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[Cancer Research 66, 8994-9001, September 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

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

Line S. Reinert1, Bo Shi3, Suvobroto Nandi1, Krystyna Mazan-Mamczarz2, Michele Vitolo1, Kurtis E. Bachman1, Huili He1 and Ronald B. Gartenhaus1

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

Requests for reprints: Ronald B. Gartenhaus, University of Maryland, Marlene and Stewart Greenebaum Cancer Center, 9-011 BRB, 655 West Baltimore Street, Baltimore, MD 21201. Phone: 410-328-3691; Fax: 410-328-6559; E-mail: rgartenhaus{at}som.umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis. (Cancer Res 2006; 66(18): 8994-9001)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The oncogene MCT-1 is amplified in certain human T cell lymphomas and has been mapped to chromosome Xq22-24 (1). This region is amplified in a subset of primary B cell non–Hodgkin lymphomas, suggesting that increased copy number of a gene(s) located in this region confers a growth advantage to certain primary human lymphomas (2–4). Lymphoma cell lines overexpressing MCT-1 exhibited increased growth rates and displayed increased protection against serum starvation–induced apoptosis when compared with matched controls (1, 5, 6). Recently, we showed that MCT-1 impairs cell cycle checkpoint control after exposure to DNA-damaging agents and transforms human mammary epithelial cells (7). Furthermore, MCT-1 may also play a role in the pathogenesis of human breast cancer by inhibiting apoptosis and promoting angiogenesis (8). The molecular mechanism(s) underlying the broad activity of MCT-1 is currently unknown. MCT1 contains a PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes (9, 10), suggesting that MCT1 might have an RNA-binding function.

In the past several years, it has become increasingly evident that mRNA translation is also an important control point, and this is particularly the case in embryonic development and during the regulation of cell growth and differentiation (11, 12). The efficiency of expression of key proteins involved in cell growth regulation, proliferation, or cell death may be controlled at the translational level by changes in the activity of components of the protein synthesis machinery. Experimentally, aberrant expression of translation factors has been shown to induce the malignant transformation of cells. The eIF4E protein binds to the cap structure m7GpppN (where N is any nucleotide), which is found at the 5'-terminus of all cellular eukaryotic mRNAs (except those in organelles) (13), and overexpression of eIF4E has been shown to result in the transformation of cells both in vitro and in vivo (14, 15). Typically, mRNAs coding for proteins positively involved in growth regulation are poorly translated in resting cells and it is 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 (DENR/DRP), which contains a SUI1 domain found in the translation initiation factor, eIF1 (18, 19). We showed that MCT1 is a cap-binding protein recruiting DENR and subsequently altering the mRNA translational profile. The data presented supports a role for the MCT1 oncogene in translation and suggests a mechanism for its role in transformation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructions and cell culture transfection. The cDNA of full-length MCT-1 (543 nucleotides) was amplified from total RNA isolated from normal lymphocytes and cloned into pcDNA3.1/V5-Histidine tag vector (Invitrogen, Carlsbad, CA). This was represented as V5-MCT plasmids in the text. Similar cloning was also done for MCT-1 deletion constructs: {Delta}PUA (1-351 nucleotides) and PUA (245-543 nucleotides). Full-length MCT-1 was subcloned into a retroviral vector pLXSN (Clontech, 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 constructs in pcDNA3.1 were used for in vitro translations and for transient transfection of 293HEK cell lines (human embryonic kidney cell lines), NIH3T3, Raji and Farage cells (American Type Culture Collection, Manassas, VA) with LipofectAMINE 2000 (Invitrogen) or Nucleofector Kit V (Amaxa, Inc., Köln, 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 the human DENR sequence (GenBank accession no. AF038554) was amplified and cloned into pCMV-myc (Clontech) and pLXSN vector (Clontech) which was transfected in Raji cell lines by using Nucleofector (Amaxa). DENR was also cloned into the pET28c His-tagged expression vector (Novagen, Madison, WI) to purify His-tagged DENR proteins using E. coli BL21 (DE3) strain (Invitrogen).

