Coding Region Determinant-binding Protein Research Articles

CRDBP Protein Phase Separation and Its Recruitment to β-Catenin Protein in a Single Living Cell.

The Coding Region Determinant-Binding Protein (CRDBP) is a carcinoembryonic protein, and it is overexpressed in various cancer cells in the form of granules. We speculated the formation of CRDBP granules possibly through liquid-liquid phase separation (LLPS) processes due to the existence of intrinsically disordered regions (IDRs) in CRDBP. So far, we did not know whether or how phase separation processes of CRDBP occur in single living cells due to the lack of in vivo methods for studying intracellular protein phase separation. Therefore, to develop an in situ method for studying protein phase separation in living cells is a very urgent task. In this work, we proposed an efficient method for studying phase separation behavior of CRDBP in a single living cell by combining in situ fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) with a fluorescence protein fusion technique. We first predicted and confirmed that CRDBP has phase separation in solution by conventional fluorescence imaging and FCS methods. And then, we in situ studied the phase separation behaviors of CRDBP in living cells and observed three states of CRDBP phase separation such as monomer state, cluster state, and granule state. We studied the effects of CRDBP truncated forms and its inhibitor on the CRDBP phase separation. Furthermore, we discovered the recruitment of CRDBP to β-catenin protein in living cells and investigated the effects of CRDBP structures and inhibitor on CRDBP recruitment behavior. This finding may help us to further understand the mechanism of CRDBP protein for regulating Wnt signaling pathway. Additionally, our results documented that FCS/FCCS is an efficient and alternative method for studying protein phase separation in situ in living cells.

In Situ Study of Interactions between Endogenous c-myc mRNA with CRDBP in a Single Living Cell by Combining Fluorescence Cross-Correlation Spectroscopy with Molecular Beacons.

mRNA-protein interactions play key roles in facilitating various biological functions in gene expression regulations and even the progression of diseases. However, it is still a challenge to directly monitor mRNA-protein interactions in a single living cell at present. Herein, we propose a new strategy for real-time studying of mRNA-protein interactions in a single living cell using fluorescence cross-correlation spectroscopy (FCCS) and molecular beacon (MB) labeling techniques. The c-myc mRNA and coding region determinant binding protein (CRDBP) were used as models. We first evaluated the performances of unmodified (2'-deoxy) and modified (2'-O-methyl) MBs and found that the 2'-O-methyl loop MB (2'-O-methyl loop domain, 2'-deoxy stem region) has high affinity to target mRNA and good nuclease resistance. Then we constructed stable cell line expressing mCherry-CRDBP using lentivirus infection, and on the basis of FCCS, we established an efficient method for quantifying the interaction of c-myc mRNA with CRDBP in a single living cell. The RNA binding domains of CRDBP cover two RNA recognition motifs (RRM) and four K homologies (KH). Furthermore, we constructed the truncated variants and point mutants on RNA binding domains of CRDBP, systematically studied the effects of RNA binding domains of CRDBP on c-myc mRNA-CRDBP interaction in living cells, and found that KH3-4 is indispensable for c-myc mRNA binding, KH1-2 plays a supplementary role, and RRM1-2 shows no binding ability to c-myc mRNA. Our work reveals the mechanisms of c-myc mRNA-CRDBP interactions and provides a general strategy for quantifying the interactions of endogenous mRNA with protein in a single living cell.

MYC is downregulated by a mitochondrial checkpoint mechanism.

The MYC proto-oncogene serves as a rheostat coupling mitogenic signaling with the activation of genes regulating growth, metabolism and mitochondrial biogenesis. Here we describe a novel link between mitochondria and MYC levels. Perturbation of mitochondrial function using a number of conventional and novel inhibitors resulted in the decreased expression of MYC mRNA. This decrease in MYC mRNA occurred concomitantly with an increase in the levels of tumor-suppressive miRNAs such as members of the let-7 family and miR-34a-5p. Knockdown of let-7 family or miR-34a-5p could partially restore MYC levels following mitochondria damage. We also identified let-7-dependent downregulation of the MYC mRNA chaperone, CRD-BP (coding region determinant-binding protein) as an additional control following mitochondria damage. Our data demonstrates the existence of a homeostasis mechanism whereby mitochondrial function controls MYC expression.

Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction.

Coding region determinant-binding protein (CRD-BP) binds to the 3’-UTR of microphthalmia-associated transcription factor (MITF) mRNA to prevent its targeted degradation by miR-340. Here, we aim to further understand the molecular interaction between CRD-BP and MITF RNA. Using point mutation in the GXXG motif of each KH domains, we showed that all four KH domains of CRD-BP are important for their physical association with MITF RNA. We mapped the CRD-BP-binding site in the 3’-UTR of MITF RNA from nts 1330–1740 and showed that the 49-nt fragment 1621–1669 is the minimal size MITF RNA for binding. Upon deletion of nts 1621–1669 within the nts1550-1740 of MITF RNA, there was a 3-fold increase in dissociation constant Kd, which further confirms the critical role sequences within nts 1621–1669 in binding to CRD-BP. Amongst the eight antisense oligonucleotides designed against MITF RNA 1550–1740, we found MHO-1 and MHO-7 as potent inhibitors of the CRD-BP-MITF RNA interaction. Using RNase protection and fluorescence polarization assays, we showed that both MHO-1 and MHO-7 have affinity for the MITF RNA, suggesting that both antisense oligonucleotides inhibited CRD-BP-MITF RNA interaction by directly binding to MITF RNA. The new molecular insights provided in this study have important implications for understanding the oncogenic function of CRD-BP and development of specific inhibitors against CRD-BP-MITF RNA interaction.

Abstract B22: Inhibition of the coding region determinant-binding protein (CRD-BP) to sensitize colorectal cancer cells to drug

Abstract This study aims at targeting CRD-BP to sensitize colorectal cancer cells to chemotherapeutics. Colorectal cancer is the third most common cancer in both men and women. An estimated 95,270 cases of colon cancer and 39,220 cases of rectal cancer are expected to be diagnosed in 2016. Treatment for colorectal cancer may involve surgery, chemotherapy, biological therapy, radiation therapy, or a combination of treatments. The choice of treatment of colorectal cancer depends mainly on the location of the tumor and the stage of the disease. Chemotherapy is used for advanced cancers; however advanced colorectal cancers are notoriously resistant to drugs. Two major mechanisms employed by colorectal cancer cells are thought to be responsible for insensitivity to therapeutics: i) resistance to apoptosis usually achieved by activation of anti-apoptotic pathways; ii) induction of multidrug resistance (MDR) membrane transporters that pump drugs out of the cells. Targeting factors involved in these mechanisms would overcome the resistance of colorectal cancer cells to drugs. Constitutive activation of the Wnt/β-catenin signaling pathway is one of the central drivers of the development of colorectal cancer. We have identified CRD-BP as a bona fide transcriptional target of Wnt/β-catenin signaling pathway. Induction of CRD-BP is responsible for a variety of pleiotropic effects of Wnt/β-catenin signaling pathway in human colorectal cancer cells such as up-regulation of c-myc and β-TrCP1, activation of NF-κB pathways via accelerated degradation of IκB, and inhibition of apoptosis. More over CRD-BP was shown to regulate the multidrug resistance-1 (MDR-1) membrane transporter which is responsible for the resistance to a large variety of drugs in human cells. This makes CRD-BP an attractive target for sensitization of colorectal cancer cells to chemotherapeutics. To assess the effects of CRD-BP inhibition on the sensitivity of colorectal cancer cells to chemotherapeutics, we used colorectal cancer cell lines with different status of activation of Wnt signaling pathway, and different degrees of aggressiveness. A normal epithelial colon cell line served as control in our study. Our results show that in colorectal cancer cells with activated Wnt signaling, CRD-BP inhibition potentiates their anti-growth response to chemotherapeutics. This study may potentially lead to design of the agents capable of inhibiting both cell proliferation and drug efflux from the cells. This would be more effective in the treatment of colorectal cancer. Chemotherapeutic drugs are known for their notorious side effects. This study might help in the long run in reducing the dose of chemotherapeutic agents and in lessening the side effects of treatment. This would significantly improve the management of the disease. Citation Format: Felicite Noubissi, Vladimir Spiegelman. Inhibition of the coding region determinant-binding protein (CRD-BP) to sensitize colorectal cancer cells to drug. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B22.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Inhibition of GLI1 Expression by Targeting the CRD-BP–GLI1 mRNA Interaction Using a Specific Oligonucleotide

