QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Isoform balance of the long noncoding RNA NEAT1 is regulated by the RNA-binding protein QKI, governs the glioma transcriptome, and impacts cell migration.

The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in a variety of human cancers. Two overlapping NEAT1 isoforms, NEAT1_1 and NEAT1_2, are produced through mutually exclusive alternative 3' end formation. Previous studies extensively investigated NEAT1 dysregulation in tumors, but often failed to achieve distinct quantification of the two NEAT1 isoforms. Moreover, molecular mechanisms governing the biogenesis of NEAT1 isoforms and the functional impacts of their dysregulation in tumorigenesis remain poorly understood. In this study, we employed an isoform-specific quantification assay and found differential dysregulation of NEAT1 isoforms in patient-derived glioblastoma multiforme cells. We further showed usage of the NEAT1 proximal polyadenylation site (PAS) is a critical mechanism that controls glioma NEAT1 isoform production. CRISPR-Cas9-mediated PAS deletion reduced NEAT1_1 and reciprocally increased NEAT1_2, which enhanced nuclear paraspeckle formation in human glioma cells. Moreover, the utilization of the NEAT1 PAS is facilitated by the RNA-binding protein quaking (QKI), which binds to the proximal QKI recognition elements. Functionally, we identified transcriptomic changes and altered biological pathways caused by NEAT1 isoform imbalance in glioma cells, including the pathway for the regulation of cell migration. Finally, we demonstrated the forced increase of NEAT1_2 upon NEAT1 PAS deletion is responsible for driving glioma cell migration and promoting the expression of genes implicated in the regulation of cell migration. Together, our studies uncovered a novel mechanism that regulates NEAT1 isoforms and their functional impacts on the glioma transcriptome, which affects pathological pathways of glioma, represented by migration.

Autogenous cross-regulation of Quaking mRNA processing and translation balances Quaking functions in splicing and translation.

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.

FOXP3-regulated lncRNA NONHSAT136151 promotes colorectal cancer progression by disrupting QKI interaction with target mRNAs.

The role of lncRNAs in the pathogenesis of cancer, including colorectal cancer (CRC), has repeatedly been demonstrated. However, very few lncRNAs have been well annotated functionally. Our study identified a novel lncRNA upregulated in CRC, NONHSAT136151, which was correlated with clinical progression. In functional assays, NONHSAT136151 significantly enhanced CRC cell proliferation, migration and invasion. Mechanistically, NONHSAT136151 interacted with RNA-binding protein (RBP) QKI (Quaking) to interfere with QKI binding to target mRNAs and regulate their expression. As well, FOXP3 may be causally related to the dysregulation of NONHSAT136151 in CRC cells through its transcriptional activity. In conclusion, our findings identified a novel lncRNA regulated by FOXP3 participates in CRC progression through interacting with QKI, indicating a novel lncRNA-RBP interaction mechanism is involved in CRC pathogenesis.

Involvement of circRNA Regulators MBNL1 and QKI in the Progression of Esophageal Squamous Cell Carcinoma.

