Poly rC Binding Protein Research Articles

Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity.

TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin.

Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3′ UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals.

Special Delivery: The Role of Iron Chaperones in the Distribution of Iron in Developing Red Cells

Iron is an essential nutrient for every cell in the human body, yet it can also be a potent cellular toxin. Iron is essential because enzymes that require iron co-factors (namely, heme, iron-sulfur clusters, mononuclear and diiron centers) are involved in virtually every major metabolic process in the cell. Hundreds of iron, zinc, copper, and manganese proteins are expressed in human cells, yet little is known about the mechanisms by which these metalloproteins acquire their native metal ligands and avoid mis-metallation. Significant advances have been made in understanding the delivery of iron to iron-dependent enzymes in the cytosol. Poly(rC)-binding proteins (PCBPs) are multifunctional adaptors that mediate interactions between single-stranded nucleic acids, iron cofactors, and other proteins, affecting the fate and activity of the components of these interactions. PCBP1 is an iron-binding protein that delivers iron to ferritin in human cells via a direct protein-protein interaction and can be described as an iron chaperone. PCBP2, a human paralog of PCBP1, is also involved in the delivery of iron to ferritin, both in yeast cells and in human cells, suggesting that PCBP1 and PCBP2 work together in iron delivery. PCBP1 and PCBP2 can also deliver iron to the two major families of non-heme iron enzymes: the mononuclear and dinuclear iron-dependent oxygenases. The prolyl hydroxylases (PHDs) are mononuclear iron enzymes that regulate the degradation of hypoxia inducible factor 1 (HIF1). Misregulation of the HIF transcription factors leads to the development of a variety of cancers in humans. Cells depend on the iron chaperones PCBP1 and PCBP2 to maintain iron in the enzymatic active site of PHDs and the related enzyme, asparagyl hydroxylase and to maintain proper regulation of HIF1a, especially under conditions of iron limitation. Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme that is required for the posttranslational modification of a single lysine residue on eukaryotic initiation factor 5A (eIF5A). EIF5A and the conversion of this conserved lysine to hypusine are essential in all eukaryotes, as it enables the translation of peptides containing polyproline sequences. We found that cells depleted of PCBP1 or PCBP2 exhibited reduced activity of DOHH, which was due to a loss of iron in the active site of the enzyme. Thus, PCBPs are basic components of a cytosolic iron delivery system that serves both of the major classes of non-heme iron enzymes in the cytosol. Recent work has indicated that a second type of iron delivery system in the cytosol is mediated by a monothiol glutaredoxin, Glrx3, which, in vitro, can bind and transfer iron-sulfur clusters to recipient apo-iron-sulfur proteins. We have determined that PCBP1 directly interacts with Glrx3-containing complexes and can affect the coordination of iron-sulfur clusters by Glrx3. The huge flux of iron through the developing erythroid cell represents unique challenges for the utilization of cellular iron. We have examined the role of PCBPs as iron chaperones in terminal erythroid differentiation. The role of ferritin in erythroid cell maturation is controversial, but our data indicate that ferritin, PCBPs and NCOA4 are critical factors in erythrocyte development. The flux of iron through ferritin via the lysosome appears to be critical for the transfer of iron to mitochondria for heme synthesis. DisclosuresNo relevant conflicts of interest to declare.

Poly r(C) Binding Protein-1 is Central to Maintenance of Cancer Stem Cells in Prostate Cancer Cells