The stabilization of glioma-associated oncogene 1 (GLI1) mRNA by coding region determinant binding protein (CRD-BP) through the Wnt/β-catenin signaling pathway is implicated in the proliferation of colorectal cancer and basal cell carcinoma. Here, we set out to characterize the physical interaction between CRD-BP and GLI1 mRNA so as to find inhibitors for such interaction. Studies using CRD-BP variants with a point mutation in the GXXG motif at each KH domain showed that KH1 and KH2 domain are critical for the binding of GLI1 RNA. The smallest region of GLI1 RNA binding to CRD-BP was mapped to nucleotides (nts) 320-380. A 37-nt S1 RNA sense oligonucleotide, containing two distinct stem-loops present in nts 320-380 of GLI1 RNA, was found to be effective in blocking CRD-BP-GLI1 RNA interaction. Studies using various competitor RNAs with modifications to S1 RNA oligonucleotide further displayed that both the sequences and the structure of the two stem-loops are important for CRD-BP-GLI1 RNA binding. The role of the two-stem-loop motif in influencing CRD-BP-RNA interaction was further investigated in cells. The 2'-O-methyl derivative of the S1 RNA oligonucleotide significantly decreased GLI1, c-myc, and CD44 mRNA levels, in a panel of colon and breast cancer cells. The results from this study demonstrate the potential importance of the two-stem-loop motif as a target region for the inhibition of the CRD-BP-GLI1 RNA interaction and Hedgehog signaling pathway. Such results pave the way for the development of novel inhibitors that act by destabilizing the CRD-BP-GLI1 mRNA interaction.

Abstract 2133: Investigating the molecular interaction between KRAS mRNA and RNA binding protein CRD-BP

Abstract The Coding Region Determinant-Binding Protein (CRD-BP) is an RNA-binding protein which, among other functions, provides a protective effect for select target mRNA transcripts against degradation, effectively increasing their half-life leading to elevated level of gene expression. CRD-BP was first identified due to its inherent ability to bind to a specific coding region of c-myc mRNA. It has further been demonstrated to convey a protective effect to target transcripts, including GLI1, MITF, CD44, and KRAS as well as a host of other mRNAs. CRD-BP is normally under strict spatiotemporal regulation; expressed only during early developmental stages where rapid growth is necessary. However, CRD-BP re-expression has been reported to occur in a number of human cancers, suggesting its roles as an onco-protein. Abnormal expression or function of KRAS, a CRD-BP target, is implicated in some cancers; roughly 90% of pancreatic cancers, 60% of colorectal, and 50% of lung cancers, making KRAS an attractive target for cancer therapies and highlighting the significance of understanding the CRD-BP-KRAS mRNA interaction. To help understand the CRD-BP-RNA interaction, we have previously mutated the first glycine of the G-X-X-G motif in the KH domains of CRD-BP to an aspartate. Using electrophoretic mobility shift assay, we showed that mutation at any two KH domains, except the KH3 and KH4 di-domains, completely abrogated CRD-BP-c-myc and CD44 RNAs interactions. To understand the CRD-BP-KRAS RNA interaction, we used the identical site-directed KH mutants of CRD-BP to assess their binding profiles to a 185-nt KRAS RNA corresponding to a specific coding region of KRAS mRNA. Using EMSA, additional binding assessments were carried out in a systematic fashion to determine the smallest region of KRAS mRNA bound by CRD-BP. In addition, plasmids carrying these variants of FLAG-CRD-BP were transfected into HeLa cells. Immuno-precipitation (IP) using anti-FLAG was performed, followed by RT-qPCR to measure the relative amounts of KRAS mRNA associated with each of the variants as compared to the controls. The in vitro EMSA and IP cell data were all consistent with the notion that all KH domain(s) are critical for CRD-BP-KRAS RNA interaction. These experiments and their results will be discussed in further detail upon presentation. Citation Format: Sebastian J. Mackedenski, Chow H. Lee. Investigating the molecular interaction between KRAS mRNA and RNA binding protein CRD-BP. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2133. doi:10.1158/1538-7445.AM2015-2133

