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The RNA binding protein Quaking represses splicing of the Fibronectin EDA exon and downregulates the interferon response.

ABSTRACT: Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

SUBMITTER: Liao KC

PROVIDER: S-EPMC8464043 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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 IFNβ 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 IFNβ activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNβ induction. Consistently, ectopic expression of RIG-I activates stronger IFNβ 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.

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Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Project description:Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

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HnRNP L represses exon splicing via a regulated exonic splicing silencer.

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miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

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Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats.

Project description:The fibronectin EIIIB exon is alternatively spliced in a cell-type-specific manner, and TGCATG repeats in the intron downstream of EIIIB have been implicated in this regulation. Analysis of the intron sequence from several vertebrates shows that the pattern of repeats in the 3' half of the intron is evolutionarily conserved. Point mutations in certain highly conserved repeats greatly reduce EIIIB inclusion, suggesting that a multicomponent complex may recognize the repeats. Expression of the SR protein SRp40, SRp20, or ASF/SF2 stimulates EIIIB inclusion. Studies of the interplay between mutations in the repeats and SRp40-stimulated inclusion suggest that the repeats are recognized in many, if not all, cell types, and that EIIIB inclusion may be regulated by quantitative changes in multiple factors.

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EDA-containing fibronectin increases proliferation of embryonic stem cells.

Project description:Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

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The Exon Junction Complex Core Represses Cancer-Specific Mature mRNA Re-splicing: A Potential Key Role in Terminating Splicing.

Project description:Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.

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RNA-binding protein AUF1 represses Dicer expression.

Project description:MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.

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RNA-binding Protein Quaking Stabilizes <i>Sirt2</i> mRNA during Oligodendroglial Differentiation.

Project description:Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv ) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3' untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation.

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