Screening of human HeLa cell cDNA library by the yeast two-hybrid system. A yeast two-hybrid screen was done using the MATCHMAKER system III (Clontech) and was used according to the manufacturer's instructions. The bait construct pGBK-T7-HA-MCT-1 encoding the full-length MCT-1 protein (181 amino acids) fused in frame to the GAL4 DNA-binding domain, was constructed by inserting the PCR-generated fragment into the SalI and the BamHI sites of pGBKT7 vector. The human HeLa cell cDNA library, which was fused with the GAL4 activation domain in the pACT2 vector, was obtained from Clontech. The MATa strain H109, which was stably transformed with bait construct MCT-1, was mixed with yeast strain Y187 that was pretransfected by human HeLa cell cDNA library (Clontech). The binding specificities of the isolated prey clones were checked after cotransformations by growth on high-stringency selective yeast media. Plasmid DNA from prey yeast clones was extracted using yeast plasmid isolation kit (Clontech) and sequenced.

MCT-1 and DENR coimmunoprecipitation. Raji cell lines were transiently transfected with the following plasmids: pcDNA empty vector, V5-MCT-1, V5-{Delta}PUA, V5-PUA, and pCMV-myc-DENR. Cell pellets were lysed and reciprocal coimmunoprecipitation experiments with either c-Myc monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-V5 monoclonal antibody (Invitrogen) were done using protein A beads. Eluted and denatured samples were loaded on 15% acrylamide SDS-PAGE. Western blot was carried out using standard methods. Coimmunoprecipitation with endogenous MCT-1 and transfected pCMV-myc-DENR in 293HEKs was done similarly, but used MCT-1 antibody (Sigma, Research Genetics, Inc., St. Louis, MO) and c-Myc monoclonal antibody (Santa Cruz Biotechnology).

GST pull-down assay. Ten micrograms of the E. coli expressed and purified His-DENR, GST, GST-MCT-1, GST-{Delta}PUA, and GST-PUA were used for GST pull-down assays as previously described (20). The glutathione-agarose beads were washed four times and eluted in 2x SDS buffer. The eluted proteins were loaded on 15% acrylamide SDS-PAGE and immunoblotted with GST or His antibody.

Immunostaining. 3T3 cells were transiently transfected with myc-DENR and V5-MCT-1 or mock-transfected as controls in two-chambered slides (Nunc, Roskilde, Denmark). Forty-eight hours after transfection, the cells were washed in PBS, fixed with a 1:1 mixture of ice-cold methanol and acetone, and blocked with 3% bovine serum albumin for 2 hours. The cells were then washed thrice with washing buffer (PBS containing 0.2% Tween 20) and incubated overnight with 1:200 mouse monoclonal anti-V5 antibody and 1:200 rabbit polyclonal anti-myc antibody at room temperature. The cells were then washed thrice with washing buffer and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 568 at a concentration of 1:200 each. The cells were then washed again, as described, and mounted using Vectashield mounting medium (Vector Labs, Burlingame, CA) and coverslips were placed on them. Photomicrographs were taken using a Leica DM4000B immunofluorescence microscope (64x magnification) with Improvision software.

In vitro translation of luciferase. pCMV-luciferase was translated in vitro as previously described (21), with minor modifications. The assay was done in the presence of luciferase 7mGpppG RNA and [35S]methionine and 0, 0.1, or 1 µg of purified GST-tagged MCT-1 proteins at 30°C for 30 minutes. The translated proteins were separated on a 15% SDS gel and autoradiographed.

