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[Objective] CRISPR/Cas9 technology was used to generate a mouse embryonic fibroblast cell line (NIH3T3) with a knockout of the Quaking gene and to investigate its impact on NIH3T3 cell proliferation. [Method] Initially, two sgRNAs targeting Quaking exons were designed using an online platform, and two CRISPR/Cas9 recombinant lentiviral plasmids targeting the first and second exons of the Quaking gene were constructed successfully. These constructs with pcDNA3.1-Quaking overexpression plasmids were co-transfected into HEK293T cells, and the knockout efficiency of Quaking protein was assessed through Western blot analysis. Subsequently, the recombinant lentiviral plasmid (LentiCRISPRv2-sgRNA1) with high knockout efficiency was co-transfected with auxiliary packaging plasmids into HEK293T cells for lentivirus packaging. After lentiviral transduction of NIH3T3 cells, positive monoclonal cell lines were selected using puromycin. Finally, we confirmed the knockout effect through Western blot and immunofluorescence staining, demonstrating the absence of Quaking protein in these cells. [Result] Sequencing confirmed the occurrence of a targeted gene segment deletion. CCK8 assays revealed that Quaking gene knockout significantly inhibited NIH3T3 cell proliferation. [Conclusion] This study represents the first successful utilization of CRISPR/Cas9 technology to establish a Quaking gene knockout cell line in mouse embryonic fibroblast cells (NIH3T3), providing a valuable in vitro model for exploring the mechanistic role of the Quaking gene in the regulation of mouse physiological functions. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are a class of non-coding RNA molecules that are gaining increasing attention for their roles in various pathophysiological processes. The RNA-binding protein quaking (QKI) has been identified as a regulator of circRNA formation. In this study, we investigate the role of QKI in the formation of circRNAs in the heart by performing RNA-sequencing on Qki-knockout mice. Loss of QKI resulted in the differential expression of 17% of the circRNAs in adult mouse hearts. Interestingly, the majority of the QKIregulated circRNAs (58%) were derived from genes undergoing QKIdependent splicing, indicating a relationship between back-splicing and linear splicing. We compared these QKI-dependent circRNAs with those regulated by RBM20, another cardiac splicing factor essential for circRNA formation. We found that QKI and RBM20 regulate the formation of a distinct, but partially overlapping set of circRNAs in the heart. Strikingly, many shared circRNAs were derived from the Ttn gene, and they were regulated in an opposite manner. Our findings indicate that QKI not only regulates alternative splicing in the heart but also the formation of circRNAs. [ABSTRACT FROM AUTHOR]

Aims In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. Methods and results We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. Conclusion Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes. [ABSTRACT FROM AUTHOR]

Abstract Background Sepsis is a fatal condition commonly caused by Methicillin-resistant Staphylococcus aureus (MRSA) with a high death rate. Macrophages can protect the host from various microbial pathogens by recognizing and eliminating them. Earlier we found that Quaking (QKI), an RNA binding protein (RBP), was involved in differentiation and polarization of macrophages. However, the role of QKI in sepsis caused by pathogenic microbes, specifically MRSA, is unclear. This study aimed to investigate the role of QKI in regulation of host–pathogen interaction in MRSA-induced sepsis and explored the underlying mechanisms. Methods Transmission electron microscope and immunofluorescence were used to observe the autophagy level in macrophages. Real-time PCR and western blot were used to analyzed the expression of mRNA and protein respectively. The potential protein interaction was analyzed by iTRAQ mass spectrometry and Immunoprecipitation. RNA fluorescence in situ hybridization, dual-luciferase reporter assay and RNA immunoprecipitation were used to explore the mechanism of QKI regulating mRNA of PI3K-p110β. Results The mRNA level of QKI was aberrantly decreased in monocytes and PBMCs of septic patients with the increasing level of plasma procalcitonin (PCT). Then the mice with myeloid specific knockout of QKI was challenged with MRSA or Cecal Ligation and Puncture (CLP). Mice in these two models displayed higher survival rates and lower bacterial loads. Mechanistically, QKI deletion promoted phagocytosis and autophagic degradation of MRSA via activating p110β (a member of Class IA phosphoinositide 3-kinases) mediated autophagic response. QKI expression in macrophages led to the sequestration of p110β in mRNA processing (P) bodies and translational repression. Upon infection, the direct interaction of RNF6, a RING-type E3 ligase, mediated QKI ubiquitination degradation and facilitated PI3K-p110β related autophagic removal of pathogen. The administration of nanoparticles with QKI specific siRNA significantly protected mice from MRSA infection. Conclusions This study disclosed the novel function of QKI in the P body mRNA regulation during infection. QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K-p110β dependent autophagy. It suggested that QKI may serve as a potential theranostic marker in MRSA-induced sepsis. Graphical Abstract

