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Alterative splicing patterns of qkI in QKI FL/FL;Olig2-Cre and QKI FL/FL;-mice. (a) IGV view of the alternative splicing events in qkI. All samples are represented on the same scale for each screenshot. (b) A schematic of the 3′-UTR of mouse qkI gene showing the location of Quaking Response Elements (QREs) identified. QREs are either a perfect match to consensus QRE or differ by one mismatch.
Source publication
The qkI gene encodes a family of RNA binding proteins alternatively spliced at its 3′ end, giving rise to three major spliced isoforms: QKI-5, QKI-6 and QKI-7. Their expression is tightly regulated during brain development with nuclear QKI-5 being the most abundant during embryogenesis followed by QKI-6 and QKI-7 that peak during myelination. Previ...
Citations
... We verified the induction of EMT in the mesHMLE samples by confirming downregulation of epithelial markers E-cadherin and ESRP1 and upregulation of mesenchymal markers ZEB1, Vimentin, N-cadherin, and QKI (Supplementary Figure S1C, Supplementary Table S3 and 4). Transfection of mesHMLE with the QKI-5 siRNA led to reduced expression of each of the major QKI isoforms (Supplementary Figure S1B, consistent with its known role in self-splicing its own transcript [28,29]. Therefore, while QKI-5 is the predominant isoform in these cells, the QKI-5 siRNA treatment serves effectively as a total QKI knockdown as reflected in loss of total QKI protein (see Figure 2E). ...
... While the finding that QKI regulates its own APA has not been previously reported, the ability of QKI to self-regulate is known [28,29]. Indeed, the tendency of RBPs to auto-regulate is a well-established phenomenon, and auto-regulation of APA has been reported for other proteins of this class [45]. ...
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.
... Certain studies showed no change in oligodendrocyte density in MDD, suggesting unchanged morphology despite possible organizational disruption (Williams et al., 2015;Ö ngür et al., 1998). The disruption might result from altered myelin-related gene expression or splicing (Aston et al., 2005;Klempan et al., 2009a;Darbelli et al., 2017); and consequently present in myelin related proteins, or as abnormalities in transcription factors of myelin-related genes (Tham et al., 2011). ...
... When it binds downstream of the alternative splicing region, however, it promotes skipping [14]. The QKI proteins are localized on mouse and human chromosomes 17 and 6, respectively; they encode the isoforms QKI-5, QKI-6 and QKI-7, which share the same K homology (KH) structural domain [15,16]. In mouse myoblasts, different variants display a network of autoregulating and cross-regulating QKI protein isoforms [17]. ...
... This discovery is consistent with the results of some previous studies. The QKI-5 automatically regulates its own alternative splicing through a self-splicing mechanism and was described as a major regulator of RNA metabolism in oligodendrocytes [16]. In lung cells, QKI-5 is the dominant isoform at the mRNA and protein levels. ...
Simple Summary
Meat goats are highly valued as excellent sources of lean dietary muscle-protein. However, the molecular mechanisms by which muscle cells and tissues are generated and the genes and signaling pathways regulating this process in meat goats are poorly understood. The present study applied currently conventional molecular-genetics methods and determined that the major isoform of the QKI gene drive the conversion of embryonic myoblast cells into mature muscle fiber and tissue. These genes activate other genetic factors and signaling pathways that are all implicated in the differentiation of muscle progenitor cells and the formation of mature, differentiated muscle tissue. Although future investigations will be necessary to elucidate the entire molecular mechanism involved, we believe that the discoveries reported herein will facilitate the genetic engineering of novel goat lines with enhanced musculature and, by extension, superior meat yield and quality, breeder profitability, and consumer satisfaction.
Abstract
The QKI genes encode RNA-binding proteins regulating cell proliferation, differentiation, and apoptosis. The Goat QKI has six isoforms, but their roles in myogenesis are unclear. In this study, the six isoforms of the QKI gene were overexpressed in goat myoblast. Immunofluorescence, qPCR and Western blot were used to evaluate the effect of QKI on the differentiation of goat myoblast. An RNA-Seq was performed on the cells with the gain of the function from the major isoforms to screen differentially expressed genes (DEGs). The results show that six isoforms had different degrees of deletion in exons 6 and 7, and caused the appearance of different types of encoded amino acids. The expression levels of the QKI-1 and QKI-5 groups were upregulated in the biceps femoris and latissimus dorsi muscle tissues compared with those of the QKI-4, QKI-7, QKI-3 and QKI-6 groups. After 6 d of myoblast differentiation, QKI-5 and the myogenic differentiators MyoG, MyoD, and MyHC were upregulated. Compared to the negative control group, QKI promoted myotube differentiation and the myoblasts overexpressing QKI-5 formed large, abundant myotubes. In summary, we identified that the overexpression of the QKI gene promotes goat-myoblast differentiation and that QKI-5 is the major isoform, with a key role. The RNA-Seq screened 76 upregulated and 123 downregulated DEGs between the negative control and the QKI-5-overexpressing goat myoblasts after d 6 of differentiation. The GO and KEGG analyses associated the downregulated DEGs with muscle-related biological functions. Only the pathways related to muscle growth and development were enriched. This study provides a theoretical basis for further exploring the regulatory mechanism of QKI in skeletal-muscle development in goats.
