protein levels were obtained virtually identical profiles in both human embryonic significantly reduced when MCT-1 was specifically knocked down kidney cell cultures and lymphoid cell lines, which suggests that by MCT-1-siRNA (Fig. 5D). this functional interaction of MCT-1 with the translation initiation complex is not tissue restricted. Discussion During the last few years, interest in the regulatory mechanisms that control translation and its role in tumorigenesis has gained There are several lines of evidence supporting the role of momentum. There is emerging evidence that the efficiency of MCT-1 in lymphomagenesis; stimulation of cell proliferation, expression of key proteins involved in cell growth regulation, enhancement of cell survival signaling, enhanced G cyclin/cdk 1 proliferation, or cell death may be controlled at the translational level by changes in the activity of components of the protein synthesis machinery (37, 38). In eukaryotes, most mRNAs are translated in a cap-dependent manner. One of the mechanisms that drive oncogenesis is the altered expression pattern of genes related to growth and development (39). For example, the proto-oncogene c-Myc up-regulates ribosomal proteins, eIF2a and eIF-4E, and is likely an important mechanism by which c-Myc regulates protein synthesis, cell growth, and ultimately, tumor formation (15). The prototypical cap-binding protein eIF4E is a proto-oncogene and is able to transform cells when the gene is amplified. Mutated proteins that are involved in ribosome biogenesis have also been associated with cancer and human disease. A recent example of this is another PUA domain containing the protein Dyskerin (Dck-1), which mediates the posttranscriptional modification of rRNA. Mutations in this gene result in defective rRNA modifications and putatively misfolded ribosomes, which promotes the dyskeratosis congenital disease that is associated with an increased susceptibility to cancer Figure 4. MCT-1 enhances translation in vitro. A, pCMV-luciferase was (38, 40, 41). translated in vitro using capped luciferase RNA in the presence of [35S]methionine and 0.1 or 1 Ag of the following proteins: lane 1, no additional The end result of gene expression is protein production, and protein; lanes 2 and 3, GST protein; lanes 4 and 5, GST-MCT-1 protein; that requires mRNAs to be recruited to ribosomes. Using an lanes 6 and 7, GST-DPUA; lanes 8 and 9, GST-PUA protein. B, array result of expression microarray screen, we were able to show that up- selected genes which were actively translated when MCT-1 is expressed. Mean fold change of polysome-bound mRNAs in MCT-1 samples compared with regulation of MCT-1 is able to significantly alter the mRNA the vector control samples (n = 3). translational profile. The importance of our findings is www.aacrjournals.org 8999 Cancer Res 2006; 66: (18). September 15, 2006

Figure 5. MCT-1 enhances translation in vivo. A, the real-time RT-PCR of the corresponding mRNAs from 293HEK cells revealed the same levels from both cells expressing MCT-1 (n = 3) or vector control (n = 3). B, Western blot analysis of representative total proteins from 293HEK cells expressing MCT-1 or vector control. C, Western blot analysis of lysate from Farage lymphoma cells expressing MCT-1 or vector control. D, Western blot of total proteins from Jurkat cells expressing scrambled siRNA, MCT-1 siRNA, or nontransfected control. The cyclin D1 and Dp-1 levels were unchanged in control and scrambled siRNA samples, whereas it was significantly reduced in MCT-1 siRNA expressing cells.

underscored by a recent report demonstrating that the human MCT-1 was identified in the archaea Pyrococcus abyssi (43). MCT-1 gene can complement, in yeast, the translation defects MCT-1 is the only known oncogene homologue in archaea and observed by the loss of the yeast gene, TMA20, having significant further highlights that MCT-1 is a highly conserved gene with homology (48%) to MCT-1. They also showed the physical critical biological function (44). Based on our experimental association of MCT-1 and DENR in yeast and mammalian cells data, we propose a model in which the MCT-1/DENR complex (42). Using a comparative genomics approach, a homologue of binds to cap either directly or indirectly with enhanced

Figure 6. Proposed model for role of MCT-1 in translation. MCT-1 bound to DENR binds to the cap complex either directly or indirectly through interaction with eIF4E with enhanced translation initiation by scanning and recognition of the initiation codon. MCT-1 and DENR might also recruit additional translation factors to the translation initiation complex.

Cancer Res 2006; 66: (18). September 15, 2006 9000 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. The MCT-1 Oncogene Modulates Translation translation initiation by scanning and recognition of the Acknowledgments initiation codon (Fig. 6). In conclusion, we suggest a mechanism Received 5/31/2006; revised 7/28/2006; accepted 8/2/2006. for the oncogenic activity of MCT-1, where it recruits the Grant support: A Merit Review Award from the Department of Veterans Affairs SUI1 domain containing DENR protein to the translation (R.B. Gartenhaus). initiation complex, thereby modulating the translational profile The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of a subset of mRNAs. Through increased understanding of the with 18 U.S.C. Section 1734 solely to indicate this fact. increased activation of the translation initiation machinery in We thank Dr. Z. Liu and Dr. G. Tian, Translational Core Facility, Dr. P. Phatak, and human malignancies, novel therapeutic strategies may emerge. M. Wilson from the University of Maryland, Marlene and Stewart Greenebaum Cancer Center, for assistance with statistical analysis and expert technical assistance with The data presented here further support the linkage between immunofluorescent microscopy. We also thank Drs. M. Gorospe and B. Hassel for translational control and oncogenesis. helpful discussions.

