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Coronary artery calcification (CAC) is a hallmark event in the pathogenesis of cardiovascular disease, involving the phenotypic transformation of vascular smooth muscle cells (VSMC) towards an osteogenic state. Despite this understanding, the molecular mechanisms governing the VSMC osteogenic switch remain incompletely elucidated. Here, we sought to examine the potential role of circular RNA (circRNA) in the context of CAC. Through transcriptome analysis of circRNA-seq, we identified circTOP1 as a potential candidate circRNA in individuals with CAC. Furthermore, we observed that overexpression of circTOP1 exacerbated vascular calcification in a CAC model. Subsequent pull-down assays revealed an interaction between circTOP1 and PTBP1, a putative target gene of circTOP1 in the context of CAC. In both in vivo and in vitro experiments, we observed heightened expression of circTOP1 and PTBP1 in the CAC model, and noted that reducing circTOP1 expression effectively reduced calcium salt deposits and mineralized nodules in model mice. Additionally, in vitro experiments demonstrated that overexpression of PTBP1 reversed the weakening of signaling caused by silencing circTOP1, thereby exacerbating the osteogenic transition and calcification of VSMC. Collectively, our findings suggested that circTOP1 promotes CAC by modulating PTBP1 expression to mediate VSMC transdifferentiation., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the author(s). All authors have reviewed and approve of the contents of the manuscript and its conclusions. The manuscript has not been submitted in part or in whole to another journal. That the data provided are original findings conducted in the lab of the corresponding author and that original data are available for eventual uploading as a supplemental file., (Copyright © 2024. Published by Elsevier Inc.)

The large amount of viral RNA produced during infections has the potential to interact with and effectively sequester cellular RNA binding proteins, thereby influencing aspects of post-transcriptional gene regulation in the infected cell. Here we demonstrate that the abundant 5' leader RNA region of SARS-CoV-2 viral RNAs can interact with the cellular polypyrimidine tract binding protein (PTBP1). Interestingly, the effect of a knockdown of PTBP1 protein on cellular gene expression is also mimicked during SARS-CoV-2 infection, suggesting that this protein may be functionally sequestered by viral RNAs. Consistent with this model, the alternative splicing of mRNAs that is normally controlled by PTBP1 is dysregulated during SARS-CoV-2 infection. Collectively, these data suggest that the SARS-CoV-2 leader RNA sequesters the cellular PTBP1 protein during infection, resulting in significant impacts on the RNA biology of the host cell. These alterations in post-transcriptional gene regulation may play a role in SARS-CoV-2 mediated molecular pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Gastric cancer (GC) is a common cancer with a high incidence and mortality rate. Herein, the role of hsa_circ_0002019 (circ_0002019) in GC was investigated., Methods: The molecular structure and stability of circ_0002019 were identified by RNase R, and Actinomycin D treatment. Molecular associations were verified by RIP. Proliferation, migration, and invasion were detected by CCK-8, EdU, and Transwell, respectively. The effect of circ_0002019 on tumor growth was analyzed in vivo., Results: Circ_0002019 was elevated in GC tissues and cells. Circ_0002019 knockdown inhibited the proliferation, migration, and invasion. Mechanically, circ_0002019 activated NF-κB signaling by increasing TNFAIP6 mRNA stability by PTBP1. Activation of NF-κB signaling limited the antitumor effect of circ_0002019 silencing in GC. Circ_0002019 knockdown inhibited tumor growth in vivo by reducing TNFAIP6 expression., Conclusions: Circ_0002019 accelerated the proliferation, migration, and invasion by regulating TNFAIP6/NF-κB pathway, suggesting circ_0002019 could be a key regulatory factor in GC progression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous studies have demonstrated that the aberrant expression of deubiquitinating enzymes (DUBs) is closely associated with cancer progression, including gastric cancer (GC), due to its role in maintaining protein stability. The 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), a member of the DUBs family, is reported to be highly expressed in some types of cancer and its overexpression indicates poor prognosis, but the function of PSMD14 in GC remains unclear. To investigate this issue, we first analyzed the PSMD14 expression via the TNMplot database and found that PSMD14 was up-regulated in GC tissues compared with the adjacent normal tissues (P < 0.01). PSMD14 knockdown notably inhibited cell proliferation, migration, and invasion in vitro, which was confirmed through in vivo experiments. However, PSMD14 overexpression presented the opposite effects. Additionally, we found that PSMD14 deletion inhibited the protein level of polypyrimidine tract-binding protein 1 (PTBP1), an activator of GC development. Further investigation confirmed that PSMD14 and PTBP1 presented co-localization and had an endogenous interaction. PSMD14 was revealed to promote the deubiquitination and stabilization of PTBP1, and PTBP1 knockdown reversed the effects caused by PSMD14 overexpression on cell function. Collectively, we demonstrate that PSMD14 as a deubiquitinating enzyme may promote the development of GC via stabilizing PTBP1, which provides a theoretical basis for a therapeutic target against GC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tumor necrosis-factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-superfamily that selectively induces apoptosis through death receptors (DRs) 4 and/ or DR5 in cancer cells, without affecting normal cells. Unfortunately, many clinical studies have shown that cancer cells acquire TRAIL-resistance and thus avoid TRAIL-induced apoptosis. In the current study, we newly found that PTBP1, a splicer protein that plays an important role in energy metabolism is highly expressed in TRAIL-resistant human colon cancer DLD-1. Interestingly, silencing PTBP1 by using siRNA for PTBP1 (siR-PTBP1) resulted in a significant increase in TRAIL-sensitivity along with the switching of pyruvate kinase muscle (PKM) isoforms from PKM2 to PKM1, leading to impaired Warburg effect, because the intracellular ATP levels were significantly increased and the production of lactate decreased. Notably, siR-PTBP1 canceled the resistance by increasing the expression level of DR5 and effectively inducing the translocation of DR5 to the cell surface membrane. Also, siR-PTBP1 up-regulated the expression level of CCN1, which contributed to the enhanced sensitivity to TRAIL-induced apoptosis. These findings indicate that silencing PTBP1, thus impairing the Warburg effect positively affected TRAIL-induced apoptosis and that this splicer protein may thus serve as a possible target molecule to cancel the resistance of cancer cells to TRAIL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human rhinovirus (HRV) is a major causative agent of the common cold, and thus has several important health implications. As a member of the picornavirus family, HRV has a small genomic RNA that utilizes several host cell proteins for RNA replication. Host proteins poly(rC) binding protein 2 (PCBP2) and polypyrimidine tract binding protein (PTB) are cleaved by a viral proteinase during the course of infection by the related picornavirus, poliovirus. The cleavage of PCBP2 and PTB inhibits poliovirus translation and has been proposed to mediate a switch in poliovirus template usage from translation to RNA replication. HRV RNA replication also requires a switch in template usage from translation to RNA replication; however, the mechanism is not yet known. We demonstrate that PCBP2 and PTB are differentially cleaved during HRV infection in different cell lines, suggesting that HRV utilizes a mechanism distinct from PCBP2 or PTB cleavage to mediate a switch in template usage., (© 2013 Elsevier Inc. All rights reserved.)

Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Exon 6B from the chicken β-tropomyosin pre-mRNA is alternatively spliced during myogenic differentiation. Exon 6B is excluded in mRNA from myoblasts and included in mRNA from myotubes. We investigated the regulation of exon 6B inclusion ex vivo in a quail myogenic cell line, which behaves as myoblasts in undifferentiated state and as myotubes after differentiation. We show that the β-tropomyosin exon 6B is a novel target of CUG-BP and ETR-3-like factor (CELF). Overexpression of CELF proteins in myoblasts activates splicing of exon 6B. Using a dominant-negative form of CELF4, we demonstrate that CELF proteins are involved in switching splicing from exon 6A towards exon 6B inclusion during myogenic differentiation. We also found that polypyrimidine tract binding protein (PTB) is required for splicing repression of exon 6B in myoblasts. CELF and PTB proteins exhibit antagonistic properties toward inclusion of exon 6B during myogenic differentiation. Our results suggest that a change in the protein level of CUGBP1 and PTB proteins, associated with a distinct pattern of PTB during the transition from myoblasts to myotubes is one of the parameters involved in regulating splicing of exon 6B during myogenesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Raver2 was originally identified as a member of the hnRNP family through database searches revealing three N-terminal RNA recognition motifs (RRMs) bearing highest sequence identity in the RNP sequences to the related hnRNP Raver1. Outside the RRM region, both Raver proteins are quite divergent in sequence except for conserved peptide motifs of the [S/G][I/L]LGxxP consensus sequence. The latter have been implicated in Raver1 binding to the polypyrimidine tract-binding protein (PTB) a regulatory splicing repressor and common ligand of both Raver proteins. In the present study we investigated the association of Raver2 with RNA and PTB in more detail. The isolated RRM domain of Raver2 weakly interacted with ribonucleotides, but the full-length protein failed to directly bind to RNA in vitro. However, trimeric complexes with RNA were formed via binding to PTB. Raver2 harbors two putative PTB binding sequences in the C-terminal half of the protein, whose influence on Raver2-PTB complex formation was analyzed in a mutational approach, replacing critical leucine residues with alanines. While mutation of either sequence motif alone negatively affected Raver2 binding to PTB in vitro, only mutation of the more C-terminally located SLLGEPP motif significantly reduced the recruitment of Raver2 into perinucleolar compartments (PNCs) in HeLa cells. The latter observation was also confirmed for Raver1: out of four sequence motifs matching the PTB binding consensus, mutations in the SLLGEPP motif were the only ones attenuating the recruitment of Raver1 into PNCs. The conserved mode of PTB binding suggests that Raver2, like Raver1, may function as a modulator of PTB activity., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. There is no effective therapy or vaccine available to HCV-infected patients other than interferon-ribavarin combination, which is effective in a relatively small percentage of infected patients. Our previous results have shown that a synthetic peptide (LAP) corresponding to the N-terminal 18 amino acids of the Lupus autoantigen (La) was a potent inhibitor of HCV IRES-mediated translation. We demonstrate here that LAP efficiently blocks HCV replication of infectious JFH1 virus in cell culture. Our data suggest that LAP forms complexes with IRES-transacting factors (ITAFs) PTB and PCBP2. LAP-mediated inhibition of HCV IRES-mediated translation in vitro could be fully rescued by recombinant PCB and PCBP2. Also transient expression of PTB / PCBP2 combination significantly restores HCV replication in LAP-inhibited cultures. These results suggest that ITAFs could be potential targets to block HCV replication.

