Down-regulation of PCBP2 Suppresses the Invasion and Migration of Trophoblasts via the WNT5A/ROR2 Pathway in Preeclampsia.

Impaired extravillous trophoblast (EVT) invasion and resulted poor placentation play a vital role in the development of preeclampsia (PE). However, the underlying mechanisms of dysregulated EVTs remain unclear. This study aimed to explore the role of poly (C)-binding protein 2 (PCBP2), a multifunctional RNA binding protein, in the pathogenesis of PE and to investigate the detailed signaling pathway. Using qRT-PCR, western blot, and immunohistochemistry, we confirmed that the expression of PCBP2 significantly decreased in placentas from 18 early-onset PE and 30 late-onset PE in comparison to those from 30 normotensive pregnancies. Besides, more significant suppression of PCBP2 was observed in the early-onset type. After transfection of HTR-8/SVneo with small interfering RNA (siRNA) specific to PCBP2, the cellular biological behaviors including vitality, immigration, invasiveness, and apoptosis were evaluated by CCK-8 assay, wound-healing assay, transwell assay, and flow cytometry respectively. RNA-seq was applied to screen differentially expressed genes (DEGs) in HTR-8/SVneo upon PCBP2 silencing. GO and KEGG analysis indicated that WNT signaling pathway and the related processes such as extracellular matrix remodeling and cell adhesion were among the most enriched pathways or processes. Meanwhile, the alternative splicing of WNT5A regulated by PCBP2 was also identified by RIP-seq. Based on HTR-8/SVneo and villous explant, the regulatory roles of PCBP2 on trophoblast were confirmed to be mediated by WNT5A. Besides, it revealed that ROR2/JNK/MMP2/9 pathway was a vital pathway downstream WNT5A in trophoblast cells. In conclusion, this study suggests that down-regulated PCBP2 impaired the functions of EVTs via suppression of WNT5A-mediating ROR2/JNK/MMPs pathway, which may eventually contribute to the development of PE.

Unveiling poly(rC)-binding protein 2 as the target protein for curcusone C against prostate cancer: mechanism validation through click chemistry-activity based proteomics profiling approach.

BACKGROUND: Prostate cancer is a disease that seriously troubles men. However, there are some inevitable limitations in interventional therapy for prostate cancer patients at present, most of which are caused by low selectivity and high toxic side effects due to unclear drug targets. In this study, we identified the target protein of Curcusone C with anti-prostate cancer potential activity and verified its target and mechanism of action. METHODS: Click chemistry-activity based proteomics profiling (CC-ABPP) method was used to find target protein of Curcusone C against prostate cancer. Competitive CC-ABPP, drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) methods were used to verifying the target protein. Moreover, potential mechanism was validated by western blot in vitro and by hematoxylin-eosin (HE) staining, detection of apoptosis in tumor tissue (TUNEL), and immunohistochemical (IHC) in vivo. RESULTS: We found that poly(rC)-binding protein 2 (PCBP2) was the target protein of Curcusone C. In addition, Curcusone C might disrupt the Bax/Bcl-2 balance in PC-3 cells by inhibiting the expression of the target protein PCBP2, thereby inducing mitochondrial damage and activation of the mitochondrial apoptosis pathway, and ultimately inducing apoptosis of prostate cancer cells. CONCLUSIONS: Curcusone C is a potential compound with anti-prostate cancer activity, and this effect occurs by targeting the PCBP2 protein, which in turn may affect the TGF/Smad signaling pathway and Bax/Bcl-2 balance. Our results laid a material and theoretical foundation for Curcusone C, to be widely used in anti-prostate cancer.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.

Upregulation of a novel LncRNA AC104958.2 stabilized by PCBP2 promotes proliferation and microvascular invasion in hepatocellular carcinoma.

