Quaking Protein Research Articles

The multifaceted role of quaking protein in neuropsychiatric disorders and tumor progression.

The Quaking protein (QKI) belongs to the STAR protein family and plays a significant role in the development of the nervous system. It serves as a crucial regulator in the processes of tumor progression and cardiovascular system development. Within the central nervous system, QKI has been associated with the onset and progression of numerous neuropsychiatric disorders, including schizophrenia, depression, ataxia, and Alzheimer's disease. In malignant tumors, the methylation of the QKI promoter inhibits its expression. QKI primarily involves in the generation, stability, and selective splicing of non-coding RNA, as well as in mRNA translation. The role of QKI in the tumor microenvironment should not be overlooked. Especially in Glioblastoma Multiforme (GBM), although QKI is not the primary mutation, it still plays a vital role in maintaining the stemness of GBM. However, the mechanisms and further studies on this topic demand extensive basic and clinical trials.

The splicing factor QKI inhibits metastasis by modulating alternative splicing of E-Syt2 in papillary thyroid carcinoma

Alternative splicing (AS) plays a crucial role in the hallmarks of cancer and can open new avenues for targeted therapies. However, the aberrant AS events and the metastatic cascade in papillary thyroid carcinoma (PTC) remain largely unclear. Here, we identify the splicing factor, quaking protein (QKI), which was significantly downregulated in PTC and correlated with poor survival outcomes in patients with PTC. Functional studies indicated that low expression of QKI promoted the PTC cell growth and metastasis in vitro and in vivo. Mechanistically, low QKI induced exon 14 retention of extended synaptotagmin 2 (E-Syt2) and produced a long isoform transcript (termed E-Syt2L) that acted as an important oncogenic factor of PTC metastasis. Notably, overexpression of long non-coding RNA eosinophil granule ontogeny transcript (EGOT) physically binds to QKI and suppressed its activity by inhibiting ubiquitin specific peptidase 25 (USP25) mediated deubiquitination and subsequent degradation of QKI. Collectively, these data demonstrate the novel mechanistic links between the splicing factor QKI and splicing event in PTC metastasis and support the potential utility of targeting splicing events as a therapeutic strategy for PTC.

Non-adherent culture method affects the proliferation and apoptosis of mesenchymal stem cells through inhibiting LINC00707 to promote RNF6-mediated QKI ubiquitination

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.

Qki is an essential regulator of microglial phagocytosis in demyelination.

The mechanism underpinning the regulation of microglial phagocytosis in demyelinating diseases is unclear. Here, we showed that the Quaking protein (Qki) in microglia was greatly induced by demyelination in the brains of both mice and humans. Deletion of the Quaking gene (Qk) in microglia severely impaired the clearance of myelin debris. Transcriptomic profiling indicated that depletion of Qki impaired total RNA levels and splicing of the genes involved in phagosome formation and maturation. RNA immunoprecipitation (RIP) confirmed the physical interactions between the Qki protein and the mRNAs of Qki targets that are involved in phagocytosis, indicating that Qki regulates their RNA stability. Both Qki depletion and inhibition of Qki target Cd36 greatly reduced the phagocytic activity of microglia and macrophages. The defective uptake and degradation of myelin debris caused by Qki depletion in microglia resulted in unresolved myelin debris that impaired axon integrity, oligodendrocyte maturation, and subsequent remyelination. Thus, our results demonstrate that Qki is an essential regulator of microglia's phagocytic activity under demyelinating conditions.

Understanding the binding specificities of mRNA targets by the mammalian Quaking protein.