OBJECTIVES: To investigate the role of circRNA regulators MBNL1 and QKI in the progression of esophageal squamous cell carcinoma. BACKGROUND: MBNL1 and QKI are pivotal regulators of pre-mRNA alternative splicing, crucial for controlling circRNA production - an emerging biomarker and functional regulator of tumor progression. Despite their recognized roles, their involvement in ESCC progression remains unexplored. METHODS: The expression levels of MBNL1 and QKI were examined in 28 tissue pairs from ESCC and adjacent normal tissues using data from the GEO database. Additionally, a total of 151 ESCC tissue samples, from stage T1 to T4, consisting of 13, 43, 87, and 8 cases per stage, respectively, were utilized for immunohistochemical (IHC) analysis. RNA sequencing was utilized to examine the expression profiles of circRNAs, lncRNAs, and mRNAs across 3 normal tissues, 3 ESCC tissues, and 3 pairs of KYSE150 cells in both wildtype (WT) and those with MBNL1 or QKI knockouts. Transwell, colony formation, and subcutaneous tumorigenesis assays assessed the impact of MBNL1 or QKI knockout on ESCC cell migration, invasion, and proliferation. RESULTS: ESCC onset significantly altered MBNL1 and QKI expression levels, influencing diverse RNA species. Elevated MBNL1 or QKI expression correlated with patient age or tumor invasion depth, respectively. MBNL1 or QKI knockout markedly enhanced cancer cell migration, invasion, proliferation, and tumor growth. Moreover, the absence of either MBNL1 or QKI modulated the expression profiles of multiple circRNAs, causing extensive downstream alterations in the expression of numerous lncRNAs and mRNAs. While the functions of circRNA and lncRNA among the top 20 differentially expressed genes remain unclear, mRNAs like SLCO4C1, TMPRSS15, and MAGEB2 have reported associations with tumor progression. CONCLUSIONS: This study underscores the tumor-suppressive roles of MBNL1 and QKI in ESCC, proposing them as potential biomarkers and therapeutic targets for ESCC diagnosis and treatment.

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Clinical characteristics and detection of MYB-QKI fusions in patients with angiocentric glioma.

PURPOSE: Angiocentric glioma (AG), a benign tumor identified within the last two decades, was officially included in the 2007 WHO Classification of Tumors of the Central Nervous System, WHO grade I. The tumor is relatively rare, with only approximately 100 cases reported. We aim to complement the characteristics and long-term prognosis of AG, as well as to detect MYB-QKI fusions. METHODS: The characteristics of all cases collected between 1 March 2009 and 1 March 2023 at the Beijing Sanbo Brain Hospital, Capital Medical University, were summarized and analyzed. Additionally, all fourteen patients were tested for MYB-QKI fusions. RESULTS: AG more predominantly occurs in adolescents (median age 16.5-year-old), and commonly presents with drug-resistant epilepsy. AG is frequently localized in the supratentorial regions and only one patient is in the brainstem. Brain parenchyma atrophy, and stalk-like signs can observe in imaging. Pathologically, tumor cells are perivascular pseudorosettes, presenting immunoreactivity for GFAP, S-100, Vimentin, "dot-like" staining for EMA, and low proliferative activity. Focal cortex dysplasia was observed in four patients. Twelve of fourteen (85.7%) patients were found with MYB-QKI fusions. Completely surgical resection typically has a satisfactory prognosis with long-term follow-up. CONCLUSION: AG is a rare benign tumor with a favorable prognosis after complete resection, characterized by refractory epilepsy, frequently occurring in adolescents. MYB-QKI fusions were detected in most AG patients, as a good defining genetic alteration pathologically. The potential presence of focal cortical dysplasia (FCD) may affect the prognosis of epilepsy.

RNA Binding Protein Quaking Promotes Hypoxia-induced Smooth Muscle Reprogramming in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a devastating disease characterized by progressive increases in pulmonary vascular resistance and remodeling, which eventually leads to right ventricular failure and death. The aim of this study was to identify novel molecular mechanisms involved in the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. In this study, we first demonstrated that the mRNA and protein expression amounts of QKI (Quaking), an RNA-binding protein, were elevated in human and rodent PH lung and pulmonary artery tissues and hypoxic human PASMCs. QKI deficiency attenuated PASMC proliferation in vitro and vascular remodeling in vivo. Next, we elucidated that QKI increases STAT3 (signal transducer and activator of transcription 3) mRNA stability by binding to its 3' untranslated region. QKI inhibition reduced STAT3 expression and alleviated PASMC proliferation in vitro. Moreover, we also observed that the upregulated expression of STAT3 promoted PASMC proliferation in vitro and in vivo. In addition, as a transcription factor, STAT3 bound to microRNA (miR)-146b promoter to enhance its expression. We further showed that miR-146b promoted the proliferation of smooth muscle cells by inhibiting STAT1 and TET2 (Tet methylcytosine dioxygenase 2) during pulmonary vascular remodeling. This study has demonstrated new mechanistic insights into hypoxic reprogramming that arouses vascular remodeling, thus providing proof of concept for targeting vascular remodeling by directly modulating the QKI-STAT3-miR-146b pathway in PH.

hsa_circ_0007919 induces LIG1 transcription by binding to FOXA1/TET1 to enhance the DNA damage response and promote gemcitabine resistance in pancreatic ductal adenocarcinoma.