Long non-coding RNAs (lncRNAs) were reported to be involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). Microvascular invasion (MVI) is an independent predictor for early recurrence and overall survival in postoperative patients with HCC. However, the mechanisms how lncRNAs affect HCC and MVI remain elusive. By RNA sequencing (RNA-seq) in a series of 65 HCC samples and 30 paired adjacent non-tumor liver tissue, we identified a novel lncRNA AC104958.2 that was significantly upregulated in HCC tissues and associated with MVI. Overexpression of AC104958.2 obviously elevated cell viability, metastasis, invasion and epithelial-mesenchymal transition (EMT), while knockout of AC104958.2 mediated by CRISPR/Cas9 technique showed the opposite effects. In addition, the interaction between AC104958.2 and Poly (rC) binding protein 2 (PCBP2) was identified by RNA pull down and mass spectrometry (MS), which was further validated by RNA immunoprecipitation (RIP). PCBP2 was also upregulated in HCC and associated with MVI. High expression of both AC104958.2 and PCBP2 was correlated with tumor size, TNM stage and MVI in HCC. Overexpression of PCBP2 greatly increased the cell viability, metastasis, invasion and EMT. Moreover, actinomycin D assay showed that overexpression of PCBP2 enhanced the RNA stability of AC104958.2. In conclusion, our study showed that a novel lncRNA AC104958.2 exerted oncogenic roles in HCC and might be a promising biomarker and therapeutic target.

PCBP2 knockdown promotes ferroptosis in malignant mesothelioma.

Malignant mesothelioma (MM) is still increasing worldwide. The pathogenesis depends on asbestos-induced iron accumulation, which eventually leads to ferroptosis-resistance of mesothelial cells via somatic mutations. Poly (rC)-binding proteins 1 and 2 (PCBP1/2) are recently recognized cytosolic Fe(II) chaperones. Here we studied the role of PCBP1/2 in rat/human mesothelial and MM cells as well as rat/human MM specimens. Normal peritoneal mesothelial cells in rats exhibited PCBP1 but not PCBP2 immunopositivity whereas primary/immortalized mesothelial cells showed PCBP1/2 immunopositivity. Rat MM specimens induced by intraperitoneal injection of chrysotile, including in situ lesion, revealed PCBP1/2 immunopositivity (90% for both) in the nucleus and cytoplasm with a tendency of higher expression in epithelioid subtype. Knockdown of PCBP2 but not PCBP1 significantly decreased both TfR1 and FTH expression in MM cells with inhibition of proliferation, indicating stagnation of intracellular iron transport. Erastin, a cysteine-deprivation type ferroptosis inducer, decreased the expression of both PCBP1/2 in MM cells. Furthermore, PCBP2 knockdown significantly increased the sensitivity of MM cells to erastin-induced ferroptosis with increased catalytic Fe(II). In conclusion, PCBP2 works for ferroptosis-resistance not only during mesothelial carcinogenesis but also in MM, which warrants further investigation as a novel therapeutic target.

Elusive Trans-Acting Factors Which Operate with Type I (Poliovirus-like) IRES Elements.

The phenomenon of internal initiation of translation was discovered in 1988 on poliovirus mRNA. The prototypic cis-acting element in the 5' untranslated region (5'UTR) of poliovirus mRNA, which is able to direct initiation at an internal start codon without the involvement of a cap structure, has been called an IRES (Internal Ribosome Entry Site or Segment). Despite its early discovery, poliovirus and other related IRES elements of type I are poorly characterized, and it is not yet clear which host proteins (a.k.a. IRES trans-acting factors, ITAFs) are required for their full activity in vivo. Here we discuss recent and old results devoted to type I IRESes and provide evidence that Poly(rC) binding protein 2 (PCBP2), Glycyl-tRNA synthetase (GARS), and Cold Shock Domain Containing E1 (CSDE1, also known as UNR) are major regulators of type I IRES activity.

Structural Characteristics of the 5'-Terminal Region of Mouse p53 mRNA and Identification of Proteins That Bind to This mRNA Region.

A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA's structure and its interactions with proteins in the translation process. In particular, the 5'-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5'-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5'-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5'-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.

Poly C Binding Protein 2 dependent nuclear retention of the utrophin-A mRNA in C2C12 cells.

Upregulation of utrophin, the autosomal homologue of dystrophin, can compensate dystrophin deficiency in Duchenne Muscular Dystrophy (DMD) although the therapeutic success is yet to be achieved. The present study has identified Poly (C) binding protein 2 (PCBP2) as a post-transcriptional suppresser for the expression of utrophin-A, the muscle-specific utrophin isoform. This study confirms nuclear retention of utrophin-A mRNA in C2C12 cells, which is mediated by PCBP2. Further investigation demonstrates PCBP2-dependent nuclear retention of follistatin mRNA as well. Its involvement in nuclear retention of mRNA sheds light on a novel function of PCBP2 that makes utrophin-A mRNA less available in cytosol. PCBP2, therefore, may be a target to de-repress utrophin-A expression in DMD.

The circular RNA circ_GRHPR promotes NSCLC cell proliferation and invasion via interactions with the RNA-binding protein PCBP2.