Mammalian Quaking (QKI) protein, a member of STAR family of proteins is a mRNA-binding protein, which post-transcriptionally modulates the target RNA. QKI protein possesses a maxi-KH domain composed of single heterogeneous nuclear ribonucleoprotein K homology (KH) domain and C-terminal QUA2 domain, that binds a sequence-specific QKI RNA recognition element (QRE), CUAAC. To understand the binding specificities for different mRNA sequences of the KH-QUA2 domain of QKI protein, we introduced point mutations at different positions in the QRE resulting in twelve different mRNA sequences with single nucleotide change. We carried out long unbiased molecular dynamics simulations using two different sets of recently updated forcefield parameters: AMBERff14SB+RNAχOL3 and CHARMM36 (with CMAP correction). We analyzed the changes in intermolecular dynamics as a result of mutation. Our results show that AMBER forcefields performed better to model the interactions between mRNA and protein. We also calculated the binding affinities of different mRNA sequences and found that the relative order correlates to the reported experimental studies. Our study shows that the favorable binding with the formation of stable complex will occur when there is an increase of the total intermolecular contacts between mRNA and protein, but without the loss of native contacts within the KH-QUA domain.

QKI, a miR-200 target gene, suppresses epithelial-to-mesenchymal transition and tumor growth.

The microRNA-200 (miR-200) family plays a major role in specifying epithelial phenotype by preventing expression of the transcription repressors ZEB1 and ZEB2, which are well-known regulators of the epithelial-to-mesenchymal transition (EMT) in epithelial tumors including oral squamous cell carcinoma (OSCC). Here, we elucidated whether miR-200 family members control RNA-binding protein quaking (QKI), a newly identified tumor suppressor that is regulated during EMT. We predicted that miR-200a and miR-200b could recognize QKI 3'-UTR by analyzing TargetScan and The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset. Forced expression of miR-200b/a/429 inhibited expression of ZEB1/2 and decreased cell migration in OSCC cell lines CAL27 and HSC3. QKI expression was also suppressed by miR-200 overexpression, and the 3'-UTR of QKI mRNA was directly targeted by miR-200 in luciferase reporter assays. Interestingly, shRNA-mediated knockdown of QKI led to pronounced EMT and protumor effects in both in vitro and in vivo studies of OSCC. Furthermore, high expression of QKI protein is associated with favorable prognosis in surgically resected HNSCC and lung adenocarcinoma. In conclusion, QKI increases during EMT and is targeted by miR-200; while, it suppresses EMT and tumorigenesis. We suggest that QKI and miR-200 form a negative feedback loop to maintain homeostatic responses to EMT-inducing signals.

Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein

STAR proteins are evolutionary conserved mRNA-binding proteins that post-transcriptionally regulate gene expression at all stages of RNA metabolism. These proteins possess conserved STAR domain that recognizes identical RNA regulatory elements as YUAAY. Recently reported crystal structures show that STAR domain is composed of N-terminal QUA1, K-homology domain (KH) and C-terminal QUA2, and mRNA binding is mediated by KH-QUA2 domain. Here, we present simulation studies done to investigate binding of mRNA to STAR protein, mammalian Quaking protein (QKI). We carried out conventional MD simulations of STAR domain in presence and absence of mRNA, and studied the impact of mRNA on the stability, dynamics and underlying allosteric mechanism of STAR domain. Our unbiased simulations results show that presence of mRNA stabilizes the overall STAR domain by reducing the structural deviations, correlating the ‘within-domain’ motions, and maintaining the native contacts information. Absence of mRNA not only influenced the essential modes of motion of STAR domain, but also affected the connectivity of networks within STAR domain. We further explored the dissociation of mRNA from STAR domain using umbrella sampling simulations, and the results suggest that mRNA binding to STAR domain occurs in multi-step: first conformational selection of mRNA backbone conformations, followed by induced fit mechanism as nucleobases interact with STAR domain.

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.

Abstract 260: Monocyte to Macrophage Conversion is Guided by Isoform Switching of the Actin Capping Protein Y-Adducin