References 16. Meijer HA, Thomas AA. Control of eukaryotic 31. RauM,OhlmannT,MorleySJ,PainVM.A protein synthesis by upstream open reading frames reevaluation of the cap-binding protein, eIF4E, as a 1. Prosniak M, Dierov J, Okami K, et al. A novel candidate in the 5¶-untranslated region of an mRNA. Biochem rate-limiting factor for initiation of translation in oncogene, MCT-1, is involved in cell cycle progression. J 2002;367:1–11. reticulocyte lysate. J Biol Chem 1996;271:8983–90. Cancer Res 1998;58:4233–7. 17. Wilkie GS, Dickson KS, Gray NK. Regulation of mRNA 32. Galban S, Fan J, Martindale JL, et al. von Hippel- 2. Monni O, Joensuu H, Franssila K, Knuutila S. DNA translation by 5¶- and 3¶-UTR-binding factors. Trends Lindau protein-mediated repression of tumor necrosis copy number changes in diffuse large B-cell lymphoma– Biochem Sci 2003;28:182–8. factor a translation revealed through use of cDNA comparative genomic hybridization study. Blood 1996; 18. Kasperaitis MA, Voorma HO, Thomas AA. The amino arrays. Mol Cell Biol 2003;23:2316–28. 87:5269–78. acid sequence of eukaryotic translation initiation factor 33. Swiercz R, Person MD, Bedford MT. Ribosomal 3. Scarpa A, Taruscio D, Scardoni M, et al. Nonrandom 1 and its similarity to yeast initiation factor SUI1. FEBS protein S2 is a substrate for mammalian PRMT3 chromosomal imbalances in primary mediastinal B- Lett 1995;365:47–50. (protein arginine methyltransferase 3). Biochem J 2005; cell lymphoma detected by arbitrarily primed PCR 19. Yoon HJ, Donahue TF. The suil suppressor locus in 386:85–91. fingerprinting. Genes Chromosomes Cancer 1999;26: Saccharomyces cerevisiae encodes a translation factor 34. Herbert GB, Shi B, Gartenhaus RB. Expression and 203–9. that functions during tRNA(iMet) recognition of the stabilization of the MCT-1 protein by DNA damaging 4. Werner CA, Dohner H, Joos S, et al. High-level DNA start codon. Mol Cell Biol 1992;12:248–60. agents. Oncogene 2001;20:6777–83. amplifications are common genetic aberrations in B-cell 20. Sambrook J, Russell DW. Detection of protein-protein 35. Pestova TV, Kolupaeva VG. The roles of individual neoplasms. Am J Pathol 1997;151:335–42. interactions using the GST fusion protein pull-down eukaryotic translation initiation factors in ribosomal 5. Dierov J, Prosniak M, Gallia G, Gartenhaus RB. technique. Nat Methods 2004;1:275. scanning and initiation codon selection. Genes Dev Increased G1 cyclin/cdk activity in cells overexpressing 21. Yang HS, Jansen AP, Komar AA, et al. The transfor- 2002;16:2906–22. the candidate oncogene, MCT-1. J Cell Biochem 1999;74: mation suppressor Pdcd4 is a novel eukaryotic transla- 36. Becker HF, Motorin Y, Planta RJ, Grosjean H. The 544–50. tion initiation factor 4A binding protein that inhibits yeast gene YNL292w encodes a pseudouridine synthase 6. Shi B, Hsu HL, Evens AM, Gordon LI, Gartenhaus RB. translation. Mol Cell Biol 2003;23:26–37. (Pus4) catalyzing the formation of psi55 in both Expression of the candidate MCT-1 oncogene in B- and 22. Kumar V, Sabatini D, Pandey P, et al. Regulation of mitochondrial and cytoplasmic tRNAs. Nucleic Acids T-cell lymphoid malignancies. Blood 2003;102:297–302. the rapamycin and FKBP-target 1/mammalian target of Res 1997;25:4493–9. 7. Hsu HL, Shi B, Gartenhaus RB. The MCT-1 oncogene rapamycin and cap-dependent initiation of translation 37. Graff JR, Zimmer SG. Translational control and product impairs cell cycle checkpoint control and by the c-Abl protein-tyrosine kinase. J Biol Chem 2000; metastatic progression: enhanced activity of the mRNA transforms human mammary epithelial cells. Oncogene 275:10779–87. cap-binding protein eIF-4E selectively enhances trans- 2005;24:4956–64. 23. Galban S, Martindale JL, Mazan-Mamczarz K, et al. lation of metastasis-related mRNAs. Clin Exp Metastasis 8. Levenson AS, Thurn KE, Simons LA, et al. MCT-1 Influence of the RNA-binding protein HuR in pVHL- 2003;20:265–73. in vivo oncogene contributes to increased tumorigenic- regulated p53 expression in renal carcinoma cells. Mol 38. Ruggero D, Pandolfi PP. Does the ribosome translate ity of MCF7 cells by promotion of angiogenesis and Cell Biol 2003;23:7083–95. cancer? Nat Rev Cancer 2003;3:179–92. inhibition of apoptosis. Cancer Res 2005;65:10651–6. 24. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A 39. Hahn WC, Weinberg RA. Rules for making human 9. Aravind L, Koonin EV. Novel predicted RNA-binding comparison of normalization methods for high density tumor cells. N Engl J Med 2002;347:1593–603. domains associated with the translation machinery. oligonucleotide array data based on variance and bias. 40. Heiss NS, Knight SW, Vulliamy TJ, et al. X-linked J Mol Evol 1999;48:291–302. Bioinformatics 2003;19:185–93. dyskeratosis congenita is caused by mutations in a 10. Ishitani R, Nureki O, Fukai S, et al. Crystal structure 25. Bachman KE, Park BH, Rhee I, et al. Histone highly conserved gene with putative nucleolar functions. of archaeosine tRNA-guanine transglycosylase. J Mol modifications and silencing prior to DNA methylation Nat Genet 1998;19:32–8. Biol 2002;318:665–77. of a tumor suppressor gene. Cancer Cell 2003;3:89–95. 41. Ruggero D, Grisendi S, Piazza F, et al. Dyskeratosis 11. Cormier P, Pyronnet S, Salaun P, Mulner-Lorillon O, 26. Altman DG. Practical statistics for medical research. congenita and cancer in mice deficient in ribosomal Sonenberg N. Cap-dependent translation and control of Chapman and Hall/CRC; 1991. RNA modification. Science 2003;299:259–62. the cell cycle. Prog Cell Cycle Res 2003;5:469–75. 27. Deyo JE, Chiao PJ, Tainsky MA. drp, a novel protein 42. Fleischer TC, Weaver CM, McAfee KJ, Jennings JL, 12. Holcik M, Sonenberg N. Translational control in expressed at high cell density but not during growth Link AJ. Systematic identification and functional stress and apoptosis. Nat Rev Mol Cell Biol 2005;6: arrest. DNA Cell Biol 1998;17:437–47. screens of uncharacterized proteins associated with 318–27. 28. Fletcher CM, Pestova TV, Hellen CU, Wagner G. eukaryotic ribosomal complexes. Genes Dev 2006;20: 13. Andersen G, Busso D, Poterszman A, et al. The Structure and interactions of the translation initiation 1294–307. structure of cyclin H: common mode of kinase factor eIF1. EMBO J 1999;18:2631–7. 43. Matte-Tailliez O, Zivanovic Y, Forterre P. Mining activation and specific features. EMBO J 1997;16: 29. Hiremath LS, Hiremath ST, Rychlik W, Joshi S, archaeal proteomes for eukaryotic proteins with 958–67. Domier LL, Rhoads RE. In vitro synthesis, phosphory- novel functions: the PACE case. Trends Genet 2000; 14. De Benedetti A, Graff JR. eIF-4E expression and its lation, and localization on 48 S initiation complexes of 16:533–6. role in malignancies and metastases. Oncogene 2004;23: human protein synthesis initiation factor 4E. J Biol 44. Nandi S, Shi B, Perreault J, Gartenhaus RB. 3189–99. Chem 1989;264:1132–8. Characterization of the MCT-1 pseudogene: Identifica- 15. Rosenwald IB. The role of translation in neoplastic 30. Hiremath LS, Webb NR, Rhoads RE. Immunological tion and implication of its location in a highly transformation from a pathologist’s point of view. detection of the messenger RNA cap-binding protein. amplified region of chromosome 20. Biochim Biophys Oncogene 2004;23:3230–47. J Biol Chem 1985;260:7843–9. Acta 2006;1759:234–9.

www.aacrjournals.org 9001 Cancer Res 2006; 66: (18). September 15, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles

Line S. Reinert, Bo Shi, Suvobroto Nandi, et al.

Cancer Res 2006;66:8994-9001.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/66/18/8994

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2006/09/19/66.18.8994.DC1

Cited articles This article cites 43 articles, 17 of which you can access for free at: http://cancerres.aacrjournals.org/content/66/18/8994.full#ref-list-1

Citing articles This article has been cited by 14 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/66/18/8994.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/66/18/8994. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.