Two Isoforms of the RNA Binding Protein, Coding Region Determinant-binding Protein (CRD-BP/IGF2BP1), Are Expressed in Breast Epithelium and Support Clonogenic Growth of Breast Tumor Cells

CRD-BP/IGF2BP1 has been characterized as an "oncofetal" RNA binding protein typically highly expressed in embryonic tissues, suppressed in normal adult tissues, but induced in many tumor types. In this study, we show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. Although CRD-BP mRNA expression is induced in breast tumor cells, levels remain ∼1000-fold lower than in embryonic tissues. Despite low expression levels, CRD-BP is required for clonogenic growth of breast cancer cells. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is therefore missing antibody epitopes, CRD-BP expression has been under-reported by previous studies. We show that a CRD-BP mutant mouse strain retains expression of the shorter transcript (ΔN-CRD-BP), which originates in intron 2, suggesting that the impact of complete ablation of this gene in mice is not yet known. Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform. In summary, although CRD-BP expression levels are low in breast cancer cells, this protein is necessary for clonogenic activity.

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.

MicroRNA-340-mediated Degradation of Microphthalmia-associated Transcription Factor (MITF) mRNA Is Inhibited by Coding Region Determinant-binding Protein (CRD-BP)

Alternative cleavage and polyadenylation generates multiple transcript variants producing mRNA isoforms with different length 3'-UTRs. Alternative cleavage and polyadenylation 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 that melanoma cells favor the expression of MITF mRNA with a shorter 3'-UTR. We also establish that this isoform is regulated by a micro RNA (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, coding region determinant-binding protein, 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.

Abstract 2338: Functional investigation of the CRD-BP hnRNP-K-Homology domains for C-myc messenger RNA interaction

Abstract The coding region determinant binding protein (CRD-BP) is an oncofetal protein, being ubiquitously expressed during embryogenesis, yet highly repressed in adult tissues, except in cancerous tissues [Bell et al, 2013; Mueller-Pillasch et al, 1999]. CRD-BP was inherently identified by its ability to physically associate with c-myc mRNA, and has been shown to protect the transcript from degradation, hence prolonging its mRNA half-life [Prokipcak et al, 1994, Noubissi et al, 2006]. In addition to c-myc mRNA, several other mRNAs bound by CRD-BP have gene products implicated in the development and progression of cancer [Bell et al, 2013]. As an RNA binding protein (RBP), CRD-BP is the Mus musculus variant and homologous to the Homo sapiens IMP-1/Insulin-Like Growth Factor 2 with 99.31% similarity, both categorized into a larger family named VICKZ (Vera/Vg1 RBP, Insulin-Like Growth Factor 1,2,3/IMP-1,2,3, CRD-BP, KOC, and ZBP-1). The VICKZ family of RBPs contains two RNA Recognition Motifs (RRM) and four heterogeneous nuclear ribonucleoprotein [hnRNP]-K-Homology (KH) domains. Deletion analysis had shown that the KH domains, and not the RRM domains, of IMP1 are important for binding RNA substrates. To understand which of the KH domains are critical for interaction with RNA, site-directed mutagenesis was performed to introduce an aspartic acid (D) at the first glycine (G) of the G-X-X-G region in the KH domains of CRD-BP. This D-X-X-G variation was individually introduced to the four KH domains of CRD-BP. These four CRD-BP KH domain variants were expressed, purified and assessed for ability to bind c-myc mRNA using electromobility shift assays (EMSA). In addition, plasmids carrying these variants of FLAG-CRD-BP were transfected into HeLa cells. Immunoprecipitation (IP) using anti-FLAG was performed, followed by RT-qPCR to measure the relative amounts of c-myc mRNA associated with each of the variants as compared to the controls. The in vitro EMSA and IP cell data were all consistent with the notion that all KH domain(s) are critical for CRD-BP-c-myc RNA interaction. Using EMSA, additional binding assessments were carried out in a systematic fashion to determine the smallest region of c-myc mRNA bound by CRD-BP. These results will be presented and discussed, as they provide insight into the role played by the KH domains of CRD-BP in binding RNA, as well as what RNA characteristics are important for binding to CRD-BP. Citation Format: Gerrit van Rensburg, Mark Barnes, Chow Lee. Functional investigation of the CRD-BP hnRNP-K-Homology domains for C-myc messenger RNA interaction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2014-2338

