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MicroRNA-340-Mediated Degradation of Microphthalmia-Associated Transcription Factor (MITF) mRNA is Inhibited by Coding Region Determinant Binding Protein (CRD-BP)
Srikanta Goswami1, Rohinton S. Tarapore1,2, Ashley M. Poenitzsch Strong1,2, Jessica J. TeSlaa3, Yevgenya Grinblat2,3, Vijayasaradhi Setaluri1, and Vladimir S. Spiegelman1,2*
Running title: CRD-BP prevents miR-340-mediated degradation of MITF mRNA 1Department of Dermatology and Paul P. Carbone Comprehensive Cancer Center, 2Molecular and Environmental Toxicology Center, 3Departments of Zoology and Anatomy; University of Wisconsin School of Medicine and Public Health, Madison, WI 53706 Address correspondence to: Vladimir S. Spiegelman, M.D., Ph.D. Department of Dermatology, University of Wisconsin School of Medicine and Public Health, 1300 University Ave., MSC-417, Madison, WI 53706. Telephone: (608) 265-8197; Fax: (608) 263-5223; Email: [email protected] Keywords: MITF, CRD-BP, IGF2BP1, melanoma, miRNA, alternative cleavage and polyadenylation.
Background: MITF is paramount for melanocyte development and melanoma pathogenesis. Results: CRD-BP restricts the action of miR-340 by preventing its access to the mRNA of MITF, thereby establishing a novel mode for the regulation of MITF. Conclusion: Regulation of MITF by CRD-BP contributes to the effects of CRD-BP on melanoma survival and progression. Significance: CRD-BP is a potential target for the prevention and treatment of melanoma.
ABSTRACT
Alternative cleavage and polyadenylation (APA) generates multiple transcript variants producing mRNA isoforms with different length 3’-UTRs. APA enables differential post-transcriptional regulation via the availability of different cis-acting elements in 3’UTRs. Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and melanogenesis. This central transcription factor is also implicated in melanoma development. Here, we show melanoma cells favor the expression of MITF mRNA with a shorter 3’-UTR. We also establish that this isoform is regulated by microRNA (miRNA/miR), miR-340. miR-340 interacts with two of its target sites on the
MITF 3’-UTR, causing mRNA degradation as well as decreased expression and activity of MITF. Conversely, the RNA binding protein (RBP), Coding region determinant binding protein (CRD-BP) was shown to be highly expressed in melanoma, directly binds to the 3’-UTR of MITF mRNA and prevents the binding of miR-340 to its target sites resulting in the stabilization of MITF transcripts, elevated expression and transcriptional activity of MITF. This regulatory interplay between RNA-binding protein and miRNA highlights an important mechanism for the regulation of MITF in melanocytes and malignant melanomas.
MITF is a basic helix-loop-helix leucine
zipper dimeric transcription factor belonging to the MYC superfamily of proteins (1). MITF forms dimers and binds to specific sequence motifs present in the promoter region of its target genes to activate their transcription. Various studies documented the role of MITF in the induction of genes required for normal melanocyte development as well as melanin formation. MITF regulates the transcription of three major pigmentation enzymes, tyrosinase (TYR), tyrosine related protein-1 (TYRP-1) and dopachrome-tautomerase (DCT) (2-4). It is also an amplified
http://www.jbc.org/cgi/doi/10.1074/jbc.M114.590158The latest version is at JBC Papers in Press. Published on November 20, 2014 as Manuscript M114.590158
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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oncogene in a subset of melanomas and was reported to regulate a distinct set of target genes which in turn are responsible for a neoplasia-related phenotype in these cancers. Primarily, this action of MITF in melanoma is mediated by increasing cell proliferation and triggering cell cycle progression. T-Box transcription factor 2 (TBX-2) is one such target that suppresses senescence via down-regulation of p21 (5,6). CDK2 is another target, regulation of which modulates cell cycle progression required for melanoma clonogenic growth (7). MITF was also reported to inhibit apoptosis and thus promote oncogenesis by increasing the expression of its direct target, the anti-apoptotic factor BCL2 (8). Furthermore, MITF contributes towards the metastasis of melanoma through transcriptional activation of the c-MET proto-oncogene (9). Therefore, understanding the mechanisms controlling the expression and function of MITF is extremely important as it will identify key components for melanocyte development and may uncover novel targets for the treatment and/or prevention of melanoma.
Previous research demonstrated that regulation of MITF expression occurs at multiple levels. Transcriptional regulation of MITF expression takes place through the MITF-M promoter via Wnt (10-13) and cAMP-CREB (14) pathways and also PAX3 (15). There is also evidence demonstrating post-translational regulation of MITF affects the availability of functional protein. Specifically, MAPK components phosphorylate MITF protein at Ser73 resulting in ubiquitination and subsequent degradation of the protein (16,17). On the other hand, phosphorylation of the Ser298 residue by GSK3β is believed to increase the DNA binding activity of MITF (18). However, apart from transcriptional and post-translational regulation, another important mode of regulating gene expression is to manipulate the stability of mature mRNA. Recent reports indicate MITF is a target of several miRNAs including the miR-96/183/182 cluster (19) and miR-137 (20), suggesting the importance of a post-transcriptional regulatory step in the expression of MITF.
miRNAs are small ~22 nucleotide non-coding RNAs that are an important class of gene regulators. They are present in both plant and animal genomes and after being transcribed and processed, the final form of mature miRNAs bind
to sites on target mRNAs typically in the 3’-UTR and may also bind to sites in the coding region (21). Additional protein components participate in the process of forming the miRNA induced silencing complex (miRISC), ultimately leading to translational repression and/or degradation of the target mRNA.
