scholarly journals Phosphor-IWS1-dependent U2AF2 splicing regulates trafficking of CAR-E-positive intronless gene mRNAs and sensitivity to viral infection

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Georgios I. Laliotis ◽  
Adam D. Kenney ◽  
Evangelia Chavdoula ◽  
Arturo Orlacchio ◽  
Abdul Kaba ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Rna Splicing ◽  
Target Genes ◽  
Apoptotic Cell ◽  
Oncolytic Viruses ◽  
Poly I:C ◽  
Type I ◽  
Exon 2 ◽  
Intronless Gene ◽  
Enhanced Sensitivity

AbstractAKT-phosphorylated IWS1 promotes Histone H3K36 trimethylation and alternative RNA splicing of target genes, including the U2AF65 splicing factor-encoding U2AF2. The predominant U2AF2 transcript, upon IWS1 phosphorylation block, lacks the RS-domain-encoding exon 2, and encodes a protein which fails to bind Prp19. Here we show that although both U2AF65 isoforms bind intronless mRNAs containing cytoplasmic accumulation region elements (CAR-E), only the RS domain-containing U2AF65 recruits Prp19 and promotes their nuclear export. The loading of U2AF65 to CAR-Elements was RS domain-independent, but RNA PolII-dependent. Virus- or poly(I:C)-induced type I IFNs are encoded by genes targeted by the pathway. IWS1 phosphorylation-deficient cells therefore, express reduced levels of IFNα1/IFNβ1 proteins, and exhibit enhanced sensitivity to infection by multiple cytolytic viruses. Enhanced sensitivity of IWS1-deficient cells to Vesicular Stomatitis Virus and Reovirus resulted in enhanced apoptotic cell death via caspase activation. Inhibition of this pathway may therefore sensitize cancer cells to oncolytic viruses.

scholarly journals IWS1 phosphorylation by AKT3 controls nuclear export of type I IFN mRNAs and sensitivity to oncolytic viral infection, by regulating the alternative RNA splicing of U2AF2

2020 ◽  
Author(s):  
Georgios I. Laliotis ◽  
Adam D. Kenney ◽  
Evangelia Chavdoula ◽  
Arturo Orlacchio ◽  
Abdul K. Kaba ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Viral Infection ◽  
Nuclear Export ◽  
Rna Splicing ◽  
Proper Function ◽  
Type I ◽  
Exon 2 ◽  
Type I Ifns ◽  
Intronless Genes ◽  
In Cells

AbstractType I IFNs orchestrate the antiviral response. Interestingly, IFNA1 and IFNB1 genes are naturally intronless. Based on previous work, the splicing factor U2 Associated Factor 65 (U2AF65), encoded by U2AF2, and pre-mRNA Processing factor 19 (Prp19) function on the Cytoplasmic Accumulation Region Elements (CAR-E), affecting the nuclear export of intronless genes. We have previously shown that the loss of IWS1 phosphorylation by AKT3, promotes the alternative RNA splicing of U2AF2, resulting in novel transcripts lacking exon 2. This exon encodes part of the Serine-Rich (RS) domain of U2AF65, which is responsible for its binding with Prp19. Here, we show that IWS1 phosphorylation and the U2AF2 RNA splicing pattern affect the nuclear export of introless mRNAs. We also demonstrate that the same axis is required for the proper function of the CAR-Es. Mechanistically, whereas both U2AF65 isoforms bind CAR-E, the recruitment of Prp19 occurs only in cells expressing phosphorylated IWS, promoting intronless genes export. Moreover, analysis of Lung adenocarcinoma patients showed that high p-IWS1 activity correlates with the assembly of the U2AF65/Prp19 complex and export of intronless genes, in vivo. Accordingly, the expression of type I IFNs was decreased in cells deficient in IWS1 phosphorylation and the viral infection was increased. Furthermore, following infection with oncolytic virus, we observed reduced activation of p-STAT1 and expression of Interferon Stimulated Genes (ISG), in cells stimulated by shIWS1-derived supernatant, or cells treated with the pan-AKT inhibitor, MK2206. Consistently, killing curves and apoptosis assays after infection with oncolytic viruses, revealed increased susceptibility upon the loss of IWS1, with subsequent activation of Caspase-mediated death. The treatment of the lung adenocarcinoma cells with MK2206, phenocopied the loss of IWS1 phosphorylation. These data identify a novel mechanism by which the AKT/p-IWS1 axis, by hijacking the epigenetic regulation of RNA splicing and processing, contributes to the resistance to oncolytic viral infection, suggesting that combined inhibition of the splicing machinery and AKT/p-IWS1 signals would sensitize tumors to oncolytic viral treatment.

scholarly journals Overexpression of the SETD2 WW domain inhibits the phosphor-IWS1/SETD2 interaction and the oncogenic AKT/IWS1 RNA splicing program.