As the most common malignancy, lung cancer is characterised by high rates of occurrence and mortality. Although circular RNAs (circRNAs) are known to act as important regulators in cancer, their role in lung cancer remains poorly understood. In this study, circ_GRHPR expression was found to be significantly upregulated in the serum of five patients with non-small cell lung cancer (NSCLC), compared to that in healthy controls. It is expressed at high levels in NSCLC cell lines, as revealed by qRT-PCR analysis. Functionally, we demonstrated that circ_GRHPR promotes NSCLC proliferation and invasion in vitro and in vivo by cell proliferation, transwell, cell cycle, and tumour-forming assays. Mechanistically, RNA pull-down and RNA immunoprecipitation assays showed that circ_GRHPR interacts with the RNA-binding protein poly(rC)-binding protein 2 (PCBP2) and regulates its subcellular localisation by forming the circ_GRHPR/PCBP2 complex, localizing PCBP2 mainly in the cytoplasm and reducing the proportion found in the nucleus. Furthermore, we demonstrated that four-and-a-half LIM-only protein 3 (FHL3) is a tumour-stimulating factor in NSCLC that interacts with and is influenced by PCBP2. Circ_GRHPR increased FHL3 expression in the nucleus of NSCLC cells by decreasing PCBP2 expression therein and promoting the proliferation and invasion of NSCLC cells. Therefore, our study identified that circ_GRHPR promotes NSCLC proliferation and invasion, providing a possible explanation for its mechanism of action.

LINC02535 co-functions with PCBP2 to regulate DNA damage repair in cervical cancer by stabilizing RRM1 mRNA.

Cervical cancer (CC) is one of the commonest malignant cancers among women with high morbidity and mortality. Despite encouraging advances had been found in diagnostic and therapeutic strategies, effective therapeutic strategy and further exploration of the mechanism underlying in CC is still needed. We searched The Cancer Genome Atlas database and found that long noncoding RNA LINC02535 was highly expressed in CC. LINC02535 has not been studied in CC, and its molecular regulation mechanism remains unknown. Based on starBase database, LINC02535 could potentially bind poly (rC) binding protein 2 (PCBP2). In the present study, we discovered a significant increase of the LINC02535 and PCBP2 expression in CC tissues and cells as compared with the adjacent normal tissues and normal cervical epithelial cells. LINC02535 and PCBP2 can bind with each other and were colocated in cytoplasm. LINC02535 and PCBP2 promoted cell proliferation, migration, invasion, and suppressed apoptosis in CC. LINC02535 and PCBP2 facilitated the repair of DNA damage to promote CC progression. LINC02535 cooperated with PCBP2 to enhance the stability of RRM1 messenger RNA (mRNA). RRM1 promoted the repair of DNA damage and epithelial-to-mesenchymal transition (EMT) process in CC cells. LINC02535 regulated tumorigenesis in vivo. In conclusion, LINC02535 cooperated with PCBP2, regulated stability of RRM1 mRNA to promote cell proliferation and EMT process in CC cells by facilitating the repair of DNA damage, providing a potential biomarker for CC.

PCBP2 promotes the development of glioma by regulating FHL3/TGF-ß/Smad signaling pathway.

The purpose of this study was to investigate the role of Poly (C)-binding protein 2 (PCBP2) and the related signaling pathway in glioma progression. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were performed to measure PCBP2 messenger RNA and protein expression in glioma tissues or cells. Cell transfection was completed using Lipofectamine 2000. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay and flow cytometry assay were used to explore the effects of PCBP2 expression on biological behaviors of glioma cells. Western blot assay was used for the detection of pathway related proteins. Expression of PCBP2 in glioma tissues and cells were higher than that in paracancerous tissues and normal cells (both p < .01). Moreover, the elevated expression of PCBP2 was significantly correlated with tumor size (p = .001) and WHO stage (p = .010). Knockdown of PCBP2 could suppress proliferation, migration and invasion of glioma cells and promote apoptosis. Besides, the expression of transforming growth factor-ß (TGF-ß) pathway related proteins TGF-ß1, p-Smad2 and p-Smad7 were decreased following the downregulation of PCBP2. PCBP2 also inhibited FHL3 expression by binding to FHL3-3'UTR. The inhibition of FHL3 could reverse the antitumor action caused by PCBP2 silencing. In vivo assay, PCBP2 was also found to inhibit the tumor growth of glioma. PCBP2 activates TGF-ß/Smad signaling pathway by inhibiting FHL3 expression, thus promoting the development and progression of glioma.

