Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor

A lot of evidence has been found on the link between tumorigenesis and the aberrant expression of splicing factors. A number of splicing factors have been reported to be either oncogenic or overexpressed in cancer cells. However, splicing factors can also play negative roles in tumorigenesis. In the current review, we focus on splicing factor poly(rC)-binding protein 1 (PCBP1), a novel tumor suppressor that is characterized by downregulation in many cancer types and shows inhibition of tumor formation and metastasis. Notably, the messenger RNA levels of PCBP1 are not significantly decreased in most cancer types. In fact, PCBP1 protein is often degraded or shows a loss-of-function through phosphorylation in cancer cells. PCBP1 is highly homologous to its family member, PCBP2. Interestingly, PCBP2 appears to be an oncogenic splicing factor. A growing body of evidence has shown that PCBP1 regulates alternative splicing, translation, and RNA stability of many cancer-related genes. Taking together, PCBP1 has distinctive tumor suppressive functions, and increasing PCBP1 expression may represent a new approach for cancer treatment.     

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.     

Foreign RNA Induces the Degradation of Mitochondrial Antiviral Signaling Protein (MAVS): The Role of Intracellular Antiviral Factors

Mitochondrial antiviral signaling protein (MAVS) is an essential adaptor molecule that is responsible for antiviral signaling triggered by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), leading to the induction of type I interferon in innate immunity. Previous studies have shown that certain viruses evade the innate immune response by cleaving the MAVS protein. However, little is known about how MAVS is regulated in response to foreign RNA, including both single-stranded (ss) and double-stranded (ds) RNA, because most previous reports have shown that the cleavage of MAVS is executed by proteases that are induced or activated by the invading RNA viruses. Here, we report that MAVS mRNA is degraded in response to polyinosinic-polycytidylic acid (polyI:C), a synthetic dsRNA, in A549 cells. RNA interference (RNAi) experiments revealed that both ssRNA- and dsRNA-associated pattern-recognition receptors (PRRs) were not involved in the degradation of MAVS mRNA. Foreign RNA also induced the transient degradation of the MAVS protein. In the resting state, the MAVS protein was protected from degradation by interferon regulatory factor 3 (IRF3); moreover, the dimerization of IRF3 appeared to be correlated with the rescue of protein degradation in response to polyI:C. The overexpression of MAVS enhanced interferon-β (IFN-β) expression in response to polyI:C, suggesting that the degradation of MAVS contributes to the suppression of the hyper-immune reaction in late-phase antiviral signaling. Taken together, these results suggest that the comprehensive regulation of MAVS in response to foreign RNA may be essential to antiviral host defenses.     

Poly(C)-Binding Protein 1, a Novel Npro-Interacting Protein Involved in Classical Swine Fever Virus Growth

N^pro is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N^pro protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N^pro protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N^pro. Our results suggest that cellular PCBP1 positively modulates CSFV growth.     

Spatiotemporal Expression of Poly(rC)-Binding Protein PCBP2 Modulates Schwann Cell Proliferation After Sciatic Nerve Injury

Poly(C)-binding proteins (PCBPs), also known as RNA-binding proteins, interact in a sequence-specific fashion with single-stranded poly(C). It was reported that PCBP2 contributed to gastric cancer proliferation and survival through miR-34a, and knockdown of PCBP2 inhibited glioma proliferation through inhibition of cell cycle progression. In addition, PCBP2 might play a critical role in the regulation of cortical neurons apoptosis induced by hypoxia or ischemia. Because of the essential role of PCBP2 in nervous system and cell growth, we investigated the spatiotemporal expression of PCBP2 in a rat sciatic nerve crush (SNC) model. We detected the upregulated expression of PCBP2 in Schwann cell after SNC. Besides, the peak expression of PCBP2 was in parallel with proliferation cell nuclear antigen. In vitro, we observed increased expression of PCBP2 during the process of TNF-α-induced Schwann cell proliferation. Specially, PCBP2-specific siRNA-transfected Schwann cell showed significantly decreased ability for proliferation. Together, all these data indicated that the change of PCBP2 protein expression was associated with Schwann cell proliferation after the trauma of the peripheral nervous system.     

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.     

PCBP1在基因表达过程中的功能和作用机理

Poly（C）-binding protein 1 （PCBP 1）是一种RNA结合蛋白,其蛋白质相对分子质量约为38000。PCBP1含三个KH（hnRNP K homology）结构域,这些结构域对其结合RNA有重要作用。PCBP1的功能主要是参与基因转录及转录后调节,如前体mRNA的剪切、mRNA稳定性、mRNA翻译过程的沉默或增强等。本文主要对PCBP1参与的信号通路以及与人类疾病的关系进行综述,试图阐明PCBP1的生物学功能和作用机理。     

Poly(C)-binding protein 2 interacts with sequences required for viral replication in the hepatitis C virus (HCV) 5' untranslated region and directs HCV RNA replication through circularizing the viral genome

Sequences in the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. The translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5'UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of the 5'UTR and different sets of RNA-binding proteins. Here, we showed that the 5'-most 157 nucleotides of HCV RNA is the minimum 5'UTR for RNA replication, and it partially overlaps with the IRES. Stem-loops 1 and 2 of the 5'UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of the 5'UTR and associated with detergent-resistant membrane fractions, which are the sites of the HCV replication complex. The knockdown of PCBP2 by short hairpin RNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5'- and 3'UTR RNA fragments to form an RNA-protein complex and induces the circularization of HCV RNA, as revealed by electron microscopy. This study thus demonstrates the mechanism of the participation of PCBP2 in HCV translation and replication and provides physical evidence for HCV RNA circularization through 5'- and 3'UTR interaction.     