Cap-binding assay. The cap-binding assay was done as previously described (22), with minor modifications. Nontagged and linearized MCT-1 plasmid was in vitro–translated using [35S]methionine, 20 µL of this rabbit reticulocyte lysate (Promega, Madison, WI) with 300 µL of buffer D [50 mmol/L HEPES (pH 7.4), 40 mmol/L NaCl, 2 mmol/L EDTA, 0.1% Triton X-100] and 30 µL of a 50% slurry of 7-methyl-GTP-sepharose (Amersham, Buckingham, United Kingdom) and incubated for 1 hour at 4°C. After washing the resin four times with buffer D, bound proteins were eluted with 2x SDS sample buffer, resolved by 15% SDS-PAGE, and autoradiographed. Competition reaction was carried out as described above but with the indicated amount of Cap analogue (m7GpppG; Ambion) or GTP (Ambion). The cap-binding assay was also done in the presence of 1 µg of purified His-DENR. The eluted His-DENR protein was visualized by performing Western blot with anti-His antibody.

Depletion of eIF4E from reticulocyte lysate. The V5-control, V5-MCT-1, V5-{Delta}PUA, and V5-PUAs plasmids were transcribed and translated according to the manufacturer in a TNT Quick coupled transcription/translation system (Promega). Half of this reaction (50 µL) was used for overnight depletion of eIF4E by using 4 µg of eIF4E antibody (Santa Cruz Biotechnology), 20 µL of protein A/G agarose beads and protease inhibitor. Prior to depletion, 4 µL of the extract, 7 µL of the supernatant which was depleted for eIF4E, and 10% of the eluate from the beads, i.e., the depleted eIF4E, was loaded on a SDS gel followed by Western blot with EIF4E antibody to control the depletion level. The cap-binding assay was done as described above but also with an equal amount of the extract after depletion of eIF4E.

Polysome isolation. 293HEK cells were transiently transfected with vector, V5-MCT-1, or pCMV-c-Myc-DENR plasmids using Lipofectamine (Invitrogen) according to the manufacturer's protocol. Thirty-six hours later, they were collected and washed with PBS containing 100 µg/mL of cycloheximide. The cells were lysed in a buffer containing 0.3 mol/L of KCl, 5 mmol/L of MgCl2, 10 mmol/L of Tris (pH 7.4), 0.5% NP-40, 5 mmol/L of DTT, proteinase K inhibitors, RNase inhibitors, and 1 mg of heparin per mL at 4°C. Centrifugation was done to pellet nuclei and unlysed cells. The supernatant was centrifuged through a 10% to 50% sucrose gradient, as described in ref. (23). Equal volumes of the fractions were loaded on a 15% SDS gel and blotted with antibody against eIF2{alpha}, eIF4E, rS6p, ß-actin, V5 for detecting MCT-1, and c-Myc for detecting DENR (Santa Cruz Biotechnology). Similar experiments were carried out on Farage and Daudi B cell lines that were stably transfected with vector or V5-MCT-1 plasmid.

Microarray and data analysis. 293HEK cells were transfected with V5-MCT and V5-vector plasmids, and the polysomes were separated as described above. Polysome-associated RNA from vector and MCT-1-transfected cells were purified by RNeasy mini kit (Qiagen, Valencia, CA) and pooled separately. Five micrograms of polysome-associated RNA were used to generate cDNA and thereafter generate cRNA probes labeled with Biotin-16-UTP (Roche Molecular Biochemicals, Basel, Switzerland) using the AmpoLabeling-LPR kit (SuperArray, Frederick, MD). The cRNA probe was hybridized to GEArray-human apoptosis and cell cycle gene arrays (SuperArray) and the hybridization signal was detected with a chemiluminescence detection kit (SuperArray). The relative mRNA level of each gene was analyzed using GEArray Analyzer software. From the quantified signals, we calculated the log2 ratio and then normalized it with a quantile normalization method (24). After the normalization, we calculated the mean log-fold change in MCT-1 samples compared with the vector control samples. The fold changes were calculated by 2(log2 ratio). The assays were independently carried out in triplicate.