Aims: To investigate global proteomic changes induced in CD44+CD24- stem cells isolated from the prostate cancer cell lines, LNCaP and DU145, post prolonged TGF-β treatment in order to understand underlying mechanisms that promote stemness in prostate cancer cells. Methods: CD44+CD133+α2β1Integrin+CD24- population was isolated from mock or TGF-β treated (7 days) prostate cancer cell line, LNCaP, through fluorescent activated cell sorting. Cell lysates were obtained from the ±TGF-β cell population and proteomics profiling (MS/MS) was performed by mass spectrometry. Relative enrichment or depletion in the CD44+CD24-population post-TGF-β treatment was determined relative to mock-treated CD44+CD24- cells post normalization to GAPDH expression levels. Results obtained from MS/MS were validated using immunoblotting. Functional validation of one putative regulator was performed using gain-of-function strategy to investigate its role in rendering stemness in LNCaP and DU145 cells in vitro and in promoting tumorigenicity in vivo. Results: TGF-β treatment caused significant enrichment of CD44+CD24- population in LNCaP cells (22.35 ± 0.94% in mock treated vs 95.23 ± 2.34% in TGF-β treated cells; P < 0.01), which were also positive for CD133 and α2β1Integrin. Mass spectrometry analysis of the enriched cell population revealed that sixty-three proteins were either up- or down-regulated greater than five folds, out of which the poly r(C) binding protein (PCBP)-1 was the most down-regulated (9.31 ± 0.05 folds). Ectopic overexpression of PCBP1 in LNCaP and DU145 cells not only attenuated enrichment of CD44+CD133+CD24- population in these cells following TGF-β treatment, but also significantly decreased tumorigenicity of the stem cell subset, as assessed by in vitro soft agar colony formation and in vivo xenograft assays. Conclusion: Our proteomic profiling and subsequent validation indicate that PCBP1 is central to CSCs enrichment and functionality in prostate cancer.

PCBP1 is an important mediator of TGF-β-induced epithelial to mesenchymal transition in gall bladder cancer cell line GBC-SD.

Gall bladder carcinoma (GBC) is the seventh most common cancer across the globe and the most common malignancy of the biliary tract. Most GBC related deaths occur due to secondary progression and metastasis to distant organs. Epithelial-mesenchymal transition (EMT) is an important pre-requisite for tumor metastasis, however its mechanism in GBC has not yet been defined. Using the GBC-SD cell line, we have uncovered an important mediator, poly r(C) binding protein-1 (PCBP1), of transforming growth factor-beta (TGF-β)-induced EMT in GBC. Our results show that TGF-β treatment resulted in PCBP1 phosphorylation in accordance with similar observation in other model systems. We further showed through gain- and loss-of-function assays that PCBP1 expression levels regulate the capacity of GBC-SD cells to migrate and invade in vitro. Finally, our results showed that PCBP1 expression levels also regulate generation of CD44(+)CD24(-) progenitor cell population in GBC-SD cells after TGF-β treatment. Cumulatively, our results indicate, pending further validation, that PCBP1 might be a prognostic marker for GBC metastasis.

Abstract P6-04-10: Components of cap-independent translation as novel targets in breast cancer

Abstract Background: The majority of cellular mRNAs are translated in a cap-dependent manner, involving ribosome assembly at 5’ 7-methylguanylate cap structures followed by scanning and initiation at a favorable AUG codon. Alternatively, mRNA structural elements termed internal ribosome entry sites (IRESs) facilitate translation in a cap-independent manner, and have been identified in mRNAs encoding many canonical cellular oncogenes, including c-MYC, c-SRC, XIAP, Aurora-A kinase, and IGF1R. IRES trans-activating factors (ITAFs) are accessory proteins that augment or inhibit translation mediated by specific IRES sequences, via binding to IRES structures and/ or other translational machinery. In addition, conversion of ribosomal RNA (rRNA) uracil bases to pseudouracil enhances IRES-mediated translation. Methods: Analysis of publically available microarray datasets of breast cancer and normal breast tissue clinical specimens revealed that expression of several ITAFs is upregulated at the mRNA level in malignant tissues compared to normal controls. mRNAs encoding the ITAFs polypyrimidine tract binding protein 1 (PTBP1), splicing factor proline/ glutamine rich (SFPQ), poly-RC binding protein 2 (PCBP2), and interleukin enhancer binding factor 2 (ILF2, also NF45) were significantly upregulated (p &amp;lt; 0.05), as well as mRNA encoding the dyskeratosis congenita 1 (DKC1) rRNA-modifying enzyme. Independent analysis of an 855 human breast tumor dataset revealed that high mRNA expression of DKC1 and ILF2 are enriched in basal-like breast tumors compared to other intrinsic subtypes (t-test, p&amp;lt;0.01). Kaplan-Meier analyses revealed that high expression of DKC1 or ILF2 mRNAs were both significantly associated with decreased relapse-free survival duration (p = 0.0007 and p = 0.002, respectively). To investigate the biological impact of ITAF and DKC1 silencing, PTBP1, PCBP2, SFPQ, and DKC1 knockdown was accomplished using pGIPZ and pTRIPZ lentiviral shRNA vectors. Silencing of these genes and concomitant reduction in protein levels (measured by western blot) led to significant proliferation impairment in MDA-MB-231, MDA-MB-436, and MCF7 breast cancer cells in culture, using Alamar Blue assay. In addition, 4T1 mouse mammary carcinoma cells express DKC1, PTBP1 and SFPQ, and may serve as a useful model in immunocompetent mice. Conclusions and future directions: Future experiments will include investigation of protein-level expression in archival human specimens using immunohistochemistry, as well as investigation of the effects of ITAF/ DKC1 silencing in human xenografts using NOD/ SCID mice. Based on their increased expression in malignant breast tissues and activity in translation of oncoproteins, cellular ITAF genes and DKC1 gene may represent novel therapeutic targets for intervention. In particular, the increased expression of DKC1 enzyme and ILF2 in basal-like breast cancers implies a possible therapeutic opportunity in triple-negative breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-10.