Background: Modulation of cellular function necessitates a versatile utilization of the transcriptome, thereby altering the proteome. These functional adaptations are post-transcriptionally guided by RNA-binding proteins (RBP), which confer cells with the capacity to rapidly respond to various stimuli and stressors. We recently discovered a pivotal role for the RBP Quaking (QKI) in directing the conversion of monocytes into macrophages, with in particular alternative splicing of pre-mRNAs critically impacting the acquisition of a pro-inflammatory phenotype. Computational analysis indicated that this differentiation process was dependent on dynamic mobilization of the actin cytoskeletal network, which prompted us to focus our attention on an exclusion event of exon 13 at the C-terminus of the actin-capping protein gamma-Adducin (ADD3). At present, the functional consequences of ADD3 isoform switching are unknown. Methods and Results: Stimulation of primary human monocytes with GM-CSF initially led to a decrease in ADD3 mRNA expression while ADD3 exon 13 usage was preserved. However, mRNA expression was restored upon differentiation towards the pro-inflammatory macrophage phenotype (t=3 days), while exon 13 inclusion strikingly dropped from 47.2% ± 10.5 to 7.7% ± 2.6 (p=0.0387, n=3). Having previously shown that QKI protein expression increases upon conversion to a macrophage, we sought to pinpoint whether QKI specifically regulates this ADD3 splicing event. Therefore, we mutated the QKI binding site (ACUAA → ACGAA) proximal to exon 13 using an ADD3 minigene. These studies revealed that disrupting the capacity for QKI to bind at this intronic sequence almost completely abolished exon 13 splicing (ACGAA: 94.9% ± 0.0025 inclusion vs. ACUAA: 2.0% ± 0.0009 inclusion; p<0.0001, n=3). Using predictive bioinformatics tools, exclusion of exon 13 was found to induce conformational changes in ADD3 protein structure. We subsequently utilized atomic force microscopy to study actomyosin dynamics and cell stiffness, pressure and tension in monocytes overexpressing the distinct ADD3 isoforms. Conclusions: QKI-mediated splicing of ADD3 triggers an isoform switch that impacts monocyte and macrophage function by altering the actin cytoskeleton.

Cross-talk between macrophages and atrial myocytes in atrial fibrillation.

Increased macrophage accumulation occurs in the atria of patients with atrial fibrillation (AF). However, the phenotype and functions of the macrophages in AF remain unclear. We investigated the macrophage-atrial myocyte interaction in AF patients and found that the increased macrophages were mainly pro-inflammatory macrophages (iNOS+, Arg1−). Tachypacing of HL-1 atrial myocytes also led to pro-inflammatory macrophage polarization. In addition, lipopolysaccharide (LPS)-stimulated pro-inflammatory macrophages-induced atrial electrical remodeling, evidenced by increased AF incidence and decreased atrial effective refractory period and L-type calcium currents (I Ca-L) in both canine and mouse AF models. Depletion of macrophages relieved LPS-induced atrial electrical remodeling, confirming the role of pro-inflammatory macrophages in the pathogenesis of AF. We also found that the effect of LPS-stimulated macrophages on atrial myocytes was mediated by secretion of interleukin 1 beta (IL-1β), which inhibited atrial myocyte quaking protein (QKI) expression. IL-1β knockout in macrophages restored the LPS-stimulated macrophage-induced inhibition of QKI and CACNA1C (α1C subunit of L-type calcium channel) in atrial myocytes. Meanwhile, QKI overexpression in atrial myocytes restored the LPS-stimulated macrophage-induced electrical remodeling through enhanced binding of QKI to CACNA1C mRNA, which upregulated the expression of CACNA1C as well as I Ca-L. In contrast, QKI knockout inhibited CACNA1C expression. Finally, using transcription factor activation profiling plate array and chromatin immunoprecipitation, we revealed that special AT-rich sequence binding protein 1 activated QKI transcription. Taken together, our study uncovered the functional interaction between macrophages and atrial myocytes in AF. AF induced pro-inflammatory macrophage polarization while pro-inflammatory macrophages exacerbated atrial electrical remodeling by secreting IL-1β, further inhibiting QKI expression in atrial myocytes, which contributed to I Ca-L downregulation. Our study demonstrates a novel molecular mechanism underlying the pathogenesis and progression of AF and suggests that QKI is a potential therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0584-z) contains supplementary material, which is available to authorized users.