BACKGROUND: Circular RNAs (circRNAs) play important roles in the occurrence and development of cancer and chemoresistance. DNA damage repair contributes to the proliferation of cancer cells and resistance to chemotherapy-induced apoptosis. However, the role of circRNAs in the regulation of DNA damage repair needs clarification. METHODS: RNA sequencing analysis was applied to identify the differentially expressed circRNAs. qRT-PCR was conducted to confirm the expression of hsa_circ_0007919, and CCK-8, FCM, single-cell gel electrophoresis and IF assays were used to analyze the proliferation, apoptosis and gemcitabine (GEM) resistance of pancreatic ductal adenocarcinoma (PDAC) cells. Xenograft model and IHC experiments were conducted to confirm the effects of hsa_circ_0007919 on tumor growth and DNA damage in vivo. RNA sequencing and GSEA were applied to confirm the downstream genes and pathways of hsa_circ_0007919. FISH and nuclear-cytoplasmic RNA fractionation experiments were conducted to identify the cellular localization of hsa_circ_0007919. ChIRP, RIP, Co-IP, ChIP, MS-PCR and luciferase reporter assays were conducted to confirm the interaction among hsa_circ_0007919, FOXA1, TET1 and the LIG1 promoter. RESULTS: We identified a highly expressed circRNA, hsa_circ_0007919, in GEM-resistant PDAC tissues and cells. High expression of hsa_circ_0007919 correlates with poor overall survival (OS) and disease-free survival (DFS) of PDAC patients. Hsa_circ_0007919 inhibits the DNA damage, accumulation of DNA breaks and apoptosis induced by GEM in a LIG1-dependent manner to maintain cell survival. Mechanistically, hsa_circ_0007919 recruits FOXA1 and TET1 to decrease the methylation of the LIG1 promoter and increase its transcription, further promoting base excision repair, mismatch repair and nucleotide excision repair. At last, we found that GEM enhanced the binding of QKI to the introns of hsa_circ_0007919 pre-mRNA and the splicing and circularization of this pre-mRNA to generate hsa_circ_0007919. CONCLUSIONS: Hsa_circ_0007919 promotes GEM resistance by enhancing DNA damage repair in a LIG1-dependent manner to maintain cell survival. Targeting hsa_circ_0007919 and DNA damage repair pathways could be a therapeutic strategy for PDAC.

Quaking 5 suppresses TGF-ß-induced EMT and cell invasion in lung adenocarcinoma.

Quaking (QKI) proteins belong to the signal transduction and activation of RNA (STAR) family of RNA-binding proteins that have multiple functions in RNA biology. Here, we show that QKI-5 is dramatically decreased in metastatic lung adenocarcinoma (LUAD). QKI-5 overexpression inhibits TGF-ß-induced epithelial-mesenchymal transition (EMT) and invasion, whereas QKI-5 knockdown has the opposite effect. QKI-5 overexpression and silencing suppresses and promotes TGF-ß-stimulated metastasis in vivo, respectively. QKI-5 inhibits TGF-ß-induced EMT and invasion in a TGFßR1-dependent manner. KLF6 knockdown increases TGFßR1 expression and promotes TGF-ß-induced EMT, which is partly abrogated by QKI-5 overexpression. Mechanistically, QKI-5 directly interacts with the TGFßR1 3' UTR and causes post-transcriptional degradation of TGFßR1 mRNA, thereby inhibiting TGF-ß-induced SMAD3 phosphorylation and TGF-ß/SMAD signaling. QKI-5 is positively regulated by KLF6 at the transcriptional level. In LUAD tissues, KLF6 is lowly expressed and positively correlated with QKI-5 expression, while TGFßR1 expression is up-regulated and inversely correlated with QKI-5 expression. We reveal a novel mechanism by which KLF6 transcriptionally regulates QKI-5 and suggest that targeting the KLF6/QKI-5/TGFßR1 axis is a promising targeting strategy for metastatic LUAD.