Background: Lacrimal adenoid cystic carcinoma (LACC) is the most common orbital malignant epithelial neoplasm. LACC with high-grade transformation (LACC-HGT) has higher rates of recurrence, metastasis, and mortality than LACC without HGT. This study investigated the effects of microRNA-29a-3p (miR-29a-3p) in the pathogenesis of LACC-HGT. Methods: An Agilent human miRNA microarray was used to screen the differentially expressed miRNAs (DEMs) in LACC and LACC-HGT tumor tissues. Then, the primary cells obtained in previous studies were used to determine the effect of miR-29a-3p. Results: The expression of miR-29a-3p was abnormally lower in LACC-HGT than in LACC. miR-29a-3p can specifically target the 3ʹ UTR of Quaking mRNA and down-regulate Quaking expression, thereby inhibiting the proliferation, migration, and epithelial–mesenchymal transition of LACC cells. Conclusions: This study illustrated that miR-29a-3p functions as a tumor suppressor by down-regulating the expression of Quaking to inhibit the tumorigenesis of LACC cells. This study may also reveal the pathogenesis of HGT in LACC cells and provide a reference for LACC-HGT targeted diagnosis. [ABSTRACT FROM AUTHOR]

Background and objectives: Cancer stem cells (CSCs) may contribute to tumor initiation, distant metastasis, and chemo-resistance. Quaking (QKI) is a RNA binding protein, a tumor suppressor, and a well-known stem cell biomarker in central nervous system (CNS) cancer. The aim of this study was to identify the potential of QKI mRNA as a prognostic marker for CNS cancer. Methods: The Cancer Genome Atlas (TCGA) was investigated for gene expression profile within CNS cancer data. Further analysis was done through cBioPortal and COSMIC to explore the QKI gene mutation(s). Moreover, QKI mRNA levels were evaluated by using SAGE Genie tools. The Kaplan-Meier Plotter was utilized to identify prognostic role of QKI mRNA levels in these cancers. Results: Higher levels of QKI mRNA were detected in brain cancer tissues. Altered QKI gene expression was observed in 2% (56/2,958) of patients. Missense QKI gene mutation rate was 35.29%. The QKI gene alterations led to deleterious amino acid changes, including P181R, Q112P, and A220G. Altered QKI gene expression was significantly correlated with reduced survival rate (p

Recognition of the intron branchpoint during spliceosome assembly is a multistep process that defines both mRNA structure and amount. A branchpoint sequence motif UACUAAC is variably conserved in eukaryotic genomes, but in some organisms more than one protein can recognize it. Here we show that SF1 and Quaking (QKI) compete for a subset of intron branchpoints with the sequence ACUAA. SF1 activates exon inclusion through this sequence, but QKI represses the inclusion of alternatively spliced exons with this intron branchpoint sequence. Using mutant reporters derived from a natural intron with two branchpoint-like sequences, we find that when either branchpoint sequence is mutated, the other is used as a branchpoint, but when both are present, neither is used due to high affinity binding and strong splicing repression by QKI. QKI occupancy at the dual branchpoint site directly prevents SF1 binding and subsequent recruitment of spliceosome-associated factors. Finally, the ectopic expression of QKI in budding yeast (which lacks QKI ) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of branchpoint sequences that closely mirror its high affinity binding site. This suggests that QKI and degenerate branchpoint sequences may have co-evolved as a means through which specific gene expression patterns could be maintained in QKI-expressing or non-expressing cells in metazoans, plants, and animals.

Background: Sepsis is a fatal condition commonly caused by Methicillin-resistant Staphylococcus aureus (MRSA) with a high death rate. Macrophages can protect the host from various microbial pathogens by recognizing and eliminating them. Earlier we found that Quaking (QKI), an RNA binding protein (RBP), was involved in differentiation and polarization of macrophages. However, the role of QKI in sepsis caused by pathogenic microbes, specifically MRSA, is unclear. This study aimed to investigate the role of QKI in regulation of host–pathogen interaction in MRSA-induced sepsis and explored the underlying mechanisms. Methods: Transmission electron microscope and immunofluorescence were used to observe the autophagy level in macrophages. Real-time PCR and western blot were used to analyzed the expression of mRNA and protein respectively. The potential protein interaction was analyzed by iTRAQ mass spectrometry and Immunoprecipitation. RNA fluorescence in situ hybridization, dual-luciferase reporter assay and RNA immunoprecipitation were used to explore the mechanism of QKI regulating mRNA of PI3K-p110β. Results: The mRNA level of QKI was aberrantly decreased in monocytes and PBMCs of septic patients with the increasing level of plasma procalcitonin (PCT). Then the mice with myeloid specific knockout of QKI was challenged with MRSA or Cecal Ligation and Puncture (CLP). Mice in these two models displayed higher survival rates and lower bacterial loads. Mechanistically, QKI deletion promoted phagocytosis and autophagic degradation of MRSA via activating p110β (a member of Class IA phosphoinositide 3-kinases) mediated autophagic response. QKI expression in macrophages led to the sequestration of p110β in mRNA processing (P) bodies and translational repression. Upon infection, the direct interaction of RNF6, a RING-type E3 ligase, mediated QKI ubiquitination degradation and facilitated PI3K-p110β related autophagic removal of pathogen. The administration of nanoparticles with QKI specific siRNA significantly protected mice from MRSA infection. Conclusions: This study disclosed the novel function of QKI in the P body mRNA regulation during infection. QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K-p110β dependent autophagy. It suggested that QKI may serve as a potential theranostic marker in MRSA-induced sepsis. [ABSTRACT FROM AUTHOR]

Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a major reason to cause viral encephalitis, with 50% patients suffering from severe neuro-inflammation and permanent neural injury. Effective anti-viral treatment is urgently needed. Here, we found RNA binding protein quaking (QKI) was involved in the progression of JE by regulating migration and anti-viral response of macrophages. After JE virus (JEV) infection, QKI-deficient mice had lower viral loads in the brain and fewer neurological symptoms. In comparison with control mice, proinflammatory cytokines in the brain of QKI-deficient animals revealed distinct patterns, with lower levels of IL-6 (interleukin-6) and IFN- β (interferon- β) at the early stage but higher levels at the end of JE. Then we found infiltration of CCR2 positive ((C-C motif) receptor 2) peripheral macrophages and CCR2 expression on macrophages were inhibited in QKI-deficient mice, while the expression of CCR2 ligands was not changed. Bioinformatical analysis showed that a QRE (quaking response element) located on 3′UTR (untranslated region) of Ccr2. We further verified that QKI was able to interact with Ccr2 mRNA and regulate its degradation in vitro. Additionally, since the IFN- β production was increased in QKI-ablation mice after JEV infection, the anti-viral response was analyzed. Results in QKI-silenced N9 cells showed that the expression of RIG-I (retinoic acid-inducible gene-I) and TBK1 (TANK binding kinase 1) was increased, thus further inducing IRF3 (interferon regulatory factor 3) phosphorylation and interferon activation. Overall, these results revealed QKI mediated the anti-viral process via interfering migration of macrophages to CNS (central nervous system) and enhancing RIG-I/IRF3/IFN- β pathway to restrict virus dissemination. • Myeloid QKI deficiency protected mice from JEV infection. • QKI deficiency inhibited local neuroinflammation by restricting infiltration of peripheral macrophages into CNS. • Recruitment of macrophages into CNS reduced in QKI-depletion mice by QKI interacting with mRNA of Ccr2 directly. • QKI silenced cells exerted enhanced anti-viral response through RIG-I/IRF3/IFN- β pathway. [ABSTRACT FROM AUTHOR]

Quaking protein isoforms arise from a single Quaking gene and bind the same RNA motif to regulate splicing, translation, decay, and localization of a large set of RNAs. However, the mechanisms by which Quaking expression is controlled to ensure that appropriate amounts of each isoform are available for such disparate gene expression processes are unknown. Here we explore how levels of two isoforms, nuclear Quaking-5 (Qk5) and cytoplasmic Qk6, are regulated in mouse myoblasts. We found that Qk5 and Qk6 proteins have distinct functions in splicing and translation, respectively, enforced through differential subcellular localization. We show that Qk5 and Qk6 regulate distinct target mRNAs in the cell and act in distinct ways on their own and each other's transcripts to create a network of autoregulatory and cross-regulatory feedback controls. Morpholino-mediated inhibition of Qk translation confirms that Qk5 controls Qk RNA levels by promoting accumulation and alternative splicing of Qk RNA, whereas Qk6 promotes its own translation while repressing Qk5. This Qk isoform cross-regulatory network responds to additional cell type and developmental controls to generate a spectrum of Qk5/Qk6 ratios, where they likely contribute to the wide range of functions of Quaking in development and cancer.

Background and objectives: Cancer stem cells (CSCs) may contribute to tumor initiation, distant metastasis, and chemo-resistance. Quaking (QKI) is a RNA binding protein, a tumor suppressor, and a well-known stem cell biomarker in central nervous system (CNS) cancer. The aim of this study was to identify the potential of QKI mRNA as a prognostic marker for CNS cancer. Methods: The Cancer Genome Atlas (TCGA) was investigated for gene expression profile within CNS cancer data. Further analysis was done through cBioPortal and COSMIC to explore the QKI gene mutation(s). Moreover, QKI mRNA levels were evaluated by using SAGE Genie tools. The Kaplan-Meier Plotter was utilized to identify prognostic role of QKI mRNA levels in these cancers. Results: Higher levels of QKI mRNA were detected in brain cancer tissues. Altered QKI gene expression was observed in 2% (56/2,958) of patients. Missense QKI gene mutation rate was 35.29%. The QKI gene alterations led to deleterious amino acid changes, including P181R, Q112P, and A220G. Altered QKI gene expression was significantly correlated with reduced survival rate (p<0.05). Conclusion: The QKI gene is most expressed in brain tissues. In patients with gliomas, altered QKI expression/mutation is associated with a shorter survival rate. The findings of this study indicate that the QKI gene mutations can be considered as a potential prognostic biomarker for brain malignancies. [ABSTRACT FROM AUTHOR]

Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms.