APA generates multiple transcript variants of a particular gene, creating mRNA isoforms with different 3’-UTR lengths depending on the position of APA signals. The exclusion of large 3’-UTR regions allows those mRNA isoforms to escape miRNA-dependent regulation. Recently, it was observed in fast proliferating cells, mRNA isoforms with shorter 3’-UTRs are favored (22). This preference helps oncogenes avoid regulation that would otherwise interfere with cell cycle progression and, ultimately, cellular transformation (22,23). There are several different isoforms of MITF-M mRNA with varying 3’-UTR lengths reported (19,20). In this study, we investigated the expression pattern of three such mRNA isoforms in melanoma cell lines and normal human melanocytes. We found MITF mRNAs with a short 3’-UTR are more abundant in melanoma cell lines and, additionally, this isoform undergoes miR-340-mediated regulation leading to the destabilization of MITF mRNA. We also elucidated the role of an RBP, CRD-BP (IMP-1, IGF2BP1), in the regulation of MITF mRNA. This protein was previously found to attenuate miRNA-dependent degradation of several mRNAs (21) and is also over-expressed in melanoma (24). Further, CRD-BP is involved in the regulation of metastatic melanoma cell proliferation and invasion by hypoxia (25) and its inhibition sensitizes melanoma cells to chemotherapeutic agents (26). Our results here show that CRD-BP restricts the action of miR-340 by preventing its access to MITF mRNA, thereby proposing a novel mode of regulation for MITF.
EXPERIMENTAL PROCEDURES
Cell Lines, Culture and Transfection conditions
Normal human melanocytes (NHMs) were maintained in Ham’s F10 media (Mediatech Inc., Manassas, VA) supplemented with Human Melanocyte Growth Supplement (HMGS, Cascade
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Biologics Inc., Portland, OR), 5% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution, containing penicillin, streptomycin and amphotericin B (PSM). 451Lu cells were maintained in Minimal Essential Media (Life Technologies, Carlsbad, CA) supplemented with 1% Sodium Pyruvate, 1% Non-Essential amino acids, 5% FBS and 1% PSM. The melanoma cell lines 928 mel, 1011 mel and 1242 mel were kindly provided by Dr. P. Robbins (Center for Cancer Research, National Cancer Institute, Bethesda, MD) and were maintained in DMEM (Life Technologies) supplemented with 10% bovine calf serum (BCS) and 1% PSM. Colorectal cancer cell lines HCT116, RKO and DLD1 along with corresponding exon 5-disrupted Dicer cell lines were kindly provided by Drs. K. Kinzler and B. Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD) (27). 293T cells were obtained from ATCC (Manassas, VA). Colorectal cancer and 293T cell lines were cultured in DMEM (Life Technologies) supplemented with 10% FBS and 1% PSM. NHMs were electroporated using the AMAXA Nucleofector™ (Lonza, Switzerland) according to the manufacturer’s protocol. Transfections of all other cells were performed using Lipofectamine 2000 according to the manufacturer’s protocol (Life Technologies).
Expression vectors
Full length MITF cDNA was purchased from Open Biosystems Inc. (Huntsville, AL; Clone ID 6066096 / Accession No. BC065243 / Cloned in pCMV-SPORT6) and amplified using Fragment 1 Forward and 3’-UTR Reverse primers (Table 1). The PCR product was then cloned into the pBI-G vector (Clontech Laboratories, Inc., Mountain View, CA), digested with NotI and SalI, end filled with Klenow enzyme (New England Biolabs, Ipswich, MA) and the clone with the correct orientation was selected. Three fragments of the MITF coding region (1-421, 422-841 and 842-1260 nt.), the full length cDNA (1-1822 nt) and 3’-UTR (1261-1822 nt.) were sub-cloned into pcDNA3.1. The 3’-UTR of MITF cDNA was PCR amplified and cloned into the pBI-GL vector (Clontech Laboratories, Inc.) just after the stop codon. To obtain the deletion mutants for miR-340 sites in pBI-G or pBI-GL constructs, we
performed blunt-end ligation of PCR products and treated with DpnI as recommended in the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
Plasmids for CRD-BP expression or the production CRD-BP shRNA were characterized in (28). The pcDNA5-CMV-d2eGFP vector and control sponge-CXCR4 construct were kindly provided by Dr. P. Sharp (MIT, Cambridge, MA). The constructs of sponge-340 and sponge-584c-3p were designed as described in (29). Separate oligonucleotides with 7 bulged binding sites for miR-340 and miR-584c-3p containing 4nt spacer sequences between them (Table 1) were annealed, gel purified and cloned into the pcDNA5-CMV-d2eGFP vector and linearized with XhoI and ApaI (New England Biolabs). Plasmids for miR-340 expression, pCMV-MIR and pCMV-MIR340, were purchased from Origene (Rockville, MD).
RNA Isolation and quantitative real-time PCR
Total RNA from cells was isolated using TRI reagent (Molecular Research Center, Cincinnati, OH). For quantitative real-time PCR (qRT-PCR) reactions, total RNA was treated with 2 units of DNase I (Promega, Madison, WI). Reverse transcription was performed using the Advantage RT-for-PCR kit (Clontech Laboratories, Inc.) and qRT-PCR was performed with Power SYBR Green PCR master mix (Life Technologies), according to the manufacturer’s recommendations. Reactions were performed on an ABI Prism 7000 machine using ABI Prism 7000 SDS software (Life Technologies). Sequences of primers used for qRT-PCR are presented in Table 2.
Stem-Loop quantitative real-time PCR
The expression of mature miR-340 was detected using a two-step process. First, using total RNA isolated by TRI reagent, the stem-loop reverse transcription (RT) primer for miR-340 (designed according to (30)) was hybridized to the miRNA molecule by incubating at 16oC for 30 min and then reverse transcribed for 30 min at 42oC using an Advantage RT-for-PCR kit (Clontech Laboratories, Inc.). After heat inactivating the reverse transcriptase at 95oC for 5 min, one tenth of the RT product was used for
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either endpoint PCR or qRT-PCR with Power SYBR Green PCR master mix (Life Technologies) using the miRNA-340 specific forward and universal reverse primers (Table 2).