Abstract 47: Quaking Post-Transcriptionally Promotes Differentiation of Monocytes Into Pro-Atherogenic Macrophages by Controling Pre-mRNA Splicing and Gene Expression

Aim: Atherosclerosis is accelerated by excessive monocyte recruitment, influx and differentiation into pro-inflammatory macrophages. While the genome-wide mRNA expression profiles of human monocytes and pro-inflammatory macrophages are well-established, their transcriptomes are ultimately defined by factors, such as RNA-binding proteins, that modulate pre-mRNA splicing patterns and mRNA transcript abundance. This prompted us to investigate the role of the RNA-binding protein Quaking in regulating global changes in pre-mRNA splicing and mature mRNA expression as human monocytes acquire the pro-inflammatory macrophage identity. Methods: We employed RNA-sequencing and splicing-sensitive microarrays to determine genome-wide changes in pre-mRNA splicing and mRNA expression upon conversion of human monocytes into pro-inflammatory macrophages, including those derived from a unique Quaking haploinsufficient patient. Results: Using laser-capture micro-dissection and immunohistochemistry, we discovered that expression levels of Quaking mRNA and protein are low in monocytes of early human atherosclerotic lesions, but abundant in macrophages of advanced plaques. Depletion of Quaking protein using both siRNA and GapmeR technology significantly impaired monocyte adhesion and migration; delayed differentiation into pro-inflammatory macrophages while maintaining the capacity to adopt the anti-inflammatory phenotype; and diminished foam cell formation in vitro and in vivo. RNA-sequencing and microarray analysis of human monocyte and macrophage transcriptomes revealed striking changes in Quaking-dependent pre-mRNA splicing and mRNA transcript levels, with gene ontology analyses identifying an enrichment in transcripts involved in cellular migration and lipid metabolism. Furthermore, these studies uncovered common as well as novel alternatively spliced transcripts with unknown biological functions in monocytes and macrophages. Conclusions: Our studies illustrate a central role for Quaking in post-transcriptionally guiding pro-inflammatory macrophage identity and function.

Methylthioadenosine (MTA) Regulates Liver Cells Proteome and Methylproteome: Implications in Liver Biology and Disease

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the adenine and methionine salvage pathways, catalyzes the hydrolysis of methylthioadenosine (MTA), a compound suggested to affect pivotal cellular processes in part through the regulation of protein methylation. MTAP is expressed in a wide range of cell types and tissues, and its deletion is common to cancer cells and in liver injury. The aim of this study was to investigate the proteome and methyl proteome alterations triggered by MTAP deficiency in liver cells to define novel regulatory mechanisms that may explain the pathogenic processes of liver diseases. iTRAQ analysis resulted in the identification of 216 differential proteins (p < 0.05) that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis led to the identification of 74 differentially methylated proteins between SK-Hep1 and SK-Hep1+ cells, including 116 new methylation sites. Restoring normal MTA levels in SK-Hep1+ cells parallels the specific methylation of 56 proteins, including KRT8, TGF, and CTF8A, which provides a novel regulatory mechanism of their activity with potential implications in carcinogenesis. Inhibition of RNA-binding proteins methylation is especially relevant upon accumulation of MTA. As an example, methylation of quaking protein in Arg(242) and Arg(256) in SK-Hep1+ cells may play a pivotal role in the regulation of its activity as indicated by the up-regulation of its target protein p27(kip1) The phenotype associated with a MTAP deficiency was further verified in the liver of MTAP± mice. Our data support that MTAP deficiency leads to MTA accumulation and deregulation of central cellular pathways, increasing proliferation and decreasing the susceptibility to chemotherapeutic drugs, which involves differential protein methylation. Data are available via ProteomeXchange with identifier PXD002957 (http://www.ebi.ac.uk/pride/archive/projects/PXD002957).