Role of CRD-BP in the Growth of Human Basal Cell Carcinoma Cells

Although the number of new cases of Basal Cell Carcinoma (BCC) has increased rapidly in the last few decades, the molecular basis of its pathogenesis is not completely understood. Activation of Hedgehog (Hh) signaling pathway has been shown to be a key factor driving the development of BCC. The Wnt/β-catenin signaling pathway was also shown to be activated in BCCs and to perhaps modulate the activity of Hh pathway. We have previously identified a novel mechanism by which Wnt signaling regulates the transcriptional outcome of Hh signaling pathway. We demonstrated that CRD-BP, a direct target of the Wnt/β-catenin signaling, binds to GLI1 mRNA, stabilizes it, and consequently upregulates its levels (mRNA and protein) and activities. We hypothesized that Wnt-induced and CRD-BP-dependent regulation of GLI1 expression and activities is important to the development of BCC. In this study, we show that CRD-BP is over-expressed in BCC and that its expression positively correlates with the activation of both Wnt and Hh signaling pathways. We also describe the generation and characterization of a human BCC cell line. This cell line was utilized to demonstrate the importance of CRD-BP-dependent regulation of GLI1 expression and activities in the development of BCC.

Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.

Igf2bp1 Is Required for Full Induction of Ptgs2 mRNA in Colonic Mesenchymal Stem Cells in Mice

Prostaglandin-endoperoxide synthase (Ptgs)2 is an enzyme involved in prostaglandin production during the response to mucosal damage. Its expression is regulated, in part, by messenger RNA (mRNA)-binding proteins that control the stability of Ptgs2 mRNA. We used a precise system of colonic injury and repair to identify Ptgs2 mRNA-binding proteins. We used endoscopy-guided mucosal excision to create focal injury sites in colons of mice. Wound beds from wild-type, Ptgs2(-/-), Ptgs2(+/-), and Myd88(-/-) mice were analyzed at 2-day intervals after injury for aspects of repair and Ptgs2 expression. We used cultured colonic mesenchymal stem cells (cMSCs) that express Ptgs2 to identify and analyze molecules that regulate Ptgs2 expression. Ptgs2(-/-) mice had defects in wound repair, validating the biopsy technique as a system to study the regulation of Ptgs2. Ptgs2(+/-) mice had similar defects in wound healing, so full induction of Ptgs2 is required for wound repair. In wild-type mice, levels of Ptgs2 mRNA increased significantly in the wound bed 2 and 4 days after injury; the highest levels of Ptgs2 were observed in cMSCs. In a functional short hairpin RNA knockdown screen, we identified Igf2bp1, a VICKZ (Vg1 RNA binding protein, Insulin-like growth factor II mRNA binding protein 1, Coding region determinant-binding protein, KH domain containing protein overexpressed in cancer, and Zipcode-binding protein-1) mRNA-binding protein, as a regulator of Ptgs2 expression in cMSCs. Igf2bp1 also interacted physically with Ptgs2 mRNA. Igf2bp1 expression was induced exclusively in wound-bed cMSCs, and full induction of Ptgs2 and Igf2bp1 during repair required Myd88. We identified Igf2bp1 as a regulator of Ptgs2 mRNA in mice. Igf2bp1 is required for full induction of Ptgs2 mRNA in cMSCs.

Inhibition of coding region determinant binding protein sensitizes melanoma cells to chemotherapeutic agents

We previously reported that malignant melanomas express high levels of the mRNA binding protein coding region determinant binding protein (CRD-BP). This molecule is important for the activation of anti-apoptotic pathways, a mechanism often linked to insensitivity to therapeutics. However, it is not known whether CRD-BP plays a role in the resistance of melanomas to anti-cancer treatment. Here we demonstrate that knockdown of CRD-BP with a specific sh-RNA enhances the effect of dacarbazine, temozolomide, vinblastine, and etoposide on both primary and metastatic melanoma cell lines. CRD-BP down-regulation contributes to cell sensitization by increasing apoptosis and diminishing melanoma cell growth in response to chemotherapeutic agents. Furthermore, inhibition of CRD-BP decreases microphthalmia-associated transcription factor (MITF) expression and reintroduction of MITF partially compensates for the absence of CRD-BP. These findings suggest that high expression of CRD-BP in melanoma cells confers resistance to chemotherapy and that these CRD-BP responses are mediated, at least in part, by MITF.