Droplet digital PCR
To quantify miR-340 RNAs at high resolution, droplet digital PCR (ddPCR) was performed as described elsewhere (31) on the QX100 ddPCR system (Bio-Rad, Hercules, CA) using TaqMan FAM labeled probes for hsa-miR-340-5p and RNU6B, as an endogenous control (Life Technologies). 20μL reactions were assembled with cDNA, probes and ddPCR Supermix for Probes (no dUTP) (Bio-Rad). Droplets were generated using the manufacturer’s suggested protocol using the QX100 Droplet Generator machine, droplet generator cartridges, gaskets and droplet generation oil for probes (Bio-Rad). Generated droplets were placed into a twin.tec PCR 96-well plate (Eppendorf, Hauppauge, NY). Plates were sealed using pierceable foil heat seal and a PX1 PCR plate sealer (Bio-Rad). A PCR reaction under the following conditions was performed on a Mastercycler Gradient thermal cycler (Eppendorf): 5 minutes at 95°C, 30 seconds at 95°C, 1 minute at 60°C for 40 cycles with a ramp speed of 2°C/second, 98°C for 10 minutes and hold at 12°C overnight. FAM-generated fluorescence in the droplets was evaluated using the QX100 Droplet Reader machine and QuantaSoft Software version 1.3.2.0 (Bio-Rad). ddPCR data presented are representative of absolute copies of transcripts in reaction samples.
mRNA Degradation In Vivo
To investigate the stability of mRNA transcripts, we used the Tet-Off gene expression system (Clontech Laboratories, Inc.) as described elsewhere (28). Melanoma, colorectal cell lines or 293T cells were transfected with 4–6μg of Tet-Off and 2–3μg of pBI-G response plasmid expressing the mRNA transcripts of interest. Transcription was stopped by adding doxycycline (1 mg/ml) into media 48hr after co-transfection. Cells were harvested at various time points after treatment and total RNA was extracted as described above. mRNA levels were analyzed using qRT-PCR.
Antibodies and Immunoprecipitation Techniques
Mouse anti-FLAG M2 antibody (Sigma- Aldrich, St. Louis, MO), anti--actin and anti-MITF (Santa Cruz Biotechnology, Inc., Dallas, TX) primary antibodies and secondary antibodies conjugated with horseradish peroxidase (Chemicon, Billerica, MA) were all purchased. Immunoblotting procedures are described elsewhere (32).
Protein Purification
To obtain whole-cell lysates for western blot analysis, cells were lysed using denaturing RIPA buffer containing PBS (pH 7.4), 0.5% sodium deoxycholate, 0.1% SDS, 1% (v/v) NP-40, 100mM sodium orthovanadate and protease inhibitor cocktail (Sigma-Aldrich). Protein extracts for UV cross-linking reactions were made in non-denaturing lysis buffer (10mM HEPES [pH 7.6], 3mM MgCl2, 40mM KCl, 2mM DTT, 5% [v/v] glycerol, 0.5% [v/v] IGEPAL and protease inhibitor cocktail).
Preparation of RNA Substrates for UV Cross-
link Analysis
Plasmids containing full length MITF mRNA or fragments of its coding region under the control of the T7 promoter were linearized with XbaI (New England Biolabs), purified with phenol-chloroform and used for in vitro transcription using [32P]UTP RNA and the Riboprobe in vitro transcription system (Promega). Radio-labeled RNA substrates were gel purified before use in UV cross-link reactions.
UV Cross-link Reaction
Analysis of RNA-protein complexes was performed as described before (28). Briefly, protein extracts (20μg) and internally labeled RNA (1.5–2 X 106 cpm) were incubated in 20μL of reaction buffer (non-denaturing lysis buffer without protease inhibitors) for 30 min at room temperature. After incubation, RNA-protein complexes were crosslinked with a 30 min exposure to 254 nm UV light, treated with RNaseA and RNaseT1, incubated with anti-FLAG antibodies for 6hr and, lastly, incubated with M2-
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agarose beads overnight. Immunoprecipitates were washed six times in lysis buffer, boiled and separated using a 10% SDS-PAGE gel. Lastly, the gel was dried and exposed to X-ray film for 3–14 days.
Luciferase Reporter Assay
Melanoma cells were transfected with the previously described shRNA constructs (28) or miR-sponge constructs, pSV40 β-gal and pHTRPL4 plasmid (33,34) or p-BI-GL-MITFwt or p-BI-GL-MITFmiR-340 plasmids. 48hr after transfection luciferase activity was measured using the luciferase reporter assay according to the manufacturer’s protocol (Promega).
Senescence-associated β-galactosidase staining
Cells were fixed for 20 min using a solution of 20% formaldehyde and 2% glutaraldehyde, washed with PBS and then stained with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) solution overnight at 37°C. Cells stained blue were counted under a microscope (20X) and the percent of stained cells was estimated.
RESULTS
Melanoma cell lines preferentially express MITF mRNA with a short 3’-UTR
As discussed earlier, several full length cDNA isoforms of MITF-M with the same coding sequence exist, but the isoforms vary in their 3’-UTR length. Separate previous reports demonstrate MITF mRNA is regulated by two different miRNAs, however the studies used two different MITF mRNA isoforms. The study by Bemis LT et al. (20) showed a functional miR-137 binding site in the 3’-UTR using a full length cDNA containing 1,143 bp of the 3’-UTR sequence (termed hereafter as the medium 3’-UTR) (Figure 1a). In a different study, Segura MF et. al. (19) used the full length cDNA with a 3’-UTR length of ~ 3kb (termed as the long 3’-UTR) in order to find a functional miR-182 site (Figure 1a). We obtained the full length MITF cDNA clone having a 3’-UTR ~ 0.57kb in length, which we termed the MITF mRNA with a short 3’-UTR (Figure 1a). The finding of one mRNA with different 3’-UTRs being regulated by different
miRNAs led us to investigate the abundance of the various MITF mRNA isoforms in several melanoma cell lines. We designed primers unique to the short, medium and long 3’-UTRs and, using qRT-PCR, evaluated the relative abundance of the distinct mRNA isoforms in four different melanoma cell lines versus NHMs (Figure 1b). The results showed that in NHMs the levels of all three isoforms were comparable. But in all four melanoma cell lines tested, the relative proportion of MITF mRNA with a short 3’-UTR predominated. This suggested fast proliferating melanoma cells preferentially express MITF mRNA with a shorter 3’-UTR.