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

Abstract LB-289: Interaction between miR-155 and Quaking in the innate immune response and leukemia

Abstract Rationale: miR-155 is a pro-inflammatory microRNA upregulated in human and mouse macrophages exposed to lipopolysaccharide (LPS) that is required to mount an effective immune response. High levels of miR-155 are observed in different solid tumors as well as leukemias, including chronic lymphocytic leukemia (CLL). Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein. In silico analyses predict that the QKI transcript is a target of miR-155, and we hypothesized that miR-155 might carry out its pro-inflammatory and oncogenic signals at least in part by targeting QKI. Methods: Mouse RAW-264.7 macrophages were stimulated with LPS or mock PBS three times over a period of 6 days. qRT-PCR was used to monitor the expression of QKI, miR-155 and Tnf (tumor necrosis factor alpha), and Quaking protein (Qki) expression was analyzed by Western blot. Expression of miR-155 and QKI in Burkitt's lymphoma cell lines and CLL cell lines was measured by qRT-PCR; QKI expression was also determined by Western blot. Cell lines were transfected with miR-155 or miR-control, or 155-I, and Western blot analysis of QKI was performed after 48 hours. To study the in vivo effects of the cooperation between miR-155 and QKI, we analyzed Qki expression in splenic B cells from wild type C57B1/6 mice, miR-155−/- mice, and Eμ-miR-155 transgenic mice (which develop aggressive CLL), with and without LPS challenge. Finally, expression of Qki was determined in samples from patients with B-cell CLL. Results: After 8 hours of LPS challenge there was a 2-fold decrease in QKI expression, while miR-155 and Tnf both increased approximately 10-fold (p&amp;lt;0.05). QKI returned to pre-treatment levels at 2 days, while miR-155remained high for 3 days. LPS re-stimulation at 3 days (mimicking chronic inflammation) reduced QKI expression 2.3-fold (p&amp;lt;0.05) over a 48 hour period in parallel with a renewed up-regulation of miR-155, and Western blotting confirmed the above observed changes for Qki. CLL-derived cell lines showed significantly higher expression of miR-155, and lower expression of QKI, compared with Burkitt's lymphoma cell lines (p&amp;lt;0.05). Transfection of miR-155 led to decreased Qki expression in Burkitt's lymphoma lines, while 155-I transfection of MEC2 CLL cells caused Qki upregulation at 48 hours. Splenic B cells from leukemic Eμ-miR-155 mice had decreased Qki levels compared with C57B1/6 mice and miR-155−/−mice, and LPS challenge of cultured B cells resulted in a pronounced downregulation of Qki expression only in B cells coming from Eμ-miR-155 mice. Finally, reduced expression of Qki was observed in B cells coming from patients with CLL compared with healthy patients. Conclusions: The inverse relationship between miR-155 and Qki is specific, and may represent a regulatory mechanism for the immune response mounted by macrophages and B cells. This relationship also sheds light on the pro-inflammatory and oncogenic properties of miR-155 in leukemia. Citation Format: Timothy K. Richmond, Esmerina Tili, Melissa Brown, Marcela Chiabai, Dario Palmieri, Ri Cui, Tyler Sheetz, Carlo M. Croce. Interaction between miR-155 and Quaking in the innate immune response and leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2015-LB-289

Abstract 217: Quaking Post-Transcriptionally Guides Monocyte Adhesion and Differentiation into the Pro-Inflammatory Macrophage

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce new transcription necessary for macrophage identity, but post-transcriptional control of human macrophage differentiation is less well understood. Here, we detail our discovery that levels of the RNA-binding protein Quaking (QKI) are low in monocytes of early atherosclerotic lesions, but abundant in macrophages of advanced plaques. Specific depletion of QKI protein impaired monocyte adhesion, migration and differentiation into macrophages, and lesion formation. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal developmental changes in RNA levels and alternative splicing of RNA transcripts enriched in QKI-bound sequence elements. The importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in post-transcriptionally guiding macrophage identity and function. These studies implicate QKI as a novel target for therapeutic intervention in inflammatory diseases.