Foot-and-mouth disease virus (FMDV) infection induces major changes in the host cell including the shutoff of cellular protein synthesis. Here, protein extracts from FMDV-infected cells have been used to monitor changes in the profile of RNA-binding factors interacting with regulatory regions of the viral RNA. Relevant differences have been detected in the pattern of interaction with proteins prepared from either infected or uninfected cells with RNA probes encompassing the internal ribosome entry site (IRES), the 5' and 3'end regions. The binding patterns obtained for two divergent FMDV isolates showed differences depending on the viral isolate used. The identity of the host proteins giving a shifted binding pattern to RNA regulatory regions has been inferred by immunoblotting. Our results show that polypyrimidine tract-binding protein (PTB) and two subunits of translation initiation factor eIF3 interacting with the IRES undergo proteolytic processing during FMDV infection. In addition, poly(A)-binding protein (PABP), interacting with the 3'end of the viral RNA is partially processed. Proteolysis of eIF3a, eIF3b, PABP and PTB correlated with the extent of cytopathic effect induced by FMDV in infected cells.

The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem-loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle.

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.

TLS/FUS is a nucleic acid-binding protein whose N-terminal half functions as a transcriptional activator domain in fusion oncoproteins found in human leukemias and liposarcomas. Previous reports have suggested a role for TLS/FUS in transcription and splicing processes. Here we report the association of TLS/FUS with the nuclear matrix and investigate its role in splicing. Splicing of two pre-mRNAs was inhibited in a TLS/FUS-immunodepleted extract and could only be partly restored by addition of recombinant TLS/FUS or/and SR proteins, known interaction partners of TLS/FUS. The subsequent analysis of TLS/FUS immunoprecipitates revealed that, in addition to the SR proteins SC35 and SRp75, the splicing factor PTB (hnRNPI) and the splicing coactivator SRm160 are complexed with TLS/FUS, thus explaining the inability to restore splicing completely. Coimmunolocalization confirmed the nuclear matrix association and interaction of TLS/FUS with PTB, SR proteins, and SRm160. Our results suggest that the matrix protein TLS/FUS plays a role in spliceosome assembly., (Copyright 2003 Elsevier Science (USA))

Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5' ends of mouse hepatitis virus (MHV) RNA and its complementary strand. To further characterize the function of PTB in MHV replication, we generated dominant-negative mutant cell lines that express a full-length PTB or a truncated form of PTB, which includes only the N-terminal half of the protein, retaining its protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, whereas the full-length PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. We observed that both the full-length and the truncated PTB, when overexpressed, functioned in a dominant-negative manner in MHV replication. However, the truncated form exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, we dissociated the processes of viral transcription from translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA during MHV infection was greatly inhibited in these cell lines, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by PTB depletion in in vitro translation in rabbit reticulocyte lysate or by PTB overexpression in in vivo translation experiments in MHV-infected cells. Given that PTB interacts with the viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on viral replication/transcription by association with viral RNA as well as other viral and cellular factors in the replication complex.