... Its roles in alternative splicing are important for NPC differentiation and, like Rbfox1, Qki isoforms are present in specific tissues and developmental stages [187]. Remarkably, Qki in oligodendrocytes autoregulates its own splicing events [188]. Qki5 has been shown to regulate targets in early embryonic neural stem cells. ...
... Axon development and microtubule dynamics were shown to be two processes dependent on Qki5 function [189]. Isoform specificity further extends to differentiated cell types such as neurons and oligodendrocytes [188]. For example, Qki promotes a GABAergic neuron profile, and loss of Qki alters the gene expression to promote a glutamatergic neuron profile [189]. ...
... A comprehensive review of the role of RBPs in neuronal differentiation can be found in [190]. In oligodendrocytes, mRNAs associated with myelination are significantly downregulated [188]. Interestingly, expression and function of the RBPs themselves are also regulated, with many RBPs being essential in certain tissues and stages of development. ...
Diverse cell types in the central nervous system (CNS) are generated by a relatively small pool of neural stem cells during early development. Spatial and temporal regulation of stem cell behavior relies on precise coordination of gene expression. Well-studied mechanisms include hormone signaling, transcription factor activity, and chromatin remodeling processes. Much less is known about downstream RNA-dependent mechanisms including posttranscriptional regulation, nuclear export, alternative splicing, and transcript stability. These important functions are carried out by RNA-binding proteins (RBPs). Recent work has begun to explore how RBPs contribute to stem cell function and homeostasis, including their role in metabolism, transport, epigenetic regulation, and turnover of target transcripts. Additional layers of complexity are provided by the different target recognition mechanisms of each RBP as well as the posttranslational modifications of the RBPs themselves that alter function. Altogether, these functions allow RBPs to influence various aspects of RNA metabolism to regulate numerous cellular processes. Here we compile advances in RNA biology that have added to our still limited understanding of the role of RBPs in neurodevelopment.
... Expression levels were estimated using HOMER V4.10 (Heinz et al., 2010). Afterwards, we employed DESeq2 to normalize the raw counts as rlog variance stabilized values, as well as to perform the differential expression analysis as previously described (Darbelli et al., 2017). For the volcano plot, genes were considered differentially expressed if they had an adjusted p value <0.05, a base mean higher than 100 and an absolute fold-change greater than 2. For the heat map, genes were considered differentially expressed if the samples with the highest and lowest expression are more than 2-fold different and one of the samples has 25 normalized reads (as in the HOMER tutorial). ...
Despite the success of immune checkpoint inhibitor (ICI) therapy for cancer, resistance and relapse are frequent. Combination therapies are expected to enhance response rates and overcome this resistance. Herein, we report that combining PRMT7 inhibition with ICI therapy induces a strong anti-tumor T cell immunity and restrains tumor growth in vivo by increasing immune cell infiltration. PRMT7-deficient B16.F10 melanoma exhibits increased expression of genes in the interferon pathway, antigen presentation, and chemokine signaling. PRMT7 deficiency or inhibition with SGC3027 in B16.F10 melanoma results in reduced DNMT expression, loss of DNA methylation in the regulatory regions of endogenous retroviral elements (ERVs) causing their increased expression. PRMT7-deficient cells increase RIG-I and MDA5 expression with a reduction in the H4R3me2s repressive histone mark at their gene promoters. Our findings identify PRMT7 as a regulatory checkpoint for RIG-I, MDA5, and their ERV-double-stranded RNA (dsRNA) ligands, facilitating immune escape and anti-tumor T cell immunity to restrain tumor growth.