DIPG-like MYB-altered diffuse astrocytoma with durable response to intensive chemotherapy.

Pontine gliomas represent difficult to treat entity due to the location and heterogeneous biology varying from indolent low-grade gliomas to aggressive diffuse intrinsic pontine glioma (DIPG). Making the correct tumor diagnosis in the pontine location is thus critical. Here, we report a case study of a 14-month-old patient initially diagnosed as histone H3 wild-type DIPG. Due to the low age of the patient, the MRI appearance of DIPG, and anaplastic astrocytoma histology, intensive chemotherapy based on the HIT-SKK protocol with vinblastine maintenance chemotherapy was administered. Rapid clinical improvement and radiological regression of the tumor were observed with nearly complete remission with durable effect and excellent clinical condition more than 6.5 years after diagnosis. Based on this unexpected therapeutic outcome, genome-wide DNA methylation array was employed and the sample was classified into the methylation class "Low-grade glioma, MYB(L1) altered." Additionally, RT-PCR revealed the presence of MYB::QKI fusion. Taken together, the histopathological classification, molecular-genetic and epigenetic features, clinical behavior, and pontine location have led us to reclassify the tumor as a pontine MYB-altered glioma. Our case demonstrates that more intensive chemotherapy can achieve long-term clinical effect in the treatment of MYB-altered pontine gliomas compared to previously used LGG-based regimens or radiotherapy. It also emphasizes the importance of a biopsy and a thorough molecular investigation of pontine lesions.

miR-196b-5p-mediated downregulation of TSPAN12 and GATA6 promotes tumor progression in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) accounts for over 80% of lung cancer cases. The RNA binding protein, QKI, belongs to the STAR family and plays tumor-suppressive functions in NSCLC. QKI-5 is a major isoform of QKIs and is predominantly expressed in NSCLC. However, the underlying mechanisms of QKI-5 in NSCLC progression remain unclear. We found that QKI-5 regulated microRNA (miRNA), miR-196b-5p, and its expression was significantly up-regulated in NSCLC tissues. Up-regulated miR-196b-5p promotes lung cancer cell migration, proliferation, and cell cycle through directly targeting the tumor suppressors, GATA6 and TSPAN12. Both GATA6 and TSPAN12 expressions were down-regulated in NSCLC patient tissue samples and were negatively correlated with miR-196b-5p expression. Mouse xenograft models demonstrated that miR-196b-5p functions as a potent onco-miRNA, whereas TSPAN12 functions as a tumor suppressor in NSCLC in vivo. QKI-5 bound to miR-196b-5p and influenced its stability, resulting in up-regulated miR-196b-5p expression in NSCLC. Further analysis showed that hypomethylation in the promoter region enhanced miR-196b-5p expression in NSCLC. Our findings indicate that QKI-5 may exhibit novel anticancer mechanisms by regulating miRNA in NSCLC, and targeting the QKI5∼miR-196b-5p∼GATA6/TSPAN12 pathway may enable effectively treating some NSCLCs.

QKI regulates adipose tissue metabolism by acting as a brake on thermogenesis and promoting obesity.