RNA-binding proteins (RBPs) form complexes with RNA, changing how the RNA is processed and thereby regulating gene expression. RBPs are important sources of gene regulation during organogenesis, including the development of lungs. The RBP called Quaking (QK) is critical for embryogenesis, yet it has not been studied in the developing lung. Here, we show that QK is widely expressed during rat lung development and into adulthood. The QK isoforms QK5 and QK7 colocalize to the nuclei of nearly all lung cells. QK6 is present in the nuclei and cytoplasm of mesenchymal cells and is only present in the epithelium during branching morphogenesis. QK knockdown in embryonic lung explants caused a greater number of multiciliated cells to appear in the airways, at the expense of basal cells. The mRNA of multiciliated cell genes and the abundance of FOXJ1/SOX2 + cells increased after knockdown, whereas P63/SOX2 + cells decreased. The cytokine IL-6, a known regulator of multiciliated cell differentiation, had increased mRNA levels after QK knockdown, although protein levels remained unchanged. Further studies are necessary to confirm whether QK acts as a blocker for the IL-6-induced differentiation of basal cells into multiciliated cells, and a conditional QK knockout would likely lead to additional discoveries on QK’s role during lung development. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) is an effective chemotherapy. However, its usage has been associated with adverse effects. Salidroside (SAL) is an antioxidative drug, which confers protective effects against several diseases. Salidroside can attenuate cardiac dysfunction induced by DOX. Quaking (QKI) is identified as a protective factor that can inhibit cardiotoxicity medicated by DOX through the regulation of cardiac circular RNA expression. The present study investigated the role of QKI on the protective effect of SAL in the DOX-induced cardiotoxicity model. Results indicated that SAL attenuated DOX-induced adverse effects, including cardiac dysfunction, weight loss, and reactive oxygen species (ROS) production, and decreased the expression of BAX, caspase 3, and FoxO1. Also, it increased the Mn-SOD2 and QKI expression in vivo and in vitro. Furthermore, QKI knockdown suppressed anti-cardiotoxicity mediated by SAL. In conclusion, the results of the current study show that salidroside attenuates doxorubicin-induced cardiac dysfunction through activation of QKI/FoxO1 pathway. [ABSTRACT FROM AUTHOR]

In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3′UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3′UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages. [ABSTRACT FROM AUTHOR]

Background: Schizophrenia risk genes are widely investigated, but a systemic analysis of miRNAs contributing to schizophrenia is lacking. Methods: Schizophrenia-associated genetic loci profiles were derived from a genome-wide association study (GWAS) from the Schizophrenia Working Group of the Psychiatric Genomics Consortium (PGC) dataset. Experimentally confirmed relationships between miRNAs and their target genes were retrieved from a miRTarBase. A competitive gene set association analysis for miRNA-target regulations was conducted by the Multi-marker Analysis of GenoMic Annotation (MAGMA) and further validated by literature-based functional pathway analysis using Pathway Studio. The association between the targets of three miRNAs and schizophrenia was further validated using a GWAS of antipsychotic treatment responses. Results: Three novel schizophrenia-risk miRNAs, namely, miR-208b-3p, miR-208a-3p, and miR-494-5p, and their targetomes converged on calcium voltage-gated channel subunit alpha1 C (CACNA1C) and B-cell lymphoma 2 (BCL2), and these are well-known contributors to schizophrenia. Both miR-208a-3p and miR-208b-3p reduced the expression of the RNA-binding protein Quaking (QKI), whose suppression commonly contributes to demyelination of the neurons and to ischemia/reperfusion injury. On the other hand, both QKI and hsa-miR-494-5p were involved in gliomagenesis. Conclusion: Presented results point at an orchestrating role of miRNAs in the pathophysiology of schizophrenia. The sharing of regulatory networks between schizophrenia and other pathologies may explain higher cardiovascular mortality and lower odds of glioma previously reported in psychiatric patients. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) is a broad-spectrum anticancer drug with considerable cardiotoxicity. DOX can induce myocardial apoptosis by modulating multiple signalling pathways. A better understanding of the underlying mechanism of DOX's cardiotoxicity will improve its clinical application and help avoid heart failure in patients. Models of DOX cardiotoxicity in cultured cardiomyocytes and mice were used. Cell death was determined by TUNEL and caspase 3/7 activity assay. Quaking (QKI) expression was detected by immunoblotting; microRNA-31-5p and circular RNA (circRNA) levels were determined by qRT-PCR. Luciferase reporter assays were performed to validate the miR-31-5p target. We found that DOX treatment upregulated miR-31-5p expression both in cultured cardiomyocytes and in mouse heart tissue. Silencing of miR-31-5p significantly alleviated the myocardial apoptosis induced by DOX treatment both in vivo and in vitro. Further analysis indicated QKI as a direct target of miR-31-5p, which has been reported to influence circRNA expression in a series of cell types. We found that circPan3 was specifically downregulated in cardiomyocytes upon DOX treatment. We further confirmed that the downregulation of circPan3 was due to the silencing of QKI by miR-31-5p. Our data reveal links among miR-31-5p, QKI and circPan3 in the apoptotic programme of cardiomyocytes. MiR-31-5p acted as a negative regulator of circPan3 by directly suppressing QKI, which may be a potential therapeutic target and strategy for DOX-induced cardiotoxicity. • MiR-31-5p involves in cardiotoxicity and promotes DOX-induced cardiotoxicity. • QKI participates in cardiotoxicity as a direct target of miR-31-5p. • QKI can facilitate circPan3 formation, thereby, participates in cardiotoxicity. • We reveal that miR-31-5p-QKI-circPan3 axis can regulate DOX-induced cardiotoxicity. [ABSTRACT FROM AUTHOR]