Abstract 8: RNA-binding Protein Quaking Maintains Endothelial Barrier Function Through β-catenin and VE-cadherin

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is determined largely by adherens junctions. Remarkably little is known about their regulation at the post[[Unable to Display Character: &amp;#8208;]]transcriptional level. We find that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent adult endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently we aimed to investigate the role of QKI in EC vascular integrity. Silencing of QKI markedly impaired (0.65 fold ±0.13; p&lt;0.05) the capacity to form a high resistance endothelial monolayer, as measured using Electric cell-substrate impedance sensing. To confirm a role for QKI in maintaining EC barrier function in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p&lt;0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to wild type littermates. Interestingly, the mRNA of both β-catenin and VE-cadherin, the prime adhesion proteins in EC adherens junctions, contain conserved QKI-binding sites. Moreover the targeted reduction of QKI resulted in a reduction of β-catenin and VE-cadherin protein expression. Importantly, we identified a direct role for QKI in regulating mRNA biology of β-catenin and VE-cadherin, as RNA immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the mRNA and induces transcript translation, respectively. This effects was perturbed upon reduced QKI expression. In conclusion, we show that QKI functions as a critical regulator of β-catenin and VE-cadherin in endothelial cells, and the modulation of QKI expression affects endothelial monolayer integrity, in vitro and in vivo. These studies provide novel insight into the importance of post-transcriptional regulation of components of the endothelial adherens junction, and may have wide ranging implications for the preservation of vascular integrity in disease.

Abstract 513: Endothelial Barrier Function is Maintained by the RNA-Binding Protein Quaking

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is maintained largely by adherens junctions. Remarkably, little is known about their regulation at the post–transcriptional level. We found that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar, atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently, we aimed to investigate the role of QKI in EC vascular integrity. Interestingly, the mRNA of VE-cadherin, the prime adhesion protein in EC adherens junctions, contains a conserved QKI-binding site. We identified that the targeted reduction of QKI results in a reduction of VE-cadherin expression and organization at the cell periphery. These studies revealed a direct role for QKI in regulating VE-cadherin mRNA biology, as RNA-immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the VE-cadherin mRNA and induce transcript translation (4 fold ± 0.4; p&lt;0.01), respectively. This effect was perturbed when the QKI-binding site was mutated. These results suggest that QKI acts to enhance barrier function. Overexpression of QKI markedly increased the capacity to form a high resistance endothelial monolayer (1.3 fold ± 0.96), while silencing of QKI markedly impaired EC barrier function (0.65 fold ± 0.13; p&lt;0.05). To validate in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p&lt;0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to WT littermates. In conclusion, we show that QKI functions as a critical regulator of VE-cadherin, and the modulation of QKI expression affects endothelial monolayer integrity. These studies provide novel insight into the importance of post-transcriptional regulation on endothelial barrier function, and may have wide ranging implications for the preservation of vascular integrity in disease.

Solution Structure of the QUA1 Dimerization Domain of pXqua, the Xenopus Ortholog of Quaking

The STAR protein family member Quaking is essential for early development in vertebrates. For example, in oligodendrocyte cells it regulates the splicing, localization, translation and lifetime of a set of mRNAs that code for crucial components of myelin. The Quaking protein contains three contiguous conserved regions: a QUA1 oligomerization element, followed by a single-stranded RNA binding motif comprising the KH and QUA2 domains. An embryonic lethal point mutation in the QUA1 domain, E48G, is known to affect both the aggregation state and RNA-binding properties of the murine Quaking ortholog (QKI). Here we report the NMR solution structure of the QUA1 domain from the Xenopus laevis Quaking ortholog (pXqua), which forms a dimer composed of two perpendicularly docked α-helical hairpin motifs. Size exclusion chromatography studies of a range of mutants demonstrate that the dimeric state of the pXqua QUA1 domain is stabilized by a network of interactions between side-chains, with significant roles played by an intra-molecular hydrogen bond between Y41 and E72 (the counterpart to QKI E48) and an inter-protomer salt bridge between E72 and R67. These results are compared with recent structural and mutagenesis studies of QUA1 domains from the STAR family members QKI, GLD-1 and Sam68.

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to naïve control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the naïve ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.

STAR RNA-binding protein Quaking suppresses cancer via stabilization of specific miRNA

Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFβR2 and the TGFβ signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFβ pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.