miRNA regulates the short 3’-UTR MITF
mRNA isoform
As discussed before, miRNAs act on MITF mRNAs with medium and long 3’-UTRs. As we found the mRNA isoform with the short 3’-UTR most prevalent in melanomas, we investigated whether this isoform is also being regulated by miRNAs. We measured the half-life of the short 3’UTR MITF mRNA in cells defective in Dicer1 (DicerEx5/Ex5) function and, thus, deficient in miRNA maturation (27). In all three Dicer1Ex5/Ex5 hypomorphic cell lines examined, the half-life of short 3’-UTR MITF mRNA was 2 to 3- fold higher compared to their normal counterpart (DicerWT) where functional miRNAs were present (Figure 2a-c). This indicated that miRNAs also regulate the MITF isoform with a short 3’-UTR. Next, we used a bioinformatics approach (http://www.microrna.org/microrna/home.do) to determine possible miRNA binding sites in this region. Two miRNAs, miR-340 and miR-548c-3p, with sites located in the short 3’-UTR of MITF mRNA were selected for further investigation (Figure 2d). To inhibit the function of these miRNAs in melanoma cells we used miR-sponge constructs as described previously (29) for both miR-340 and miR-548c-3p. Our results show the amount of short 3’-UTR MITF mRNA increased significantly only when the function of miR-340 was inhibited (Figure 2e). Moreover, inhibition of miR-340 function in 451Lu cells also increased MITF protein levels (Figure 2f). These data demonstrate that MITF mRNA with a short 3’-UTR is regulated by miR-340.
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miR-340 destabilizes MITF mRNA
After initial observations regarding miR-340 involvement in the regulation of MITF mRNA, we set out to investigate whether miR-340 regulates the turnover of MITF mRNA. The result shows a two-fold increase in mRNA half-life upon miR-340 inhibition (Figure 3a) indicating miR-340 is responsible for the destabilization of MITF mRNA. We further tested the efficacy of miR-340 action using a reporter construct. We cloned the short 3’-UTR of MITF mRNA just after the luciferase coding region into pBI-GL (Clontech Laboratories, Inc.). Inhibition of miR-340 function in melanoma cell lines significantly increased the half-life of the chimeric RNA as well as luciferase enzyme activity (Figure 3b,c). This finding suggested the short 3’-UTR of MITF mRNA was sufficient for miR-340-mediated inhibition. Next, both sites of miR-340 were deleted from the full length MITF mRNA as well as from the reporter construct to generate corresponding mutant versions. The half-life of the deleted full length MITF mRNA increased significantly (Figure 3d). The luciferase activity of the mutated chimeric mRNA was also increased about two and a half-fold, comparable to the outcome when miR-340 function was inhibited (Figure 3c). On the other hand, miR-340 inhibition had no additional effect on the luciferase activity of mutant mRNA (Figure 3c). Together these data suggest miR-340 acts through its target sites on the 3’-UTR of MITF mRNA leading to its destabilization.
CRD-BP stabilizes MITF mRNA
CRD-BP is a multifunctional mRNA binding protein that modulates the stability, localization and translation of several RNAs (c-myc, IGF-II, H19, CD4, MDR-1, Gli1, etc.) (35-42). As discussed earlier, previous research shows CRD-BP protects another mRNA, TrCP1, from miRNA mediated destabilization (21). Moreover, CRD-BP is over-expressed in melanoma cell lines and human melanoma samples, thus, contributing toward oncogenesis (24). Here we show knockdown of CRD-BP in 451Lu cells decreased the level of endogenous MITF mRNA by more than five-fold (Figure 4a). Further, when CRD-BP was knocked down, MITF-dependent luciferase activity was drastically reduced (Figure 4b). On
the other hand, the levels of endogenous MITF mRNA were increased three-fold when CRD-BP was over-expressed in NHMs (Figure 4c). Over-expression of CRD-BP in NHMs also resulted in the significant inhibition of senescence, consistent with the function of MITF during cell cycle progression in melanocytes (Figure 4d). Interestingly, the effect of CRD-BP on melanocyte senescence was similar to the effect of a powerful senescence inhibitor, hTERT (Figure 4d). Together these data indicate CRD-BP regulates MITF expression as well as its function. CRD-BP over-expression also dramatically increased the half-life of MITF mRNA (Figure 4e) indicating MITF expression is regulated post-transcriptionally by CRD-BP. This result underscores previous reports of how CRD-BP regulates its other target mRNAs. Lastly, to test whether this regulation is mediated through a direct interaction of CRD-BP protein with MITF mRNA, we performed a UV cross-linking and immunoprecipitation experiment using radio-labeled fragments or full length MITF mRNA with protein extracts from FLAG-CRD-BP expressing cells. The results show CRD-BP directly interacts with the short 3’-UTR of MITF mRNA (Figure 4f). Altogether, these experiments demonstrate CRD-BP directly interacts with MITF mRNA and stabilizes it.