KCNQ1OT1 aggravates cell proliferation and migration in bladder cancer through modulating miR-145-5p/PCBP2 axis.

BACKGROUND: The large involvement of long non-coding RNAs (LncRNAs) in the biological progression of numerous cancers has been reported. The function of lncRNA KCNQ1OT1 in bladder cancer (BC) remains largely unknown. This study aimed to explore the critical role of KCNQ1OT1 in BC. MATERIALS AND METHODS: The qRT-PCR was applied to test the expression of RNAs. Cell proliferation was detected by CCK-8 and colony formation assays. Cell apoptosis was measured by TUNEL and flow cytometry experiments. Wound healing and transwell assays were employed to evaluate cell migration and invasion ability respectively. Western blot assay was used to measure relevant protein expression. Immunofluorescence (IF) staining was used to observe EMT process in BC. RESULTS: KCNQ1OT1 was significantly overexpressed in BC tissue and cell lines. KCNQ1OT1 depletion repressed cell proliferation, migration and invasion, whereas encouraged cell apoptosis. KCNQ1OT1 was a negatively/positively correlated with miR-145-5p/PCBP2 in respect with expression. Mechanically, KCNQ1OT1 was sponge of miR-145-5p and up-regulated the expression of PCBP2. MiR-145-5p inhibition and PCBP2 up-regulation could countervail the tumor-inhibitor role of KCNQ1OT1 knockdown in BC. CONCLUSION: KCNQ1OT1 serves as competing endogenous RNA (ceRNA) to up-regulate PCBP2 via sponging miR-145-5p in BC progression.

How iron is handled in the course of heme catabolism: Integration of heme oxygenase with intracellular iron transport mechanisms mediated by poly (rC)-binding protein-2.

Heme and iron are essential to almost all forms of life. The strict maintenance of heme and iron homeostasis is essential to prevent cellular toxicity and the existence of systemic and intracellular regulation is fundamental. Cytosolic heme can be catabolized and detoxified by heme oxygenases (HOs). Interestingly, free heme detoxification through HOs results in the production of free ferrous iron, which can be potentially hazardous for cells. Recently, the intracellular iron chaperone, poly (rC)-binding protein 2 (PCBP2), has been identified, which can be involved in accepting iron after heme catabolism as well as intracellular iron transport. In fact, HO1, NADPH-cytochrome P450 reductase, and PCBP2 form a functional unit that integrates the catabolism of heme with the binding and transport of iron by PCBP2. In this review, we provide an overview of our understanding of the iron chaperones and discuss the mechanism how iron chaperones bind iron released during the process of heme degradation.

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2.

Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine.

The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer.

Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)-NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1-CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.

NLRX1 Mediates MAVS Degradation To Attenuate the Hepatitis C Virus-Induced Innate Immune Response through PCBP2.

Activation of innate immunity is essential for host cells to restrict the spread of invading viruses and other pathogens. However, attenuation or termination of signaling is also necessary for preventing immune-mediated tissue damage and spontaneous autoimmunity. Here, we identify nucleotide binding oligomerization domain (NOD)-like receptor X1 (NLRX1) as a negative regulator of the mitochondrial antiviral signaling protein (MAVS)-mediated signaling pathway during hepatitis C virus (HCV) infection. The depletion of NLRX1 enhances the HCV-triggered activation of interferon (IFN) signaling and causes the suppression of HCV propagation in hepatocytes. NLRX1, a HCV-inducible protein, interacts with MAVS and mediates the K48-linked polyubiquitination and subsequent degradation of MAVS via the proteasomal pathway. Moreover, poly(rC) binding protein 2 (PCBP2) interacts with NLRX1 to participate in the NLRX1-induced degradation of MAVS and the inhibition of antiviral responses during HCV infection. Mutagenic analyses further revealed that the NOD of NLRX1 is essential for NLRX1 to interact with PCBP2 and subsequently induce MAVS degradation. Our study unlocks a key mechanism of the fine-tuning of innate immunity by which NLRX1 restrains the retinoic acid-inducible gene I-like receptor (RLR)-MAVS signaling cascade by recruiting PCBP2 to MAVS for inducing MAVS degradation through the proteasomal pathway. NLRX1, a negative regulator of innate immunity, is a pivotal host factor for HCV to establish persistent infection.IMPORTANCE Innate immunity needs to be tightly regulated to maximize the antiviral response and minimize immune-mediated pathology, but the underlying mechanisms are poorly understood. In this study, we report that NLRX1 is a proviral host factor for HCV infection and functions as a negative regulator of the HCV-triggered innate immune response. NLRX1 recruits PCBP2 to MAVS and induces the K48-linked polyubiquitination and degradation of MAVS, leading to the negative regulation of the IFN signaling pathway and promoting HCV infection. Overall, this study provides intriguing insights into how innate immunity is regulated during viral infection.