Iron homeostasis regulates the activity of the microRNA pathway through poly(C)-binding protein 2

MicroRNAs (miRNAs) control gene expression by promoting degradation or repressing translation of target mRNAs. The components of the miRNA pathway are subject to diverse modifications that can modulate the abundance and function of miRNAs. Iron is essential for fundamental metabolic processes, and its homeostasis is tightly regulated. Here we identified iron chelators as a class of activator of the miRNA pathway that could promote the processing of miRNA precursors. We show that cytosolic iron could regulate the activity of the miRNA pathway through poly(C)-binding protein 2 (PCBP2). PCBP2 is associated with Dicer and promotes the processing of miRNA precursors. Cytosolic iron could modulate the association between PCBP2 and Dicer, as well as the multimerization of PCBP2 and its ability to bind to miRNA precursors, which can alter the processing of miRNA precursors. Our findings reveal a role of iron homeostasis in the regulation of miRNA biogenesis.     

Cellular protein modification by poliovirus: the two faces of poly(rC)-binding protein

During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-DeltaKH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.     

IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1

Intracellular RNA viruses are sensed by receptors retinoic acid-inducible gene I/MDA5, which trigger formation of the mitochondrial antiviral signaling (MAVS) complex on mitochondria. Consequently, this leads to the activation of TNFR-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) and phosphorylation of IFN regulatory factor 3 (IRF3). It remains to be elucidated how MAVS activates TBK1/IRF3. In this study, we report that IFN-induced protein with tetratricopeptide repeats 3 (IFIT3) is significantly induced upon RNA virus infection. Ectopic expression or knockdown of IFIT3 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, the tetratrico-peptide repeat motif (E164/E165) of IFIT3 interacts with the N terminus (K38) of TBK1, thus bridging TBK1 to MAVS on the mitochondrion. Disruption of this interaction markedly attenuates the activation of TBK1 and IRF3. Furthermore, host antiviral responses are significantly boosted or crippled in the presence or absence of IFIT3. Collectively, our study characterizes IFIT3 as an important modulator in innate immunity, revealing a new function of the IFIT family proteins (IFN-induced protein with tetratricopeptide repeats).     

Structural Insights into Mitochondrial Antiviral Signaling Protein (MAVS)-Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Signaling

In response to viral infection, cytosolic retinoic acid-inducible gene I-like receptors sense viral RNA and promote oligomerization of mitochondrial antiviral signaling protein (MAVS), which then recruits tumor necrosis factor receptor-associated factor (TRAF) family proteins, including TRAF6, to activate an antiviral response. Currently, the interaction between MAVS and TRAF6 is only partially understood, and atomic details are lacking. Here, we demonstrated that MAVS directly interacts with TRAF6 through its potential TRAF6-binding motif 2 (T6BM2; amino acids 455–460). Further, we solved the crystal structure of MAVS T6BM2 in complex with the TRAF6 TRAF_C domain at 2.95 Å resolution. T6BM2 of MAVS binds to the canonical adaptor-binding groove of the TRAF_C domain. Structure-directed mutational analyses in vitro and in cells revealed that MAVS binding to TRAF6 via T6BM2 instead of T6BM1 is essential but not sufficient for an optimal antiviral response. Particularly, a MAVS mutant Y460E retained its TRAF6-binding ability as predicted but showed significantly impaired signaling activity, highlighting the functional importance of this tyrosine. Moreover, these observations were further confirmed in MAVS^−/− mouse embryonic fibroblast cells. Collectively, our work provides a structural basis for understanding the MAVS-TRAF6 antiviral response.     

Selection and cloning of poly(rC)-binding protein 2 and Raf kinase inhibitor protein RNA activators of 2',5'-oligoadenylate synthetase from prostate cancer cells

The antiviral and antitumor functions of RNase L are enabled by binding to the allosteric effectors 5′-phosphorylated, 2′,5′-linked oligoadenylates (2-5A). 2-5A is produced by interferon-inducible 2′,5′-oligoadenylate synthetases (OAS) upon activation by viral double-stranded RNA (dsRNA). Because mutations in RNase L have been implicated as risk factors for prostate cancer, we sought to determine if OAS activators are present in prostate cancer cells. We show that prostate cancer cell lines (PC3, LNCaP and DU145), but not normal prostate epithelial cells (PrEC), contain RNA fractions capable of binding to and activating OAS. To identify the RNA activators, we developed a cDNA cloning strategy based on stringent affinity of RNAs for OAS. We thus identified mRNAs for Raf kinase inhibitor protein (RKIP) and poly(rC)-binding protein 2 (PCBP2) that bind and potently activate OAS. In addition, human endogenous retrovirus (hERV) envelope RNAs were present in PC3 cells that bind and activate OAS. Analysis of several gene expression profiling studies indicated that PCBP2 RNA was consistently elevated in metastatic prostate cancer. Results suggest that OAS activation may occur in prostate cancer cells in vivo stimulated by cellular mRNAs for RKIP and PCBP2.     

PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2

Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.