CRD-BP interferes with miR-340 function
resulting in the stabilization of MITF mRNA
Based on the results that CRD-BP directly interacts with the short 3’-UTR of MITF mRNA, we investigated whether CRD-BP prevents miR-340-dependent regulation of MITF mRNA in a similar fashion to what was reported earlier for different mRNAs (21). Munro et al. characterized the sequence motif required for IMP binding in Drosophila as UUUAY (43) and we found this motif either within or juxtaposed to both miR-340 binding sites. Subsequently, when CRD-BP was over-expressed the half-life of MITF mRNA increased similar to the effect on MITF mRNA when miR-340 function was inhibited (Figure 5a). However, CRD-BP over-expression could not further increase the half-life of short 3’-UTR MITF mRNA with concurrent inhibition of miR-340 function, as miR-340 inhibition already stabilized the mRNA (Figure 5a). Similarly, when
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miR-340 function was inhibited there was an increase in the endogenous MITF mRNA level which was not further altered due to CRD-BP overexpression (Figure 5b). Further, overexpression of CRD-BP increased the levels of MITF mRNA in a dose-dependent manner, but failed to do so when miR-340 was also ectopically overexpressed (Figure 5c). Lastly, knock down of CRD-BP decreased MITF-dependent transcription by more than two-fold, but had no effect when miR-340 function was already inhibited (Figure 5d). These data suggest CRD-BP exerts its effect on MITF mRNA by preventing the access of miR-340 to its sites. When miR-340 action is blocked, the mRNA is stable by itself and becomes unresponsive to the availability of CRD-BP. In order to further confirm the mechanism of CRD-BP interference with the function of miR-340, we measured the half-life of MITF mRNAs where miR-340 sites were deleted. We found the half-life of mutated MITF mRNA increased significantly compared to wild type MITF mRNA and over-expression of CRD-BP had no effect on the stability of MITF mRNAs lacking miR-340 binding sites (Figure 5e). The finding that CRD-BP could stabilize MITF mRNA only when miR-340 is functional demonstrates that CRD-BP acts by blocking miR-340-dependent regulation of MITF mRNA. Additionally, ectopic expression of CRD-BP does not affect the levels of miR-340 and over-expression of miR-340 failed to change CRD-BP levels (Figure 5f-g) ruling out the possibility that the observed changes to MITF expression are due to the effects of CRD-BP and miR-340 on each other. To analyze the effect of miR-340 inhibition on cell proliferation we performed colony formation assays and found that inhibition of miR-340 function using specific sponge constructs resulted in a significant increase in the number of colonies formed by both 928 mel and 451Lu melanoma cells (Figure 5h). A similar increase was detected when CRD-BP was over-expressed, but no additive effect on colony formation was observed when CRD-BP and sponge-340 were co-expressed. As expected, knockdown of CRD-BP resulted in a drastic inhibition in the number of colonies; however inhibition of miR-340 rescued the effect of CRD-BP knockdown on the growth of 928 mel and 451Lu melanoma cells (Figure 5h).
DISCUSSION
MITF is widely regarded as the master regulator of melanocyte biology because of its involvement in the regulation of important melanogenic proteins as well as its contribution towards melanoblast survival, melanocyte lineage commitment and melanoma pathogenesis (reviewed in (44)). Therefore, it is the importance of MITF which potentiates the necessity to study its regulatory mechanisms. Post-transcriptional regulation of gene expression involving RBPs is pivotal in regulating the expression of several genes and the discovery of miRNAs added a new dimension to this regulatory scheme. MITF is also post-transcriptionally regulated and two recent studies show evidence that miR-137 and miR-182 target MITF mRNA (19,20). In the context of this study, we set out to investigate the post-transcriptional regulation of MITF mRNA expression in further detail.
One key mechanism underlying the process of
malignant transformation is the activation of proto-oncogenes. Loss of miRNA binding sites from the 3’-UTRs of oncogenic mRNAs is considered an important mode of oncogene activation (45,46). This was supported by recent observations demonstrating cancer cells and other proliferating cells favor the expression of mRNA isoforms containing shorter 3’-UTRs in order to escape miRNA-mediated regulation (22,23). MITF mRNA also has several isoforms with different length 3’-UTRs and, most interestingly, the isoform with a short 3’-UTR does not have binding sites for the previously reported miRNAs (Figure 1a). Here, we show melanoma cells preferentially express MITF mRNA isoforms with short 3’-UTRs (Figure 1b) and comply with other fast proliferating cells in terms of the regulation of oncogenic mRNAs. This finding, thus, designates the MITF mRNA isoform with a short 3’-UTR as the most prevalent isoform among melanoma cell lines. Moreover, our results show that although the MITF mRNA isoform with a short 3’-UTR escapes regulation by previously reported miRNAs, it is still being regulated by a different miRNA, miR-340. Interaction of miR-340 with its two target sites present on the short 3’-UTR of MITF mRNA results in the destabilization of MITF mRNA and a decrease in MITF expression
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and transcriptional activity (Figures 2, 3 and 4b). Interestingly, miR-340 was found to be expressed in primary melanoma cell lines suggesting its importance in the regulation of melanoma-specific target mRNAs (47). Since the fragment containing the miR-340 binding sites is part of all three known MITF 3’-UTRs, the regulation of MITF by miR-340 appears to be independent of APA and therefore more universal (shared by both melanocytes and melanoma cells).
Results of this study establish CRD-BP as an important positive regulator of MITF expression and function. CRD-BP stabilizes MITF mRNA and increases MITF expression as well as its transcriptional activity (Figure 4b). This effect of CRD-BP is mediated by counteracting the miR-340-mediated degradation of MITF mRNA (Figure 5). This is not surprising as we demonstrated previously that CRD-BP also interferes with miR-183 function resulting in the stabilization of TrCP1 mRNA (21). Our findings here are in line with recent reports describing an interplay between RBPs and miRNAs, where RBPs interfere with miRNA function, thereby,
highlighting a novel mode of post-transcriptional regulation of gene expression (Figure 6) (48,49).