Real-time RT-PCR analysis. Total RNA was isolated from V5-MCT-1 and V5 control vector expressing 293HEK cells and using the RNeasy system (Qiagen). Single-stranded cDNA was generated using first-strand cDNA synthesis kit (Amersham) following the manufacturer's directions. Control template reactions were prepared in parallel without the addition of reverse transcriptase. Real-time RT-PCR was then done as previously described (25). Each reaction was carried out in triplicate; in addition, three independent reactions were run simultaneously. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA served as the internal control template. The change in mRNA level was calculated by: {Delta}{Delta}cycle threshold (Ct) = ({Delta}Ct MCT-1 expressing sample) – ({Delta}Ct vector expressing sample), where {Delta}Ct = (Ct for the target gene) – (Ct for GAPDH gene). ANOVAs (P values) were calculated by using a null hypothesis H_0 that all the three experiments had the same mean as reviewed in ref. (26). We let x_1, x_2, and x_3 denote the means of the three independent {Delta}{Delta}Ct, respectively. If P values were >0.05, we could not reject the null hypothesis H_0 (i.e., all three {Delta}{Delta}Ct means are equal).

Western blots. We did Western blots to quantify the protein levels of several mRNAs that were actively recruited to the polysomes. The protein levels of MCT-1, Mre11, cyclin D1, E2F1, Bcl-w, Dp-1, and GAPDH were examined from either stably transfected Farage or Daudi cell lines, or transiently transfected 293HEK whole cell lysates that were obtained at three different time points: 24, 36, and 48 hours after transfection (n = 3). Western blot for total proteins from Jurkat cell lines that expressed pSIREN-RetrpQ vector containing random (NT) small interfering RNA (siRNA) scrambled oligo or siRNA against MCT-1 (NT 503-523; ref. 7) were transfected by using Nucleofector (Amaxa). Controls were nontransfected Jurkat cells. The cells were harvested 72 hours posttransfection and 40 µg of total cell extract was loaded on 15% SDS gel followed by Western blot. Quantitative analysis was carried out using Alpha Innotech quantitation software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MCT-1 physically interacts with DENR. The NH2 terminus of MCT-1 contains a domain with significant homology to the protein-protein binding domain found in cyclin H (1, 13). To identify which proteins physically interact with MCT-1, we used a yeast two-hybrid approach. A total of 16 positive clones were found (data not shown), the plasmids from these clones were sequenced and all of them were found to encode for the same protein known as density-regulated protein (DENR/DRP) or smooth muscle cell–associated protein 3 (SMAP-3) (27). The search for a conserved domain database revealed that DENR contained a SUI1 domain similar to the archetypal translation initiation factor eIF1 (Fig. 1A ). The SUI1 domain in eIF1 has a secondary structure fold corresponding 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 stably transfected lymphoid cell lines. The two proteins were shown to physically interact in reciprocal coimmunoprecipitation assays in vivo in transfected Raji cells (Fig. 1B) and in 293HEK when only DENR was overexpressed (Fig. 1C). Using a GST pull-down assay, we attained similar results (Fig. 1D). Subcellular localization of MCT-1 and DENR proteins was examined using immunohistochemistry. Both MCT-1 and DENR were shown to colocalize predominantly in the cytoplasm of 3T3 cells (Fig. 1E). Very little background staining was found in the merge slide of mock-transfected control 3T3 cells (data not shown).


Figure 1
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  		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.