Abstract 3609: Generation and characterization of a novel monoclonal antibody recognizing both the blood and tissue form of human PCBP-1

Abstract Background: Poly(rC)-binding protein-1 (PCBP-1) is a nucleic-acid-binding protein that localizes in nucleus and/or cytoplasm of cells. Shuttling between nucleus and cytoplasm has been related to its function in regulation of RNA transcription, translation and also implicated its involvement in cancer metastasis, progression and chemoresistance. In the present study, we investigated the levels and expression of PCBP-1 in breast carcinoma and its potential use as a biomarker in breast carcinoma differential diagnosis. Methods/Results: We generated a novel monoclonal antibody (mAb), AB-PCBP-1 using hybridoma technology and native protein in cell culture medium as immunogen. This mAb is of IgG1 (kappa) type. It specifically recognized both native and denatured human PCBP-1 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry applications. Using this mAb in competitive ELISA, we showed for the first time that soluble PCBP-1 level is significantly increased in the plasma of metastatic breast cancer patients’ plasma. PCBP-1 is also expressed in metastatic breast carcinoma tissue by immunohistochemistry. Seventy percent of normal breast ducts and lobules expressed strong nuclear staining whereas cytoplasmic staining was less specific and found in most cancer tissues. We examined 90 cases of breast carcinoma tissues classified as either ductal and lobular carcinoma. To evaluate the immunoreactivity of PCBP-1, the staining intensity and intracellular distribution were compared and scored as 0, 1+, 2+ and 3+ based on intensity. Seventy nine % (60 out of 76) of ductal carcinomas showed strong cytoplasmic staining (2+ and 3+) and 45% also exhibited strong nuclear localization for PCBP-1. Lobular carcinomas (n=14), on the other hand, showed an exclusively nuclear staining pattern.Conclusion: AB-PCBP-1 mAb showed high performance in blood and tissue assays. PCBP-1 levels were significantly increased in blood and demonstrated a distinct pattern of expression related to histological tumor types, suggesting that PCBP-1 may be involved in a regulatory mechanism that differs in various subtypes of breast carcinoma. The results suggest the potential clinical use of PCBP-1 as a therapeutic target and/or a biomarker in blood and tissue for the diagnosis and prognosis of breast carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3609. doi:1538-7445.AM2012-3609

Suppressor of cytokine signaling 4 (SOCS4): Moderator of ovarian primordial follicle activation

Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.

Activation of the HIF Prolyl Hydroxylase by the Iron Chaperones PCBP1 and PCBP2

Mammalian cells express dozens of iron-containing proteins, yet little is known about the mechanism of metal ligand incorporation. Human poly (rC) binding protein 1 (PCBP1) is an iron chaperone that binds iron and delivers it to ferritin, a cytosolic iron storage protein. We have identified the iron-dependent prolyl hydroxylases (PHDs) and asparaginyl hydroxylase (FIH1) that modify hypoxia-inducible factor α (HIFα) as targets of PCBP1. Depletion of PCBP1 or PCBP2 in cells led to loss of PHD activity, manifested by reduced prolyl hydroxylation of HIF1α, impaired degradation of HIF1α through the VHL/proteasome pathway, and accumulation of active HIF1 transcription factor. PHD activity was restored invitro by addition of excess Fe(II), or purified Fe-PCBP1, and PCBP1 bound to PHD2 and FIH1 invivo. These data indicated that PCBP1 was required for iron incorporation into PHD and suggest a broad role for PCBP1 and 2 in delivering iron to cytosolic nonheme iron enzymes.