CRD-BP is important for the growth and survival of many types of cancer cells (28,42,50). It is reported to be involved in mammalian development and linked to effects on cellular adhesion and invasion taking place during development and malignancy (39,51). These effects of CRD-BP are attributed to its regulation of different target mRNAs (c-myc, TrCP1, Gli1, etc.). We reported previously that CRD-BP is over-expressed in a majority of malignant melanomas (24). However, knockdown of CRD-BP in melanoma cells had a much more robust effect than in other tumor cells and thus it was hypothesized an additional melanoma-specific factor must be involved. Data provided in this manuscript suggest the regulation of MITF by CRD-BP may contribute to the observed effects of CRD-BP on melanoma survival and progression as well as indicate CRD-BP is a potential target for the prevention and treatment of this deadly disease.
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FOOTNOTES
* We thank Drs. K. Kinzler, P. Robbins, J. Ross, P. Sharp, T. Tuschl and B. Vogelstein for their generous gifts of reagents and Dr. N. Sanek for help with data collection. This work was supported by NCI grants AR063361 and CA121851 (to V.S.S.), CA12509 (to V.S.) and by NIH GM076244 (to Y. G).
The abbreviations used are: APA, Alternative cleavage and polyadenylation; CRD-BP; Coding
Region Determinant-Binding Protein; MITF, Microphthalmia-associated transcription factor; miRNA, miR, microRNA; NHMs, normal human melanocytes.
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FIGURE LEGENDS Figure 1: MITF mRNA with a short 3’-UTR is more abundant in melanoma cell lines. A, Graphical representation of different MITF mRNA isoforms with varying 3’-UTR lengths.
Approximate position of reported miRNA target sites are shown on long and medium 3’-UTRs. B, Expression of MITF mRNA isoforms containing various 3’-UTR lengths evaluated by qRT-PCR
using primers specific for the long, medium and short 3’-UTR. Figure 2: The abundant short 3’-UTR MITF mRNA is also regulated by miRNA. A, Dicerwt and DicerEx5/Ex5 HCT116 cells were co-transfected with Tet–Off plasmid and p-BI-G-MITF
plasmid with a short 3’-UTR. Transcription was stopped by treatment with doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with qRT-PCR and normalized to GAPDH expression.
B, p-BI-G-MITF plasmid with short 3’-UTR was expressed in DLD1 cells, Dicerwt and DicerEx5/Ex5, under the control of the Tet-Off system. The stability of MITF mRNA was analyzed as in panel A.
C, The stability of MITF transcripts with short 3’-UTR expressed in RKOwt and RKO DicerEx5/Ex5 cells was analyzed as in panel A.
All results are representative of three separate experiments and expressed as the mean ± S.D. (error bars). The average half-lives of mRNAs are presented in Table 3.
D, Sequence of the short 3’-UTR of MITF showing binding sites for miR-340 (in bold) and miR-548c-3p (underlined).
E, The levels of endogenous MITF mRNA in 451Lu cells, transfected with the indicated miR-sponge constructs, were estimated by qRT-PCR after normalization to GAPDH expression. Results are representative of three separate experiments and presented in percentage to control (SP-CXCR4) as the mean ± S.D. (error bars).
F, Immunoblot analysis of MITF expression in 451Lu cells transfected with indicated miR-sponge constructs (upper panel). Lower panel shows -actin expression.
Figure 3: MITF mRNA is a target of miR-340. A, 451Lu cells were co-transfected with Tet–Off plasmid, p-BI-G-MITF plasmid with short 3’-UTR
and indicated miR-sponge construct. Transcription was stopped by treatment doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with qRT-PCR, normalized to GAPDH expression.
B, 451Lu cells were co-transfected with Tet–Off plasmid, p-BI-GL-MITF-Short 3’-UTR and indicated miR-sponge construct. The turnover of chimeric Luciferase-MITF-3’-UTR transcripts was analyzed as in panel A.
C, 451Lu cells were co-transfected with -Gal expressing plasmid, Tet–Off plasmid, p-BI-GL-MITFwt or p-BI-GL-MITFmiR-340 and indicated miR-sponge construct. After 24hrs luciferase activity was measured. Values represent luciferase activity normalized to -gal.
D, 451Lu cells were co-transfected with Tet–Off plasmid, p-BI-G-MITFwt or p-BI-G-MITFmiR-340. The stability of MITF transcripts was analyzed as in panel A.
All results are representative of three separate experiments and expressed as the mean ± S.D. (error bars). The average half-lives of mRNAs are presented in Table 3.
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Figure 4: CRD-BP is a positive regulator of MITF expression. A, 451Lu cells were transfected with either control shRNA or shRNA against CRD-BP. 48hrs after
transfection cells were collected and assayed for levels of MITF mRNA by qRT-PCR, normalized to GAPDH expression and presented in percentage to control (scrambled shRNA).
B, 1241 Mel and Mel IM cells were co-transfected with indicated shRNA expressing plasmids, -Gal expressing plasmid and pHTRPL4, where the luciferase gene is expressed under an MITF-dependent promoter. Values represent luciferase activity normalized to -Gal expression.
C, NHMs were electroporated using the AMAXA Nucleofector™ with either CRD-BP over-expressing plasmid or empty vector. 72hrs after transfection cells were collected and assayed for levels of MITF mRNA by qRT-PCR, normalized to GAPDH expression and presented in percentage to control (pBABE).
D, NHMs were electroporated using the AMAXA Nucleofector™ with the indicated plasmids. 48hrs after electroporation, cells were stained for senescence-associated -gal and the percentage of β-gal positive cells were calculated.
E, 451Lu cells were co-transfected with Tet–Off plasmid, p-BI-G-MITF and either pcDNA control or CRD-BP over-expressing plasmid. The turnover of MITF transcripts was analyzed by qRT-PCR after stopping transcription by doxycycline treatment for the indicated time points. Normalization was done with respect to GAPDH expression.