Adipose tissue controls numerous physiological processes, and its dysfunction has a causative role in the development of systemic metabolic disorders. The role of posttranscriptional regulation in adipose metabolism has yet to be fully understood. Here, we show that the RNA-binding protein quaking (QKI) plays an important role in controlling metabolic homeostasis of the adipose tissue. QKI-deficient mice are resistant to high-fat-diet (HFD)-induced obesity. Additionally, QKI depletion increased brown fat energy dissipation and browning of subcutaneous white fat. Adipose tissue-specific depletion of QKI in mice enhances cold-induced thermogenesis, thereby preventing hypothermia in response to cold stimulus. Further mechanistic analysis reveals that QKI is transcriptionally induced by the cAMP-cAMP response element-binding protein (CREB) axis and restricts adipose tissue energy consumption by decreasing stability, nuclear export, and translation of mRNAs encoding UCP1 and PGC1α. These findings extend our knowledge of the significance of posttranscriptional regulation in adipose metabolic homeostasis and provide a potential therapeutic target to defend against obesity and its related metabolic diseases.

Multiple circRNAs regulated by QKI5 conjointly spongemiR-214-3p to antagonize bisphenol A-inducedspermatocyte toxicity.

Although circular RNAs (circRNAs) are found to play important roles in many pathophysiological processes, the canonical theory that they act as microRNA sponges is now more and more challenged, given that most circRNAs only have few binding sites in a particular microRNA. Our previous study revealed that some up-regulated circRNAs play protective roles in bisphenol A (BPA)-induced toxicity in GC-2 germ cells. Here by CCK-8 assay, apoptosis assay, qRT-PCR and western blot analysis, we further discover that circRNAs (represented by circDcbld2, circMapk1 and circTbcld20) can cooperatively sponge miR-214-3p and then up-regulate AKT1 in ameliorating BPA-induced reproductive toxicity. They share binding sites with miR-214-3p and collectively reinforce the sponging effects. In addition, the upstream regulation mechanism, proven by bioinformatics analysis and in vitro gain- and loss-of-function study, shows that down-regulation of RNA binding protein QKI5 after BPA exposure can increase the expressions of these protective circRNAs, and thus activate the cell protective process. The QKI5-circDcbld2/circMapk1/circTblcd20-miR-214-3p-AKT1 axis ameliorates the toxic effect of BPA on GC-2 cells. Many other circRNAs up-regulated upon BPA treatment and QKI5 down-regulation also show binding sites with miR-214-3p. Thus the above axis may also be extrapolated to other circRNAs. Our results enrich the context of circRNA sponge mode and may provide new ideas in future multiple nucleic acid therapy.

NRP1 and MMP9 are dual targets of RNA-binding protein QKI5 to alter VEGF-R/ NRP1 signalling in trophoblasts in preeclampsia.

Preeclampsia (PE) is characterized by placental ischemia and hypoxia, resulting in abnormal casting of the uterine spiral artery, which is mainly caused by insufficient trophoblastic cell infiltration. A reduction in levels of growth factor-based signalling via Neuropilin-1 (NRP1) has been shown to contribute to dysfunctional trophoblast development. In this study, we showed that the RNA-binding protein, QKI5, regulated NRP1 expression and significantly improved trophoblast proliferation in vitro and in vivo. QKI5 and NRP1 expressions were significantly reduced in human PE placentas and in trophoblasts during hypoxia. Overexpression of these factors significantly improved cell proliferation and migration in vitro, in contrast to a decrease upon siRNA knockdown of QKI5 and NRP1 in HTR-8/SVneo cells. Using RIP and RNA pull-down assays, we further showed that QKI5 directly interacted with the 3'-UTR region of NRP1, to mediate cell proliferation and migration via matrix metalloprotease-9. Further, similar to NRP1, QKI5 also targets matrix metalloproteinase 9 (MMP9) involved in secretion of growth factors and its effects can be counteracted by NRP1 overexpression. In vivo studies using a PE mouse model revealed that QKI5 overexpression alleviated PE-related symptoms such as elevated blood pressure and proteinuria. Taken together, we found that QKI5 was a novel regulator, of VEGF-R/NRP1 signalling pathway functioning in trophoblast proliferation and migration, resulting in major contributors to the pathogenesis of PE. While careful evaluation of the broad implications of QKI5 expression is still necessary, this study identified QKI5 as a promising target for treatment strategies in acute PE patients.