Exploration of the molecular mechanisms of mesenchymal stem cell (MSC) growth has significant clinical benefits. Long non-coding RNAs (lncRNAs) have been reported to play vital roles in the regulation of the osteogenic differentiation of MSCs. However, the mechanism by which lncRNA affects the proliferation and apoptosis of MSCs is unclear. In this study, sequencing analysis revealed that LINC00707 was significantly decreased in non-adherent human MSCs (non-AC-hMSCs) compared to adherent human MSCs. Moreover, LINC00707 overexpression promoted non-AChMSC proliferation, cell cycle progression from the G0/G1 phase to the S phase and inhibited apoptosis, whereas LINC00707 silencing had the opposite effect. Furthermore, LINC00707 interacted directly with the quaking (QKI) protein and enhanced the E3 ubiquitin-protein ligase ring finger protein 6 (RNF6)-mediated ubiquitination of the QKI protein. Additionally, the overexpression of QKI rescued the promotive effects on proliferation and inhibitory effects on apoptosis in non-AC-hMSCs induced by the ectopic expression of LINC00707. Thus, LINC00707 contributes to the proliferation and apoptosis in non-AChMSCs by regulating the ubiquitination and degradation of the QKI protein. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

The mammalian homolog of Drosophila empty spiracles 2 (Emx2) is a homeobox transcription factor that plays central roles in early development of the inner ear, pelvic and shoulder girdles, cerebral cortex, and urogenital organs. The role for Emx2 is best understood within the context of the development of the neocortical region of the cortex, where Emx2 is expressed in a high posterior-medial to low anterior-lateral gradient that regulates the partitioning of the neocortex into different functional fields that perform discrete computational tasks. Despite several lines of evidence demonstrating an Emx2 concentration-dependent mechanism for establishing functional areas within the developing neocortex, little is known about how Emx2 physically carries out this role. Although several binding partners for Emx2 have been identified (including Sp8, eIF4E, and Pbx1), no screens have been used to identify potential protein binding partners for this protein. We utilized a yeast two-hybrid screen using a library constructed from embryonic mouse cDNA in an attempt to identify novel binding partners for Emx2. This initial screen isolated two potential Emx2-binding partner proteins, Cnot6l and QkI-7. These novel Emx2-binding proteins are involved in multiple levels of mRNA metabolism that including splicing, mRNA export, translation, and destruction, thus making them interesting targets for further study. [ABSTRACT FROM AUTHOR]

In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1) quaking viable mice (qkv) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre (QKIFL/FL;LysM-Cre mice), we demonstrate that the abrogation of QKI expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The qkv and QKIFL/FL;LysM-Cre mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from QKIFL/FL;LysM-Cre mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases.

Mutations in human tuberous sclerosis complex (TSC) genes TSC1 and TSC2 are the leading causes of developmental brain abnormalities and large tumors in other tissues. Murine Tsc1/2 have been shown to negatively regulate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in most tissues, and this pathway has been shown to be essential for proper oligodendrocytes/Schwann cell differentiation and myelination. Here, we report that ablation of Tsc1 gene specifically in oligodendrocytes/Schwann cells activates mTORC1 signaling resulting in severe motor disabilities, weight loss, and early postnatal death. The mutant mice of either sex showed reduced myelination, disrupted paranodal domains in myelinated axons, and disorganized unmyelinated Remak bundles. mRNA and protein expression analyses revealed strong reduction in the RNA–binding protein Quaking (Qk) and the 155 kDa glial Neurofascin (NfascNF155). Re-introduction of exogenous Qk gene in Tsc1 mutant oligodendrocytes restored NfascNF155 protein levels indicating that Qk is required for the stabilization of NfascNF155 mRNA. Interestingly, injection of Rapamycin, a pharmacological mTORC1 inhibitor, to pregnant mothers increased the lifespan of the mutant offspring, restored myelination as well as the levels of Qk and NfascNF155, and consequently the organization of the paranodal domains. Together our studies show a critical role of mTORC1 signaling in the differentiation of myelinating glial cells and proper organization of axonal domains and provide insights into TSC-associated myelinated axon abnormalities.