 
The PUA domain of MCT-1 is required for Cap complex–binding. The above data pointed to MCT-1 and DENR forming a functional complex involved in translation initiation. The PUA domain present in MCT-1 at the COOH-terminal end is a predicted RNA-binding domain (Fig. 2A ). Here, we show that MCT-1 is able to bind a cap complex, which is present at the 5'-ends of mRNA. Equal amounts of in vitro–translated MCT-1 proteins were incubated with m7GTP-sepharose beads and the bound proteins were eluted, run on SDS gel and autoradiographed (Fig. 2B). Full-length MCT-1 and the PUA domain both exhibited strong binding to the m7GTP-sepharose, whereas the binding of {Delta}PUA was very modest. This indicated that the PUA domain of MCT-1 was required for the interaction with m7GTP-sepharose. To confirm the specificity of the cap binding by MCT-1, a competition assay with sepharose beads alone or together with a soluble cap analogue was carried out and the binding was successfully competed out in a dose-dependent manner (Fig. 2C). The free GTP does not compete the cap complex-MCT-1 binding, consistent with MCT-1 binding specifically to GTP in the m7GTP-sepharose beads (Fig. 2D). Furthermore, MCT-1 does not bind to other RNA structures including uncapped 5'-untranslated region, open reading frame, or 3'-untranslated region RNA fragments (Supplementary Fig. S1). To identify if DENR binds directly to the cap complex, MCT-1 and {Delta}PUA proteins were incubated separately with m7GTP-sepharose beads in the presence of His-DENR protein. DENR only came down with the cap in the presence of full-length MCT-1. The cap complex incubated with the {Delta}PUA deletion mutant was not able to bring down the DENR protein (Fig. 2E).


Figure 2
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  		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, {Delta}PUA (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.

 
MCT-1 interacts with the translation machinery. Next, we asked if MCT-1 binding to the cap complex requires eIF4E. In order to address this question, we expressed MCT-1 in reticulocyte lysates and then depleted eIF4E (Fig. 3A ). Reticulocyte lysates containing equal amounts of MCT-1 proteins, before and after the depletion of eIF4E were then used for the cap binding assay (Fig. 3B). Both full-length MCT-1 and PUA-containing mutant lose their cap-binding function in eIF4E-depleted extracts, consistent with eIF4E being required for their interaction with the cap complex.


Figure 3
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  		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 eIF2{alpha}, eIF4E, Myc-DENR, V5-MCT-1, ß-actin, and rS6p (bottom).

 
Because MCT-1 interacts with the cap complex, we asked whether the MCT-1/DENR complex interacts with the translation machinery by analyzing polysomal fractions isolated through a sucrose gradient. This separates the unbound mRNA from translationally active mRNAs bound to polysomes of increasing size. Low-passage 293HEK cells (human embryonic kidney cell lines) that readily achieve highly efficient transient transfections (>80% transfection rate) were used for this assay. Cytosolic extracts from transiently transfected 293HEK cells expressing empty vector or V5-MCT-1 and pCMV-Myc-DENR were applied to a linear 10% to 50% sucrose gradient. The ribosomal units were monitored at an absorbance of 260 nm and the profile is shown in Fig. 3C (top). In order to examine if MCT-1 and DENR interact with ribosomes, we did Western blot analyses of the polysome fractions from cells expressing both V5-MCT-1 and Myc-DENR. The eIF4E and eIF2 components of the translation initiation complex are found in the RNP sedimentation fraction and in 40S fractions as previously reported (29–31). Our data showed that MCT-1 sediments in the same fractions as eIF4E and the eIF2{alpha} under our assay conditions (Fig. 3C, bottom). The DENR protein was detected in similar fractions as both MCT-1 and eIF2{alpha}. Both ß-actin and a component of ribosomal subunit, rpS6, were fractionated with monosomes and polysomes as expected (refs. 32, 33; Fig. 3C). We obtained nearly identical profiles and Western blots employing Farage and Daudi B cell lines (data not shown), that were stably transfected with either MCT-1 or empty vector plasmids.

MCT-1 is involved in translational regulation. Having established that MCT-1 sediments predominantly with the RNP and 40S sedimentation fraction, we asked whether MCT-1 was able to enhance translation. We showed that MCT-1 enhances the translation of the luciferase reporter gene when GST-MCT-1 protein was added in excess to the rabbit reticulocyte lysate translation system in a dose-dependent manner (Fig. 4A ). This function was lost, when GST alone, GST-{Delta}PUA, or only GST-PUA protein was substituted for GST-MCT-1. This established that the full-length MCT-1 enhanced translation in vitro and that the PUA RNA-binding domain was critical for cap binding, but not sufficient to function as a translation enhancer. This indicated that MCT-1 required both PUA to bind to the cap RNA, and {Delta}PUA to possibly interact with other proteins to enhance translation.