Iron Export through the Transporter Ferroportin 1 Is Modulated by the Iron Chaperone PCBP2.

Ferroportin 1 (FPN1) is an iron export protein found in mammals. FPN1 is important for the export of iron across the basolateral membrane of absorptive enterocytes and across the plasma membrane of macrophages. The expression of FPN1 is regulated by hepcidin, which binds to FPN1 and then induces its degradation. Previously, we demonstrated that divalent metal transporter 1 (DMT1) interacts with the intracellular iron chaperone protein poly(rC)-binding protein 2 (PCBP2). Subsequently, PCBP2 receives iron from DMT1 and then disengages from the transporter. In this study, we investigated the function of PCBP2 in iron export. Mammalian genomes encode four PCBPs (i.e. PCBP1-4). Here, for the first time, we demonstrated using both yeast and mammalian cells that PCBP2, but not PCBP1, PCBP3, or PCBP4, binds with FPN1. Importantly, iron-loaded, but not iron-depleted, PCBP2 interacts with FPN1. The PCBP2-binding domain of FPN1 was identified in its C-terminal cytoplasmic region. The silencing of PCBP2 expression suppressed FPN1-dependent iron export from cells. These results suggest that FPN1 exports iron received from the iron chaperone PCBP2. Therefore, it was found that PCBP2 modulates cellular iron export, which is an important physiological process.

RNA-binding Protein PCBP2 Regulates p73 Expression and p73-dependent Antioxidant Defense.

TAp73, a member of the p53 family tumor suppressors, plays a critical rule in tumor suppression and neuronal development. However, how p73 activity is controlled at the posttranscriptional level is not well understood. Here, we showed that TAp73 activity is regulated by RNA-binding protein PCBP2. Specifically, we found that knockdown or knock-out of PCBP2 reduces, whereas ectopic expression of PCBP2 increases, TAp73 expression. We also showed that PCBP2 is necessary for p73 mRNA stability via the CU-rich elements in p73 3'-UTR. To uncover the biological relevance of PCBP2-regulated TAp73 expression, we showed that ectopic expression of PCBP2 inhibits, whereas knockdown or knock-out of PCBP2 increases, the production of reactive oxygen species (ROS) in a TAp73-dependent manner. Additionally, we found that glutaminase 2 (GLS2), a modulator of p73-dependent antioxidant defense, is also involved in PCBP2-regulated ROS production. Moreover, we generated PCBP2-deficient mice and primary mouse embryonic fibroblasts (MEFs) and showed that loss of PCBP2 leads to decreased p73 expression and, subsequently, increased ROS production and accelerated cellular senescence. Together, our data suggest that PCBP2 regulates p73 expression via mRNA stability and p73-dependent biological function in ROS production and cellular senescence.

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.

Meis1 promotes poly (rC)-binding protein 2 expression and inhibits angiotensin II-induced cardiomyocyte hypertrophy.

The poly(rC)-binding protein 2 (PCBP2) is currently reported to inhibit cardiac hypertrophy. However, how PCBP2 is regulated at transcriptional level remains unknown. Here, we show that Meis1, a PBX1-related homeobox gene, binds to PCBP2 promoter and promotes its transcription. In human failing heart tissues and murine hypertrophic heart tissues, the mRNA and protein levels of Meis1 are markedly downregulated, and the level of Meis1 significantly correlates with levels of Nppa, Myh7, and PCBP2. In neonatal rat cardiomyocytes, angiotensin II (Ang II) treatment induces hypertrophic growth of the cells (increase in cell size, enhanced protein synthesis, and hyperexpression of hypertrophic fetal genes), which are significantly inhibited by Meis1 overexpression or promoted by Meis1 knockdown. Meis1 also reduces Ang II-induced activation of Akt-mTOR pathway. Finally, we show that PCBP2 overexpression rescues the Meis1 effects of Akt-mTOR pathway and hypertrophy of cardiomyocytes. © 2015 IUBMB Life, 68(1):13-22, 2016.