Odontoblastic differentiation of human dental pulp stem cells (hDPSCs) is essential for the formation of reparative dentin after dental caries or injury. Our previous studies have demonstrated that krüppel‐like factor 4 (KLF4) is a critical transcription factor that promotes the odontoblastic differentiation of hDPSCs. Analysis of the microRNA binding sites within the 3′‐UTR of KLF4 revealed that QKI, an RNA‐binding protein, shared the most microRNAs with KLF4, presumably served as a “competent endogenous RNA (ceRNA)” with KLF4. Thus, we hypothesized QKI could also promote odontoblastic differentiation. In this study, we found QKI was up‐regulated during mouse odontoblast differentiation in vivo and hDPSCs odontoblastic differentiation in vitro. Overexpression or knockdown of QKI in hDPSCs led to the increase or decrease of odontoblast marker genes’ expressions, indicating its positive role in odontoblastic differentiation. We further validated that QKI served as a key ceRNA of KLF4 via interaction of the shared miRNAs in hDPSCs. Last, we found that, as an RNA binding protein, QKI protein could bind to, and stabilize dentin sialophosphoprotein (DSPP) mRNA, resulting in the augmented accumulation of DSP protein. Taken together, our study indicates that QKI promotes the odontoblastic differentiation of hDPSCs by acting as a ceRNA of KLF4 and as a binding protein of DSPP mRNA to stabilize its level. These two mechanisms of QKI will together positively regulate the downstream pathways and hence potentiate odontoblastic differentiation. [ABSTRACT FROM AUTHOR]

Mutations in human tuberous sclerosis complex (TSC) genes TSC1 and TSC2 are the leading causes of developmental brain abnormalities and large tumors in other tissues. Murine Tsc1/2 have been shown to negatively regulate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in most tissues, and this pathway has been shown to be essential for proper oligodendrocytes/Schwann cell differentiation and myelination. Here, we report that ablation of Tsc1 gene specifically in oligodendrocytes/Schwann cells activates mTORC1 signaling resulting in severe motor disabilities, weight loss, and early postnatal death. The mutant mice of either sex showed reduced myelination, disrupted paranodal domains in myelinated axons, and disorganized unmyelinated Remak bundles. mRNA and protein expression analyses revealed strong reduction in the RNA–binding protein Quaking (Qk) and the 155 kDa glial Neurofascin (NfascNF155). Re-introduction of exogenous Qk gene in Tsc1 mutant oligodendrocytes restored NfascNF155 protein levels indicating that Qk is required for the stabilization of NfascNF155 mRNA. Interestingly, injection of Rapamycin, a pharmacological mTORC1 inhibitor, to pregnant mothers increased the lifespan of the mutant offspring, restored myelination as well as the levels of Qk and NfascNF155, and consequently the organization of the paranodal domains. Together our studies show a critical role of mTORC1 signaling in the differentiation of myelinating glial cells and proper organization of axonal domains and provide insights into TSC-associated myelinated axon abnormalities. [ABSTRACT FROM AUTHOR]

Abstract: Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing. [ABSTRACT FROM AUTHOR]

The androgen receptor (AR) has a crucial role in prostate cancer. RNA‑binding protein‑mediated post‑transcriptional regulation is important in the initiation and development of cancer. The present study attempted to elucidate the mutual association of AR and RNA‑binding protein quaking (QKI) in the development of prostate cancer. Dual‑luciferase reporter demonstrated that AR can positively regulate the expression of QKI in prostate cancer cell lines due to its effective transcription regulating function. In addition, QKI may increase expression of AR by heat shock protein 90, which is a coactivator of AR, and silencing QKI can increase the sensitive of Casodex, which is an antagonist of AR in castration‑resistant prostate cancer. This may be a new strategy for advanced prostate cancer. [ABSTRACT FROM AUTHOR]

Noise exposure causes auditory nerve (AN) degeneration and hearing deficiency, though the proximal biological consequences are not entirely understood. Most AN fibers and spiral ganglion neurons are ensheathed by myelinating glia that provide insulation and ensure rapid transmission of nerve impulses from the cochlea to the brain. Here we show that noise exposure administered to mice of either sex rapidly affects myelinating glial cells, causing molecular and cellular consequences that precede nerve degeneration. This response is characterized by demyelination, inflammation, and widespread expression changes in myelin-related genes, including the RNA splicing regulator Quaking (QKI) and numerous QKI target genes. Analysis of mice deficient in QK1 revealed that QK1 production in cochlear glial cells is essential for proper myelination of spiral ganglion neurons and AN fibers, and for normal hearing. Our findings implicate QKI dysregulation as a critical early component in the noise response, influencing cochlear glia function that leads to AN demyelination and, ultimately, to hearing deficiency. [ABSTRACT FROM AUTHOR]