... One of the most established functions of QKI is the regulation of pre-mRNA alternative splicing (Wu et al., 2002). QKI has been found to regulate many alternative splicing events in different contexts including during EMT (Conn et al., 2015;Pillman et al., 2018;Yang et al., 2016), monocyte differentiation (De Bruin, Shiue, et al., 2016), myelination (Darbelli et al., 2016;Darbelli et al., 2017;Mandler et al., 2014), microglia function (Lee et al., 2020;, muscle differentiation (Hall et al., 2013), and various aspects of cancer progression (Brosseau et al., 2014;de Miguel et al., 2016;Zong et al., 2014). The nuclear isoform, QKI-5, has been found to bind primarily within introns, suggesting that its major function is regulation of splicing (Pillman et al., 2018). ...
... A feature of the QKI isoforms that complicates their study is autoregulation. Expression of QKI-5 is required to allow the formation of QKI-6 and QKI-7, which is suggested to be controlled though alternative splicing (Darbelli et al., 2017;Fagg et al., 2017). This is consistent with the presence of QKI motifs within introns of the QKI pre-mRNA; however, the mechanism behind this phenomenon has not been determined. ...
The RNA‐binding protein Quaking (QKI) has emerged as a potent regulator of cellular differentiation in developmental and pathological processes. The QKI gene is itself alternatively spliced to produce three major isoforms, QKI‐5, QKI‐6, and QKI‐7, that possess very distinct functions. Here, we highlight roles of the different QKI isoforms in neuronal, vascular, muscle, and monocyte cell differentiation, and during epithelial–mesenchymal transition in cancer progression. QKI isoforms control cell differentiation through regulating alternative splicing, mRNA stability and translation, with activities in gene transcription now also becoming evident. These diverse functions of the QKI isoforms contribute to their broad influences on RNA metabolism and cellular differentiation.
This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications
RNA Processing > Splicing Regulation/Alternative Splicing
RNA in Disease and Development > RNA in Development
... S + O + Z-transduced ADSCs expressed high levels of Qki7 mRNA under conditions that support expansion ( Figure 2J). Quaking proteins are key regulators of the metabolism of transcription factor RNA and the expression of myelin-related genes involved in oligodendrocyte differentiation (Darbelli et al., 2017). The expression of Qki7 in converted cells (but not in untransduced ADSCs) is strong evidence supporting that the Sox10, Olig2, and Zfp536 transgene combination induces the generation of oligodendroglial cells. ...
Obtaining oligodendroglial cells from dispensable tissues would be of great interest for autologous or immunocompatible cell replacement therapy in demyelinating diseases, as well as for studying myelin-related pathologies or testing therapeutic approaches in culture. We evaluated the feasibility of generating oligodendrocyte precursor cells (OPCs) from adult rat adipose tissue by expressing genes encoding transcription factors involved in oligodendroglial development. Adipose-derived mesenchymal cells were lentivirally transduced with tetracycline-inducible Sox10 , Olig2 , Zfp536 , and/or Nkx6.1 transgenes. Immunostaining with the OPC-specific O4 monoclonal antibody was used to mark oligodendroglial induction. O4- and myelin-associated glycoprotein (MAG)-positive cells emerged after 3 weeks when using the Sox10 + Olig2 + Zfp536 combination, followed in the ensuing weeks by GFAP-, O1 antigen-, p75NTR (low-affinity NGF receptor)-, and myelin proteins-positive cells. The O4 ⁺ cell population progressively expanded, eventually constituting more than 70% of cells in culture by 5 months. Sox10 transgene expression was essential for generating O4 ⁺ cells but was insufficient for inducing a full oligodendroglial phenotype. Converted cells required continuous transgene expression to maintain their glial phenotype. Some vestigial characteristics of mesenchymal cells were maintained after conversion. Growth factor withdrawal and triiodothyronine (T 3 ) supplementation generated mature oligodendroglial phenotypes, while FBS supplementation produced GFAP ⁺ - and p75NTR ⁺ -rich cultures. Converted cells also showed functional characteristics of neural-derived OPCs, such as the expression of AMPA, NMDA, kainate, and dopaminergic receptors, as well as similar metabolic responses to differentiation-inducing drugs. When co-cultured with rat dorsal root ganglion neurons, the converted cells differentiated and ensheathed multiple axons. We propose that functional oligodendroglia can be efficiently generated from adult rat mesenchymal cells by direct phenotypic conversion.
... Expression levels were estimated using HOMER V4.10 (Heinz et al., 2010). Afterwards, we employed DESeq2 to normalize the raw counts as rlog variance stabilized values, as well as to perform the differential expression analysis as previously described (Darbelli et al., 2017). For the volcano plot, genes were considered differentially expressed if they had an adjusted p value <0.05, a base mean higher than 100 and an absolute fold-change greater than 2. For the heat map, genes were considered differentially expressed if the samples with the highest and lowest expression are more than 2-fold different and one of the samples has 25 normalized reads (as in the HOMER tutorial). ...