PCBP2 siRNA Reverses the Alcohol-induced Pro-fibrogenic Effects in Hepatic Stellate Cells

Type I collagen accumulates during liver fibrosis primarily because α-complex protein-2 (αCP(2)), encoded by the poly(rC) binding protein 2 (PCBP2) gene, binds to the 3' end of the collagen mRNA and increases its half-life. This study aimed to reverse the pro-fibrogenic effect of alcohol on hepatic stellate cells (HSCs) by silencing the PCBP2 gene with siRNA. The silencing effects of a series of predesigned PCBP2 siRNAs were evaluated in the rat hepatic stellate cell line, HSC-T6. The pro-fibrogenic effects of alcohol on the expression levels of PCBP2 and type-I collagen were examined by several methods. The effect of PCBP2 siRNA on the stability of type I collagen α1(I) mRNA was investigated by an in vitro mRNA decay assay. We identified one potent PCBP2 siRNA that reversed the alcohol-induced expression of PCBP2 in HSCs. The decay rate of the collagen α1(I) mRNA increased significantly in HSCs treated with the PCBP2 siRNA. This study provides the first evidence that alcohol up-regulates the expression of PCBP2, which subsequently increases the half-life of collagen α1(I) mRNA. Silencing of PCBP2 using siRNA may provide a promising strategy to reverse the alcohol-induced pro-fibrogenic effects in HSCs.

Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma

Although expression of non-protein-coding RNA (ncRNA) can be altered in human cancers, their functional relevance is unknown. Ultraconserved regions are noncoding genomic segments that are 100% conserved across humans, mice, and rats. Conservation of gene sequences across species may indicate an essential functional role, and therefore we evaluated the expression of ultraconserved RNAs (ucRNA) in hepatocellular cancer (HCC). The global expression of ucRNAs was analyzed with a custom microarray. Expression was verified in cell lines by real-time PCR or in tissues by in situ hybridization using tissue microarrays. Cellular ucRNA expression was modulated with siRNAs, and the effects on global gene expression and growth of human and murine HCC cells were evaluated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells compared with nonmalignant hepatocytes. Among these ucRNAs, the greatest change was noted for ultraconserved element 338 (uc.338), which was dramatically increased in human HCC compared with noncancerous adjacent tissues. Although uc.338 is partially located within the poly(rC) binding protein 2 (PCBP2) gene, the transcribed ncRNA encoding uc.338 is expressed independently of PCBP2 and was cloned as a 590-bp RNA gene, termed TUC338. Functional gene annotation analysis indicated predominant effects on genes involved in cell growth. These effects were experimentally demonstrated in both human and murine cells. siRNA to TUC338 decreased both anchorage-dependent and anchorage-independent growth of HCC cells. These studies identify a critical role for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.

Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment

BackgroundThere is increasing evidence that cancers contain their own stem-like cells, and particular attention has been paid to one subset of cancer-stem cells termed side population (SP). Stem cells under normal physical conditions are tightly controlled by their microenvironment, however, the regulatory role of the microenvironment surrounding cancer stem cells is not well characterized yet. In this study we found that the phenotype of SP can be "generated" by macrophage-like cells under conditioned culture. Furthermore the gene regulation pathway involved in cellular reprogramming process was investigated.MethodsThe selection and identification of SP in 50 CNE-2 single cell clones were performed by flow cytometry. The transwell assay and immunofluorescence staining were used to measure migration and cancer stem cell characters of non-SP single clone cells cultured with conditioned medium respectively. The subtraction suppression hybridization (SSH) technique and northern blotting analysis was applied to explore the pluripotency-associated genes under a unique epigenetic sub-microenvironment.ResultsAmong 50 clones, only one did not possess SP subpopulation while others did. The non-SP cells induced by macrophage-like cells showed more aggressive characters, which increased cell migration compared with the control cells and showed some fraction of SP phenotype. These cells expressed distinguished level of pluripotency-associated genes such as ADP-ribosylation factor-like 6 interacting protein (ARMER), poly (rC) binding protein 1 (PCBP1) and pyruvate dehydrogenase E1-β subunit (PDHB) when subjected to the environment.ConclusionTo our knowledge, this is the first study to demonstrate that non-SP single-clone cells can be induced to generate a SP phenotype when they are cultured with conditioned medium of macrophage-like cells, which is associated with the reactivation of pluripotency-associated genes.