Transcriptional Regulation of CRD-BP by c-myc: Implications for c-myc Functions

The coding region determinant binding protein, CRD-BP, is a multifunctional RNA binding protein involved in different processes such as mRNA turnover, translation control, and localization. It is mostly expressed in fetal and neonatal tissues, where it regulates many transcripts essential for normal embryonic development. CRD-BP is scarce or absent in normal adult tissues but reactivated and/or overexpressed in various neoplastic and preneoplastic tumors and in most cell lines. Its expression has been associated with the most aggressive form of some cancers. CRD-BP is an important regulator of different genes including a variety of oncogenes or proto-oncogenes (c-myc, β-TrCP1, GLI1, etc.). Regulation of CRD-BP expression is critical for proper control of its targets as its overexpression may play an important role in abnormal cell proliferation, suppression of apoptosis, invasion, and metastasis. Molecular bases of the regulatory mechanisms governing CRD-BP expression are still not completely elucidated. In this article, we have identified c-myc as a novel transcriptional regulator of CRD-BP. We show that c-myc binds to CRD-BP promoter and induces its transcription. This induction of CRD-BP expression contributes to the role of c-myc in the regulation of translation, increase in cell size, and acceleration of cell cycle progression via a mechanism involving upregulation of β-TrCP1 levels and activities and accelerated degradation of PDCD4.

MicroRNA-340-mediated Degradation of Microphthalmia-associated Transcription Factor mRNA Is Inhibited by the Coding Region Determinant-binding Protein

Alternative cleavage and polyadenylation generate multiple transcript variants of mRNA isoforms with different length of 3'-untranslated region (UTR). Alternative cleavage and polyadenylation enable differential post-transcriptional regulation of transcripts 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. It has also been implicated in melanoma development. Here we show that melanoma cells favor the expression of MITF mRNA with shorter 3'-UTR. This isoform of mRNA is regulated by microRNA, miR-340. miR-340 interacts with two of its target sites on the 3'-UTR of MITF mRNA, causing mRNA degradation and decreased expression and activity of MITF. On the other hand, the RNA-binding protein coding region determinant-binding protein, 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 stabilization of the MITF transcript and elevated expression and transcriptional activity of MITF. This interplay between RNA-binding protein and miRNA describes the important mechanism of regulation of MITF in melanocytes and malignant melanomas.

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant-binding protein (CRD-BP) in response to beta-catenin signaling leads to the stabilization of beta-TrCP1, a substrate-specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IkappaBalpha and activation of canonical nuclear factor-kappaB (NF-kappaB) pathway. Here, we show that the noncanonical NF-kappaB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with beta-catenin-mediated increased expression of CRD-BP and beta-TrCP1. In the carcinoma tissues exhibiting high levels of nuclear beta-catenin the phospho-p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF-kappaB pathway. Knockdown of CRD-BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF-kappaB inhibitory proteins, p105 and IkappaBalpha. Furthermore decreased NF-kappaB binding activity was observed in CRD-BP siRNA-transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF-kappaB1 p105 signaling in colorectal carcinoma might be attributed to beta-catenin-mediated induction of CRD-BP and beta-TrCP1.

Overexpression of mRNA-binding protein CRD-BP in malignant melanomas

Wnt/beta-catenin signaling pathway plays an important role in embryogenesis, stem cell maintenance, tumorigenesis and aging. Here, we show that RNA-binding protein, coding region determinant-binding protein (CRD-BP) (a transcriptional target of Wnt signaling pathway), is highly expressed in primary human malignant melanomas and melanoma cell lines with activated Wnt/beta-catenin signaling pathway. Upregulation of CRD-BP is associated with an elevated level of beta-TrCP1 ubiquitin ligase receptor and activation of nuclear transcriptional factors-kappa B (NF-kappaB) signaling. Knockdown of CRD-BP inhibits NF-kappaB activity, induces apoptosis, and suppresses proliferation and tumorigenic properties of melanoma cells.