Figure 4
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  		Figure 4. MCT-1 enhances translation in vitro. A, pCMV-luciferase was translated in vitro using capped luciferase RNA in the presence of [35S]methionine and 0.1 or 1 µg of the following proteins: lane 1, no additional protein; lanes 2 and 3, GST protein; lanes 4 and 5, GST-MCT-1 protein; lanes 6 and 7, GST-{Delta}PUA; lanes 8 and 9, GST-PUA protein. B, array result of selected genes which were actively translated when MCT-1 is expressed. Mean fold change of polysome-bound mRNAs in MCT-1 samples compared with the vector control samples (n = 3).

 
MCT-1 was previously shown to play a role in cell cycle progression, apoptosis, and DNA damage response (5–8, 34). In order to identify if the increased translation of genes participating in these processes was caused by the up-regulation of MCT-1, we isolated polysomal RNA fractions as previously described from 293HEK cells transiently transfected with V5-MCT-1 or vector control plasmids. The mRNAs that were actively recruited to the polysomes were analyzed on a targeted microarray containing 267 genes involved in apoptosis and cell cycle (SuperArray). For the quantified signals from three independent experiments, we calculated the log2 ratio, and then normalized it with a quantile normalization method to obtain the same background intensity (24). After the normalization, we calculated the mean fold change V5-MCT-1 polysomal samples compared with the vector polysomal samples. We identified 67 genes (25%) that were ≥2-fold increased in V5-MCT-1 polysomal fractions compared with the vector polysomal fractions (Fig. 4B; Supplementary Table 1).

In order to verify that protein levels were increased in accord with the enhanced recruitment to the polysome of mRNAs, we carried out Western blot analyses of selected proteins. To confirm that the Western blot data actually reflected an increase in the translation of these genes and was not due to differences in the transcription level, we did real-time PCR on the total mRNA from control vector and V5-MCT expressing 293HEK cells. The real-time PCR was done in triplicate for each sample from the three independent experiments (Fig. 5A ). The differences in cycle threshold (Ct) from the V5 vector and the V5-MCT-1 samples, which were normalized to GAPDH, were compared. The total mRNA levels were virtually identical in all target genes examined despite the differences in protein levels consistent with differences in translation. The MCT-1 mRNA levels were ~5-fold increased in the V5-MCT-1 sample compared with vector (Fig. 5A).


Figure 5
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  		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.

 
Total cell lysates either from stably transfected Farage B cell lymphoma cells expressing empty vector or PLXSN-V5-MCT-1 or from 293HEK cells were isolated at 24, 36, and 48 hours posttransfection with either MCT-1 or empty vector control plasmids. Western blotting was carried out using antibodies to Mre11, Bcl-w, cyclin D1, E2F1, Dp-1, GAPDH, and MCT-1 (Fig. 5B and C). After standardizing to control GAPDH protein levels, there was a consistent increase observed in the protein levels of cyclin D1 protein by 2.8-fold, Mre11 protein levels by 2-fold, Bcl-w protein by 3-fold, the transcription factors E2F1 by 4-fold, and Dp-1 by 2-fold in the MCT-1 transiently transfected cells compared with vector controls. We also showed that MCT-1 increased Mre11 protein levels by 5-fold and Dp-1 by 2-fold in Farage cells (Fig. 5C). These differences were most apparent at 48 hours posttransfection in 293HEK cells (Fig. 5B) and were reproducible in at least three independent experiments. To further support the effect of elevated MCT-1 protein levels on translation, we employed a knock-down approach in Jurkat lymphoma cells that exhibit high levels of endogenous MCT-1 (6). The cyclin D1 and Dp-1 protein levels were significantly reduced when MCT-1 was specifically knocked down by MCT-1-siRNA (Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are several lines of evidence supporting the role of MCT-1 in lymphomagenesis; stimulation of cell proliferation, enhancement of cell survival signaling, enhanced G1 cyclin/cdk kinase activity, elevated levels of MCT-1 protein in primary diffuse large B cell lymphoma, and overriding cell cycle checkpoints. The molecular mechanism(s) underlying the broad activity of MCT-1 is currently unknown. Using a yeast two-hybrid approach, MCT-1 was shown to interact with the density-regulated protein (DENR/DRP), a protein containing the SUI1 domain. This complex was shown to interact with the cap complex and to alter the mRNA translational profile of human embryonic kidney cell lines.