Riboproteomic analysis of polypeptides interacting with the internal ribosome‐entry site element of foot‐and‐mouth disease viral RNA

Initiation of translation driven by internal ribosome entry site (IRES) elements depends upon the structural organization of this mRNA region. Besides translation initiation factors (eIFs), auxiliary proteins can also affect IRES activity. With the aim to identify proteins interacting with two unrelated IRESs present in the genome of foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) we have used a proteomic approach. This procedure allowed the identification of 21 RNA-binding proteins interacting with discrete regions of the FMDV IRES, domains 3 and 5, and 16 interacting with domain III of the HCV IRES. In support of the binding specificity, the factors interacting with domain 3 differed from those interacting with domain 5, and included three poly(rC)-binding protein (PCBP) members, besides proliferation-associated 2G4 (PA2G4) and deleted-azoospermia 1 (DAZ1) protein. Around 71% of the identified factors associated with the FMDV IRES differ from those interacting with the HCV IRES. The group of proteins interacting with the FMDV or the HCV IRES includes eIF4B and 5 subunits of eIF3, respectively, known to interact with each of these RNAs, validating the results of this approach. According to the function of the identified proteins, 55% are involved in translation control, whereas 35% play a role in different aspects of RNA lifespan. Compilation of factors preferentially associated with FMDV or HCV IRES provides a basis for examining the strategies used by IRESs to recruit the translation machinery.

Interaction of poly(rC)-binding protein 2 domains KH1 and KH3 with coxsackievirus RNA

Recombinant hnRNP K-homology (KH) domains 1 and 3 of the poly(rC)-binding protein (PCBP) 2 were purified and assayed for interaction with coxsackievirus B3 RNA in electrophoretic mobility shift assays using in vitro transcribed RNAs which represent signal structures of the 5′-nontranslated region. KH domains 1 and 3 interact with the extended cloverleaf RNA and domain IV RNA of the internal ribosome entry site (IRES). KH1 but not KH3 interacts with subdomain IV/C RNA, whereas KH3 interacts with subdomain IV/B. All in vitro results are consistent with yeast three-hybrid experiments performed in parallel. The data demonstrate interaction of isolated PCBP2 KH1 and KH3 domains to four distinct target sites within the 5′-nontranslated region of the CVB3 genomic RNA.

A link between SIN1 (MAPKAP1) and poly(rC) binding protein 2 (PCBP2) in counteracting environmental stress

When SIN1 (MAPKAP1) was used as the bait in a two-hybrid screen of a human bone marrow cDNA library, its most frequent partner was poly(rC) binding protein 2 (PCBP2/hnRNP-E2), which associates with the N-terminal domain of SIN1 and can be coimmunoprecipitated with SIN1 and the cytoplasmic domain of the IFN receptor IFNAR2 from HeLa cells. SIN1, but not PCBP2, also associates with the receptors that bind TNFalpha. PCBP2 is known to bind pyrimidine-rich repeats within the 3' UTR of mRNAs and has been implicated in control of RNA stability and translation and selective cap-independent transcription. RNAi silencing of either SIN1 or PCBP2 renders cells sensitive to basal and stress-induced apoptosis. Stress in the form of TNFalpha and H(2)O(2) treatments rapidly raises the cell content of SIN1 and PCBP2, an effect reversible by inhibiting MAPK14. A meta analysis of human microarray information with an algorithm that discerns similarities in gene-regulatory profiles shows that SIN1 and PCBP2 are generally coregulated with large numbers of genes implicated in both cell survival and death and in cellular stress responses, including RNA translation and processing. We predict that SIN1 is a scaffold protein that organizes antiapoptotic responses in stressed cells, whereas PCBP2, its binding partner, provides for the selective expression of cell survival factors through posttranslational events.