Despite the success of immune checkpoint inhibitor (ICI) therapy in different cancers, resistance and relapses are frequent. Thus, combination therapies are expected to enhance response rates and overcome resistance to ICIs. Herein, we report that combining protein arginine methyltransferase 7 (PRMT7) inhibition with ICIs triggers a strong anti-tumor T cell immunity and restrains tumor growth in vivo by increasing tumor immune cell infiltration. Consistently, TCGA database analysis showed an inverse correlation between PRMT7 expression and T cell infiltration in human melanomas. Mechanistically, we show that PRMT7 has a two-prong effect on melanoma tumor immunity. On one hand, it serves as a coactivator of IRF-1 for PD-L1 expression by upregulating promoter H4R3me2s levels in melanoma cells. Next, PRMT7 prevents repetitive element expression to avoid intracellular dsRNA accumulation or 'viral mimicry'. PRMT7 deletion resulted in increased endogenous retroviral elements (ERVs), dsRNA, and genes implicated in interferon activation, antigen presentation and chemokine signaling. Our findings identify PRMT7 as factor used by melanoma to evade anti-tumor immunity and define the therapeutic potential of PRMT7 alone or in combination with PD-(L)1 blockade to enhance ICI efficiency.
... In the brain (Fig. 2a), neurons are myelinated in a Qki-dependent manner by oligodendrocytes [45,46]. The Qki gene encodes an RNA binding protein involved in alternative splicing [45,46], and conditional Qki deletion in mouse oligodendrocytes (Fig. 2a) results in severe hypomyelination and death of the animal [46]. ...
... In the brain (Fig. 2a), neurons are myelinated in a Qki-dependent manner by oligodendrocytes [45,46]. The Qki gene encodes an RNA binding protein involved in alternative splicing [45,46], and conditional Qki deletion in mouse oligodendrocytes (Fig. 2a) results in severe hypomyelination and death of the animal [46]. Analysis of RNA-seq comparing animals with Qki-deficient and Qki-proficient oligodendrocytes [45] revealed 1899 DE genes (Additional file 1: Supplementary Fig. S1B). ...
... The Qki gene encodes an RNA binding protein involved in alternative splicing [45,46], and conditional Qki deletion in mouse oligodendrocytes (Fig. 2a) results in severe hypomyelination and death of the animal [46]. Analysis of RNA-seq comparing animals with Qki-deficient and Qki-proficient oligodendrocytes [45] revealed 1899 DE genes (Additional file 1: Supplementary Fig. S1B). ...
A bottleneck in high-throughput functional genomics experiments is identifying the most important genes and their relevant functions from a list of gene hits. Gene Ontology (GO) enrichment methods provide insight at the gene set level. Here, we introduce GeneWalk (github.com/churchmanlab/genewalk) that identifies individual genes and their relevant functions critical for the experimental setting under examination. After the automatic assembly of an experiment-specific gene regulatory network, GeneWalk uses representation learning to quantify the similarity between vector representations of each gene and its GO annotations, yielding annotation significance scores that reflect the experimental context. By performing gene- and condition-specific functional analysis, GeneWalk converts a list of genes into data-driven hypotheses.
... The major isoforms QKI-5, QKI-6, and QKI-7, which differ in their C-terminal 30 amino acids, bind a specific RNA QKI response element (QRE) with the sequence ACUAAY (1-20) UAAY (Y; C/U) (Galarneau and Richard, 2005). Notably, genome-wide RNA sequencing (RNA-seq) data revealed a significant number of alternative splice defects in QKI-deficient cells (Darbelli et al., 2017;de Bruin et al., 2016;Hall et al., 2013). ...
The role of RNA binding proteins in regulating the phagocytic and cytokine-releasing functions of microglia is unknown. Here, we show that microglia deficient for the QUAKING (QKI) RNA binding protein have increased proinflammatory cytokine release and defects in processing phagocytosed cargo. Splicing analysis reveals a role for QKI in regulating microexon networks of the Rho GTPase pathway. We show an increase in RhoA activation and proinflammatory cytokines in QKI-deficient microglia that are repressed by treating with a Rock kinase inhibitor. During the cuprizone diet, mice with QKI-deficient microglia are inefficient at supporting central nervous system (CNS) remyelination and cause the recruited oligodendrocyte precursor cells to undergo apoptosis. Furthermore, the expression of QKI in microglia is downregulated in preactive, chronic active, and remyelinating white matter lesions of multiple sclerosis (MS) patients. Overall, our findings identify QKI as an alternative splicing regulator governing a network of Rho GTPase microexons with implications for CNS remyelination and MS patients.