Macrophages, characterized by considerable diversity and plasticity, play a crucial role in a broad spectrum of biological processes, including inflammation. However, the molecular mechanisms underlying the diverse phenotypes of macrophages are not well defined. Here, we show that the RNA-binding protein, quaking (QKI), dynamically modulates macrophage polarization states. After lipopolysaccharide (LPS) stimulation, QKI-silenced RAW 264.7 cells displayed a pro-inflammatory M1 phenotype characterized by increased expression of iNOS, TNF-α, and IL-6 and decreased expression of anti-inflammatory factors, such as IL-10, found in inflammatory zone (Fizz1), and chitinase-like 3 (Chil3 or Ym1). By contrast, QKI5 overexpression led to a suppressive phenotype resembling M2 macrophages, even under M1 differentiation conditions. Moreover, myeloid-specific QKI-deficient mice tended to be more susceptible to LPS-induced endotoxic shock, while the exogenous transfer of macrophages overexpressing QKI5 exerted a significant improving effect. This improvement corresponded to a higher proportion of M2 macrophages, in line with elevated levels of IL-10, and a decrease in levels of pro-inflammatory mediators, such as IL-6, TNF-α, and IL-1β. Further mechanistic studies disclosed that QKI was a potent inhibitor of the nuclear factor-kappa B (NF-κB) pathway, suppressing p65 expression and phosphorylation. Strikingly, reduced expression of the aryl hydrocarbon receptor (Ahr) and reduced phosphorylation of signal transducer and activator of transcription 1 in QKI-deficient cells failed to restrain the transcriptional activity of NF-κB and NRL pyrin domain containing 3 (NLRP3) activation, while restoring QKI expression skewed the above M1-like response toward an anti-inflammatory M2 state. Taken together, these findings suggest a role for QKI in restraining overt innate immune responses by regulating the Ahr/STAT1–NF-κB pathway.

The mouse monoclonal antibody marketed as anti-adenomatous polyposis coli clone CC1, often referred to as CC1, is the antibody most commonly used to specifically label mature oligodendrocytes without labeling myelin. Previous studies have shown that despite being raised against adenomatous polyposis coli, this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking 7, an RNA-binding protein that is highly up-regulated in myelinating oligodendrocytes in the central nervous system. [ABSTRACT FROM AUTHOR]

RNA binding proteins required for the maintenance of myelin and axoglial junctions are unknown. Herein, we report that deletion of the Quaking (QKI) RNA binding proteins in oligodendrocytes (OLs) using Olig2-Cre results in mice displaying rapid tremors at postnatal day 10, followed by death at postnatal week 3. Extensive CNS hypomyelination was observed as a result of OL differentiation defects during development. The QKI proteins were also required for adult myelin maintenance, because their ablation using PLP-CreERT resulted in hindlimb paralysis with immobility at ~30 d after 4-hydroxytamoxifen injection. Moreover, deterioration of axoglial junctions of the spinal cord was observed and is consistent with a loss of Neurofascin 155 (Nfascl55) isoform that we confirmed as an alternative splice target of the QKI proteins. Our findings define roles for the QKI RNA binding proteins in myelin development and maintenance, as well as in the generation of Nfascl55 to maintain healthy axoglial junctions. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are generated by backsplicing of immature RNA forming covalently closed loops of intron/exon RNA molecules. Pervasiveness, evolutionary conservation, massive and regulated expression, and posttranscriptional regulatory roles of circRNAs in eukaryotes have been appreciated and described only recently. Moreover, being easily detectable disease markers, circRNAs undoubtedly represent a molecular class with high bearing on molecular pathobiology. CircRNAs can be detected from RNAseq data using appropriate computational methods to identify the sequence reads spanning backsplice junctions that do not colinearly map to the reference genome. To this end, several programs were developed and critical assessment of various strategies and tools suggested the combination of at least two methods as good practice to guarantee robust circRNA detection. Here,we present CirComPara (http://github.com/egaffo/CirComPara), an automated bioinformatics pipeline, to detect, quantify and annotate circRNAs from RNAseq data using in parallel four different methods for backsplice identification. CirComPara also provides quantification of linear RNAs and gene expression, ultimately comparing and correlating circRNA and gene/transcript expression level. We applied our method to RNAseqdata of monocyte and macrophage samples in relation to haploinsufficiency of the RNAbinding splicing factor Quaking (QKI). The biological relevance of the results, in terms of number, types and variations of circRNAs expressed, illustrates CirComPara potential to enlarge the knowledge of the transcriptome, adding details on the circRNAome, and facilitating further computational and experimental studies.