All results are representative of three separate experiments and expressed as the mean ± S.D. (error
bars). The average half-lives of mRNAs are presented in Table 3. F, FLAG immunoprecipitation of UV cross-linked RNP complexes. Protein extracts from 293T cells
transfected with FLAG-CRDBP were incubated with internally [32P]-labeled RNA from three fragments of the MITF mRNA coding region, the full-length MITF mRNA and the short 3’-UTR. Fragment 1 contains nucleotide 1 to nucleotide 421 of the coding region, fragment 2 consists of nucleotides 422-841 and fragment 3 consists of nucleotide 842-1260 of the MITF coding region. RNP complexes were precipitated with anti-FLAG antibodies and analyzed on PAGE and autoradiographed.
Figure 5: CRD-BP counteracts miR-340 action and stabilizes MITF mRNA. A, Wild type MITF expressing plasmid p-BI-G-MITFwt was co-transfected with Tet–Off plasmid and
the indicated constructs in 451Lu cells. The turnover of MITF transcripts was analyzed as Fig 4E. B, 451Lu cells were transfected with indicated constructs. 48hrs after transfection cells were collected
and assayed for levels of endogenous MITF mRNA by qRT-PCR, normalized to GAPDH expression and presented in percentage to control.
C, 451Lu cells were transfected with either control pcDNA 3.1, CRD-BP over-expressing, control pCMV-MIR or overexpressing miR-340 pCMV-MIR340 plasmids as indicated. 48hrs after transfection cells were collected and assayed for levels of MITF mRNA by qRT-PCR, normalized to GAPDH expression and presented in percentage to control.
D, 451Lu cells were co-transfected with MITF-dependent luciferase expressing vector pHTRPL4, indicated shRNA, miR-sponge expressing plasmids and -Gal expressing plasmid. Values correspond to luciferase activity normalized to -Gal expression.
E, Construct expressing MITF with both of the miR-340 sites deleted (p-BI-G-MITFmiR-340) was co-transfected with Tet–Off plasmid and the indicated constructs in 451Lu cells. The turnover of MITF transcript was analyzed as Fig 4E.
All results are representative of three separate experiments and expressed as the mean ± S.D. (error bars). The average half-lives of mRNAs are presented in Table 3.
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F, 451Lu cells were transfected as indicated. 48hrs after transfection cells were collected and assayed for levels of mature miR-340 transcript by ddPCR. Results are presented as the number of copies per 1μg of total RNA.
G, 451Lu cells were transfected as indicated. 48hrs after transfection cells were collected and assayed for levels of CRD-BP mRNA by qRT-PCR, normalized to GAPDH expression and presented as fold-change compared to control.
H, 451Lu and 928 Mel cells were co-transfected with the indicated constructs and pTk-Puro. The colonies were selected for puromycin resistance, stained with crystal violet and counted.
Figure 6: Model for Interference of miR-340 Function by CRD-BP. A, In the absence of CRD-BP, the miR-340-guided miRISC interacts with the 3’UTR of MITF
mRNA and accelerates its degradation. B, When CRD-BP is present; it binds to the 3’UTR of MITF mRNA in the vicinity of miR-340
binding sites, shielding the MITF mRNA from miR-340-mediated down-regulation. The resulting elevated levels of MITF contribute to melanoma cell proliferation.
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Table 1 Name of the Oligo Sequence (5’ 3’) Fragment 1 FWD ATGCTGGAAATGCTAGAATAT Fragment 1 REV CAGGATCCATCAAGCCCAAG Fragment 2 FWD CTTTGCAAATGGCAAATACG Fragment 2 REV AATGCCGGTTGGCGTGCTCC Fragment 3 FWD TGTTGCTCAGAATACAGGAAC Fragment 3 REV CTAACAAGTGTGCTCCGTCTC 3’-UTR FWD CGAATCCTCCCTGCACTGC 3’-UTR REV ACAGAATACATATTTCTTTAAATAG pBI-GL SENSE TTACAATTTGGACTTTCCGCCC pBI-GL ANTI-SENSE AATGTAACTGTATTCAGCGATGAC miR-340 Site I Del SENSE ATAGCCCAGGATATATTTTATTTTTAG miR-340 Site I Del ANTI-SENSE TTCATATTAAAGGAAAATTATCAAGAAAAC miR-340 Site II Del SENSE TGCAAACTATTTAAAGAAATATGTATTCTG miR-340 Site II Del ANTI-SENSE GGGTACTGTATCTTTAAGGCAG sponge-340 DNA oligonucleotide TCGAGAATCAGTCTCTACCTTTATAACCGGAATCAGT
CTCTACCTTTATAACCGGAATCAGTCTCTACCTTTATAACCGGAATCAGTCTCTACCTTTATAACCGGAATCAGTCTCTACCTTTATAACCGGAATCAGTCTCTACCTTTATAACCGGAATCAGTCTCTACCTTTATAAGGGCC
sponge-548c-3p DNA oligonucleotide
TCGAGGCAAAAGTAAAACGATTTTTGCCGGGCAAAAGTAAAACGATTTTTGCCGGGCAAAAGTAAAACGATTTTTGCCGGGCAAAAGTAAAACGATTTTTGCCGGGCAAAAGTAAAACGATTTTTGCCGGGCAAAAGTAAAACGATTTTTGCCGGGCAAAAGTAAAACGATTTTTGGGGCC
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Table 2
Name of the Oligo Sequence (5’ 3’) GAPDH FWD ATGGTTGCCACTGGGGATCT GAPDH REV TGCCAAAGCCTAGGGGAAGA MITF FWD CCAGGCATGAACACACATTC MITF REV GCAGACCTTGGTTTCCATAAAG Long 3’-UTR Detection FWD CCAAGAGGCAGTGGTTTGGG Long 3’-UTR Detection REV AACCAAATGCTTTAATGAGGCTATC Medium 3’-UTR Detection FWD GAAAACCGAACTGGGCATATTTC Medium 3’-UTR Detection REV GGTTATCAATCTCCAAGAATATTGC Short 3’-UTR Detection FWD CGAATCCTCCCTGCACTGC Short 3’-UTR Detection REV ACAGAATACATATTTCTTTAAATAG Stem-loop RT primer for miR-340 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG
GATACGACAATCAGTCT Forward primer for miR-340: GCGGCGGTTATAAAGCAATG Universal reverse primer GCGGGTGCAGGGTCCGAGGT
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Table 3
Figure Group Average half-life (hrs) 2A Wt 2.5 2A Ex5 7.1 2B Wt 2.7 2B Ex5 5.6 2C Wt 2.6 2C Ex5 5.6 3A Sponge CXCR4 3.2 3A Sponge-340 6.6 3B Sponge CXCR4 2.4 3B Sponge 340 6.8 3D WT 2.8 3D 340 mutant 5.1 4E pcDNA 2.8 4E CRD-BP 14 6A Control 2.5 6A Sponge-340 5.4 6A CRD-BP 5.4 6A CRD-BP + Sponge-340 5.3 6E Control 6.4 6E Sponge-340 7.8 6E CRD-BP 6.8 6E CRD-BP + Sponge-340 7.6