LncRNA HCG11/miR-26b-5p/QKI5 feedback loop reversed high glucose-induced proliferation and angiogenesis inhibition of HUVECs.

Acute coronary syndrome caused by the rupture of atherosclerotic plaques is one of the primary causes of cerebrovascular and cardiovascular events. Neovascularization within the plaque is closely associated with its stability. Long non-coding RNA (lncRNA) serves a crucial role in regulating vascular endothelial cells (VECs) proliferation and angiogenesis. In this study, we identified lncRNA HCG11, which is highly expressed in patients with vulnerable plaque compared with stable plaque. Then, functional experiments showed that HCG11 reversed high glucose-induced vascular endothelial injury through increased cell proliferation and tube formation. Meanwhile, vascular-related RNA-binding protein QKI5 was greatly activated. Luciferase reporter assays and RNA-binding protein immunoprecipitation (RIP) assays verified interaction between them. Interestingly, HCG11 can also positively regulated by QKI5. Bioinformatics analysis and luciferase reporter assays showed HCG11 can worked as a competing endogenous RNA by sponging miR-26b-5p, and QKI5 was speculated as the target of miR-26b-5p. Taken together, our findings revered that the feedback loop of lncRNA HCG11/miR-26b-5p/QKI-5 played a vital role in the physiological function of HUVECs, and this also provide a potential target for therapeutic strategies of As.

Identification and characterization of host proteins bound to dengue virus 3' UTR reveal an antiviral role for quaking proteins.

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3' untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3' UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3' UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3' UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3' UTR. Interestingly, removal of QRE from DENV4 3' UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3' UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.

The RNA binding protein Quaking represses host interferon response by downregulating MAVS.

Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNß transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNß activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNß induction. Consistently, ectopic expression of RIG-I activates stronger IFNß induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.

QKI-6 inhibits bladder cancer malignant behaviours through down-regulating E2F3 and NF-κB signalling.

Quaking homolog (QKI) is a member of the RNA-binding signal transduction and activator of proteins family. Previous studies showed that QKI possesses the tumour suppressor activity in human cancers by interacting with the 3'-untraslated region (3'-UTR) of various gene transcripts via the STAR domain. This study first assessed the association of QKI-6 expression with clinicopathological and survival data from bladder cancer patients and then investigated the underlying molecular mechanisms. Bladder cancer tissues (n = 223) were subjected to immunohistochemistry, and tumour cell lines and nude mice were used for different in vitro and in vivo assays following QKI-6 overexpression or knockdown. QKI-6 down-regulation was associated with advanced tumour TNM stages and poor patient overall survival. QKI-6 overexpression inhibited bladder cancer cell growth and invasion capacity, but induced tumour cell apoptosis and cell cycle arrest. Furthermore, ectopic expression of QKI-6 reduced tumour xenograft growth and expression of proliferation markers, Ki67 and PCNA. However, knockdown of QKI-6 expression had opposite effects in vitro and in vivo. QKI-6 inhibited expression of E2 transcription factor 3 (E2F3) by directly binding to the E2F3 3'-UTR, whereas E2F3 induced QKI-6 transcription by binding to the QKI-6 promoter in negative feedback mechanism. QKI-6 expression also suppressed activity and expression of nuclear factor-κB (NF-κB) signalling proteins in vitro, implying a novel multilevel regulatory network downstream of QKI-6. In conclusion, QKI-6 down-regulation contributes to bladder cancer development and progression.

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR-9-5p/QKI-5 axis.

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT-PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR-9-5p and QKI-5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK-8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down-regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR-9-5p, whereas miR-9-5p down-regulated QKI-5 expression. Overexpressed MEG3 and QKI-5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR-9-5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up-regulated expression of QKI-5 in vivo. LncRNA MEG3 was a down-regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR-9-5p and its targeting gene QKI-5.