The increasing expression of microRNA-155 (miR-155) and decreasing expression of RNA-binding protein quaking (QKI) in colon cells have been observed previously. In this study, we attempted to establish the correlation between miR-155 and QKI. In addition, we assessed whether the expression of miR-155 and QKI is linked to the proliferation and invasion capabilities of colon cells. Firstly, nineteen tumor samples, divided into two groups according to the presence or absence of lymphatic metastasis, were obtained from colon cancer patients at the First Affiliated Hospital of Wenzhou Medical University, China. The expression level of miR-155 and QKI was measured by quantitative polymerase chain reaction (qPCR). Secondly, the GES-1, SW480 and COLO205 cell lines were cultured and the expression level of QKI and miR-155 was also assessed by qPCR. Thirdly, a luciferase reporter gene assay was performed to detect the association between miR-155 and QKI, and qPCR and western blot analysis were performed to confirm the effects of miR-155 on the expression of QKI at the mRNA and protein level. Subsequently, the SW480 cells were used in the following experiments. Following treatment with miR-155 inhibitor and QKI overexpression vector, western blot analysis, propidium iodide (PI) staining and a cell scratch assay were carried out to assess the effects of miR-155 on the proliferation and invasion potential of colon cancer cells. qPCR findings revealed higher miR-155 expression and lower QKI expression in colon cancer tissues as well as the colon cancer cell lines SW480 and COLO205. The relative luciferase activity of the 3' untranslated region (3'UTR) was decreased by approximately 45% when SW480 cells stimulated by mimic-miR-155 were combined with the wild-type 3'UTR constructs. In addition, when the cells were treated with mimic-miR-155, QKI expression was significantly decreased at the mRNA and protein level. These outcomes revealed that miR-155 decreased the production of QKI by acting on the 3'UTR of the QKI gene. Furthermore, PI staining and the cell scratch assay revealed that miR-155 influenced the cell cycle and invasion abilities of colon cancer cells by directly targeting QKI and decreased the production of QKI by acting on the 3'UTR of the QKI gene. This study has demonstrated the correlation between miR-155 and QKI, in which miR-155 regulates the cell cycle and invasion ability of colon cancer cells via the modulation of QKI expression. Our study provides novel therapeutic strategies for colon cancer therapy. [ABSTRACT FROM AUTHOR]

Aims Angiogenesis is a critical component of many pathological conditions in adult tissues and is essential for embryonic development. MicroRNAs are indispensable for normal vascular development, but their exact role in regulating angiogenesis remains unresolved. Previously, we have observed that miR-214 is differentially expressed in compensatory arteriogenesis. Here, we investigated the potential role of miR-214 in the process of angiogenesis. Methods and results miR-214 is expressed in all major vascular cell types, and modulation of miR-214 levels in endothelial cells significantly affected tubular sprouting. In vivo silencing of miR-214 enhanced the formation of a perfused vascular network in implanted Matrigel plugs and retinal developmental angiogenesis in mice. miR-214 directly targets Quaking, a protein critical for vascular development. Quaking knockdown reduced pro-angiogenic growth factor expression and inhibited endothelial cell sprouting similar to miR-214 overexpression. In accordance, silencing of miR-214 increased the secretion of pro-angiogenic growth factors, including vascular endothelial growth factor, and enhanced the pro-angiogenic action of the endothelial cell-derived conditioned medium, whereas miR-214 overexpression had the opposite effect. Conclusion Here, we report a novel role for miR-214 in regulating angiogenesis and identify Quaking as a direct target of miR-214. The anti-angiogenic effect of miR-214 is mediated through the down-regulation of Quaking and pro-angiogenic growth factor expression. This study presents miR-214 as a potential important target for pro- or anti-angiogenic therapies. [ABSTRACT FROM AUTHOR]

Background: A number of studies have suggested deficits in myelination and glial gene expression in different psychiatric disorders. We examined the brain expression and genetic/epigenetic regulation of QKI, an oligodendrocyte-specific RNA binding protein important for cell development and myelination. Methods: The microarray-based expression of QKI was evaluated in cortical and subcortical brain regions from suicide victims with a diagnosis of major depression (n = 16) and control subjects (n = 13). These findings were also assessed with a real-time (quantitative polymerase chain reaction [qPCR]) approach, with QKI protein levels evaluated through immunoblotting. Identification of a QKI promoter sequence was then used to examine genetic and epigenetic variation at the QKI locus. Results: The messenger RNA (mRNA) levels of multiple transcripts of QKI were evaluated on Affymetrix microarrays, revealing significant reductions in 11 cortical regions and the hippocampus and amygdala of suicide victims compared with control subjects. Microarray findings were confirmed by qPCR, and reduced expression of QKI protein was identified in orbitofrontal cortex. Analysis of promoter variation and methylation state in a subset of individuals did not identify differences at the genetic or epigenetic level between depressed suicide victims and control subjects. Conclusions: The observation of consistent reductions in multiple isoforms of QKI mRNA in depressed suicide victims supports the growing body of evidence for a role of myelination-related deficits in the etiology of psychiatric disorders. A specific role of QKI in this process is implied by its reduced expression and known interactions with genes involved in oligodendrocyte determination; however, QKI gene variation responsible for these changes remains to be identified. [Copyright &y& Elsevier]

Michele Tarter's (2004) essay, on first generation Friends and their prophecy of celestial flesh, explores the striking bodily manifestations of their spiritual experience, particularly 'quaking'. Reflecting on this, she writes: 'it is precisely what we no longer do: quake'. Using interview data from a small group of British Friends I shall show that some twenty-first-century Friends certainly do quake. I use accounts of early quaking, a variety of Quaker commentators, and historical accounts of the understanding of the body, to show the ways in which current quaking is different, and differently understood, from that of early Friends. [ABSTRACT FROM AUTHOR]