The half life of mRNA (T1/2) was calculated as follows: T1/2= T x log 2 . log AmtB/AmtE Where: T1/2=Half Life of mRNA T = Elapsed Time after addition of Doxycycline AmtB = Average Beginning Amount of mRNA AmtE = Average Ending Amount of mRNA
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Coding sequence 3’-UTR
miR-182 binding sites
miR-137 binding site
MITF- Long- 3’-UTR
MITF- Medium- 3’-UTR
MITF- Short- 3’-UTR
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Sp-CXCR4 Sp- 340 Sp-548c-3p
6%
13%
25%
50%
100%
0 4 8
wt
ex5/ex5
6%
13%
25%
50%
100%
0 4 8
wt
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0 4 8
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HCT116 DLD 1 RKO
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Figure 2
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E F
MITF
-actin
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Sp
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Sp
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c3p
cgaauccucccugcacugcauucgcacaaacugcuuccuuucuugauccguagauuuaauaacuuaccugaagggguuuucuugauaauuuuccuuuaauaugaaauuuuuuuucaugcuuuaucaauagcccaggauauauuuuauuuuuagaauuuugugaaacagacuuguauauucuauuuuacaacuacaaaugccuccaaaguauuguacaaauaagugugcaguaucugugaacugaauucaccacagacuuuagcuuucugagcaagaggauuuugcgucagagaaaugucuguccauuuuuauucaggggaaacuugauuugagauuuuuaugccugugacuuccuuggaaaucaaauguaaaguuuaauugaaagaauguaaagcaaccaaaaagaaaaaaaaaaaagaaagaaagaggaaaagaaauccauacuaacccuuuuccauuuuauaaauguauugauucauugguacugccuuaaagauacaguaccccucuagcuuuguuuagucuuuauacugcaaacuauuuaaagaaauauguauucuguaaaaaaaaaaaaaaa
B C
hr hr hr
% o
f m
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A r
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pB
IGL
-MIT
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CX
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IGL
-MIT
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T +
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pB
IGL
-MIT
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UT
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IGL
-MIT
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100%
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WT
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6%
13%
25%
50%
100%
0 4 8
Sponge CXCR4
Sponge-340
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25%
50%
100%
0 4 8
Sponge CXCR4
Sponge-340
Lu
cife
rase
Act
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Figure 3
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C D
hr
hr hr
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pBABE pBABE-CRD-BP
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50%
100%
0 4 8
pcDNA
CRD-BP
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20%
40%
60%
80%
100%
120%
scrambledshRNA
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A B
C
Figure 4
Lu
cife
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E F
~ 65 kD
3’U
TR
(12
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822)
Fu
ll L
eng
th (
1-18
22)
1-42
1
422-
841
842-
1260
Coding region
hr
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10
20
30
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pBABE pBABE-hTERT
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f m
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25%
50%
75%
100%
125%
1241 Mel Mel IM
ScrambledshRNA
MIT
F m
RN
AM
ITF
mR
NA
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0%100%200%300%400%500%600%
pcD
NA
/S
po
ng
e-C
XC
R4
pcD
NA
/S
po
ng
e-34
0
CR
DB
P /
Sp
on
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CR
4
CR
DB
P /
Sp
on
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340
25%
50%
100%
0 4 8
hr
pcDNA + Sponge CXCR4
pcDNA + Sponge 340
CRDBP + Sponge CXCR4
CRDBP + Sponge 340
6%
13%
25%
50%
100%
0 4 8
hr
pcDNA + Sponge CXCR4pcDNA + Sponge 340CRDBP + Sponge CXCR4CRDBP + Sponge 340
A BFigure 5
0
100
200
300
400
500
scra
mb
led
sh
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A+
Sp
on
ge
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CR
4
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Act
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2
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Fo
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0
100
200
300
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600
700
800
Control (pcDNA/ Sponge
CXCR4 /shscrambled)
CRDBP /Sponge-CXCR4
pcDNA /Sponge-340
CRDBP /Sponge-340
sh CRDBP /Sponge-CXCR4
sh CRDBP /Sponge-340
928 mel 451Lu
Nu
mb
er o
f co
lon
ies
per
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teF G
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Figure 5
200
250
300
350
400
450
500
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ge
Exp
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D-B
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012345
Control CRD-BP miR-340
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Yevgenya Grinblat, Vijayasaradhi Setaluri and Vladimir S. SpiegelmanSrikanta Goswami, Rohinton S. Tarapore, Ashley M. Poenitzsch Strong, Jessica J. TeSlaa,
(CRD-BP)Factor (MITF) mRNA is Inhibited by Coding Region Determinant Binding Protein
MicroRNA-340-Mediated Degradation of Microphthalmia-Associated Transcription
published online November 20, 2014J. Biol. Chem.
10.1074/jbc.M114.590158Access the most updated version of this article at doi:
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