The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation

Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5′ noncoding region (5′ NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5′ NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein–protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

Poly(rC) Binding Proteins and the 5′ Cloverleaf of Uncapped Poliovirus mRNA Function during De Novo Assembly of Polysomes

Poliovirus (PV) mRNA is unusual because it possesses a 5'-terminal monophosphate rather than a 5'-terminal cap. Uncapped mRNAs are typically degraded by the 5' exonuclease XRN1. A 5'-terminal cloverleaf RNA structure interacts with poly(rC) binding proteins (PCBPs) to protect uncapped PV mRNA from 5' exonuclease (K. E. Murray, A. W. Roberts, and D. J. Barton, RNA 7:1126-1141, 2001). In this study, we examined de novo polysome formation using HeLa cell-free translation-replication reactions. PV mRNA formed polysomes coordinate with the time needed for ribosomes to traverse the viral open reading frame (ORF). Nascent PV polypeptides cofractionated with viral polysomes, while mature PV proteins were released from the polysomes. Alterations in the size of the PV ORF correlated with alterations in the size of polysomes with ribosomes present every 250 to 500 nucleotides of the ORF. Eukaryotic initiation factor 4GI (eIF4GI) was cleaved rapidly as viral polysomes assembled and the COOH-terminal portion of eIF4GI cofractionated with viral polysomes. Poly(A) binding protein, along with PCBP 1 and 2, also cofractionated with viral polysomes. A C24A mutation that inhibits PCBP-5'-terminal cloverleaf RNA interactions inhibited the formation and stability of nascent PV polysomes. Kinetic analyses indicated that the PCBP-5' cloverleaf RNA interaction was necessary to protect PV mRNA from 5' exonuclease immediately as ribosomes initially traversed the viral ORF, before viral proteins could alter translation factors within nascent polysomes or contribute to ribonucleoprotein complexes at the termini of the viral mRNA.

Poly(rC)-binding protein 2 interacts with the oligo(rC) tract of coxsackievirus B3

The human poly(rC)-binding protein (PCBP) 2 is known to interact with enteroviral RNA. Here, the interaction of PCBP2 with RNA target sequences at the 5′ end of the coxsackievirus B3 genome was investigated. Using the electrophoretic mobility shift assay and the yeast three-hybrid system, a short oligo(rC) tract connecting cloverleaf and IRES is demonstrated to contribute to PCBP2 binding. This oligo(rC) tract is conserved among entero- and rhinoviruses. In absence of the viral 3C proteinase, an extended cloverleaf RNA (nt 1–105) containing the oligo(rC) tract interacts with PCBP2 whereas the cloverleaf (nt 1–87) lacking the oligo(rC) tract does not. In the presence of 3C proteinase, cloverleaf RNA (1–87) interacts with PCBP2.

Regulation of BAG-1 IRES-mediated translation following chemotoxic stress

There are three major isoforms of BAG-1 in mammalian cells, termed BAG-1L (p50), BAG-1M (p46) and BAG-1S (p36) that function as pro-survival proteins and are associated with tumorigenesis and chemoresistance. Initiation of BAG-1 protein synthesis can occur by both cap-dependent and cap-independent mechanisms and it has been shown that synthesis of BAG-1S is dependent upon the presence of an internal ribosome entry segment (IRES) in the 5'-UTR of BAG-1 mRNA. We have shown previously that BAG-1 IRES-meditated initiation of translation requires two trans-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB) for function. The former protein allows BAG-1 IRES RNA to attain a structure that permits binding of the ribosome, while the latter protein appears to be involved in ribosome recruitment. Here, we show that the BAG-1 IRES maintains synthesis of BAG-1 protein following exposure of cells to the chemotoxic drug vincristine but not to cisplatin and that this is brought about, in part, by the relocalization of PTB and PCBP1 from the nucleus to the cytoplasm.

Replication of Poliovirus Requires Binding of the Poly(rC) Binding Protein to the Cloverleaf as Well as to the Adjacent C-Rich Spacer Sequence between the Cloverleaf and the Internal Ribosomal Entry Site

The 5' nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CD(pro). The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C(93-95) and C(98-100)) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C(93-95)) resulted in slower viral growth, whereas the C(98-100)A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C(94-95)) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5'-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CD(pro) to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.