The SUI1 domain present in DENR is homologous to the prototypical one found in eIF1, containing a secondary structure fold corresponding to that of a number of ribosomal proteins and RNA-binding domains (28). The function of SUI1 in eIF1, is to scan along the mRNA to locate the initiation codon and to discriminate between cognate and noncognate initiation codons (35). MCT-1 contains a PUA domain, which is a recently described RNA binding domain and is found in several RNA modification enzymes. Previous studies with proteins containing a PUA domain suggested that they are involved in tRNA and rRNA modifications as exemplified by the yeast pseudouridine synthases (9, 36). We have provided experimental evidence that MCT-1 is a cap complex–binding protein interacting with m7GTP through the PUA domain and that this interaction requires the presence of eIF4E. In light of our data demonstrating that both MCT-1 and DENR sediment in the RNP sedimentation fraction and in 40S fractions, as was the case for eIF2, it is plausible to hypothesize that MCT-1 by binding to the cap structure brings the SUI1 domain containing DENR in close proximity with the translation initiation complex. We obtained virtually identical profiles in both human embryonic kidney cell cultures and lymphoid cell lines, which suggests that this functional interaction of MCT-1 with the translation initiation complex is not tissue restricted.

During the last few years, interest in the regulatory mechanisms that control translation and its role in tumorigenesis has gained momentum. There is emerging evidence that the efficiency of expression of key proteins involved in cell growth regulation, 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, eIF2{alpha} 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 (38, 40, 41).

The end result of gene expression is protein production, and that requires mRNAs to be recruited to ribosomes. Using an expression microarray screen, we were able to show that up-regulation of MCT-1 is able to significantly alter the mRNA translational profile. The importance of our findings is underscored by a recent report demonstrating that the human MCT-1 gene can complement, in yeast, the translation defects observed by the loss of the yeast gene, TMA20, having significant homology (48%) to MCT-1. They also showed the physical association of MCT-1 and DENR in yeast and mammalian cells (42). Using a comparative genomics approach, a homologue of MCT-1 was identified in the archaea Pyrococcus abyssi (43). MCT-1 is the only known oncogene homologue in archaea and further highlights that MCT-1 is a highly conserved gene with critical biological function (44). Based on our experimental data, we propose a model in which the MCT-1/DENR complex binds to cap either directly or indirectly with enhanced translation initiation by scanning and recognition of the initiation codon (Fig. 6 ). In conclusion, we suggest a mechanism for the oncogenic activity of MCT-1, where it recruits the SUI1 domain containing DENR protein to the translation initiation complex, thereby modulating the translational profile of a subset of mRNAs. Through increased understanding of the increased activation of the translation initiation machinery in human malignancies, novel therapeutic strategies may emerge. The data presented here further support the linkage between translational control and oncogenesis.


Figure 6
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  		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.

 

   Acknowledgments
 
Grant support: A Merit Review Award from the Department of Veterans Affairs (R.B. Gartenhaus).

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Z. Liu and Dr. G. Tian, Translational Core Facility, Dr. P. Phatak, and M. Wilson from the University of Maryland, Marlene and Stewart Greenebaum Cancer Center, for assistance with statistical analysis and expert technical assistance with immunofluorescent microscopy. We also thank Drs. M. Gorospe and B. Hassel for helpful discussions.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 5/31/06. Revised 7/28/06. Accepted 8/ 2/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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