2021 ◽  
Author(s):  
Georgios I. Laliotis ◽  
Evangelia Chavdoula ◽  
Vollter Anastas ◽  
Satishkumar Singh ◽  
Adam I. Kenney ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Target Genes ◽  
Therapeutic Potential ◽  
Human Cancer ◽  
Metastatic Potential ◽  
Egfr Mutations ◽  
Type I ◽  
Transcriptional Elongation ◽  
Ww Domain ◽  
Intronless Genes

Our earlier studies had shown that AKT phosphorylates IWS1, and that following phosphorylation, IWS1 recruits the histone methyltransferase SETD2 to an SPT6/IWS1/ALY complex, which assembles on the Ser2-phosphorylated CTD of RNA Pol II. Recruited SETD2 methylates histone H3 at K36, during transcriptional elongation of target genes, and this regulates multiple steps in RNA metabolism. By regulating the RNA splicing of U2AF2, it controls cell proliferation. Importantly, pathway activity correlates with grade, stage and metastatic potential of lung adenocarcinomas, especially those with EGFR mutations. By regulating nucleocytoplasmic mRNA transport of intronless genes, including those encoding type I IFNs, it regulates sensitivity to viral infection. Here, we show that SETD2 interacts with IWS1 via its WW domain, that the interaction is IWS1 phosphorylation-dependent and that WW domain overexpression blocks the interaction and inhibits the pathway and its biological outcomes. We conclude that blocking the phosphor-IWS1/SETD2 interaction is feasible and has significant therapeutic potential in human cancer.

scholarly journals AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Georgios I. Laliotis ◽  
Evangelia Chavdoula ◽  
Maria D. Paraskevopoulou ◽  
Abdul Kaba ◽  
Alessandro La Ferlita ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Splicing ◽  
Target Genes ◽  
Tumor Stage ◽  
The Body ◽  
Exon 2 ◽  
A Cell ◽  
Cycle Dependent ◽  
Egfr Mutant ◽  
Lung Adenocarcinomas

AbstractAKT-phosphorylated IWS1 regulates alternative RNA splicing via a pathway that is active in lung cancer. RNA-seq studies in lung adenocarcinoma cells lacking phosphorylated IWS1, identified a exon 2-deficient U2AF2 splice variant. Here, we show that exon 2 inclusion in the U2AF2 mRNA is a cell cycle-dependent process that is regulated by LEDGF/SRSF1 splicing complexes, whose assembly is controlled by the IWS1 phosphorylation-dependent deposition of histone H3K36me3 marks in the body of target genes. The exon 2-deficient U2AF2 mRNA encodes a Serine-Arginine-Rich (RS) domain-deficient U2AF65, which is defective in CDCA5 pre-mRNA processing. This results in downregulation of the CDCA5-encoded protein Sororin, a phosphorylation target and regulator of ERK, G2/M arrest and impaired cell proliferation and tumor growth. Analysis of human lung adenocarcinomas, confirmed activation of the pathway in EGFR-mutant tumors and showed that pathway activity correlates with tumor stage, histologic grade, metastasis, relapse after treatment, and poor prognosis.

scholarly journals PGAM5-MAVS interaction regulates TBK1/ IRF3 dependent antiviral responses

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yu-qiang Yu ◽  
Marta Zielinska ◽  
Wei Li ◽  
Dominic B. Bernkopf ◽  
Christiane Silke Heilingloh ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Cellular Response ◽  
Target Genes ◽  
Viral Infections ◽  
Mitochondrial Protein ◽  
Direct Interaction ◽  
Type I Interferons ◽  
Poly I:C ◽  
Phosphoglycerate Mutase ◽  
Type I

Abstract Viral infections trigger host innate immune responses, characterized by the production of type-I interferons (IFN) including IFNβ. IFNβ induces cellular antiviral defense mechanisms and thereby contributes to pathogen clearance. Accumulating evidence suggests that mitochondria constitute a crucial platform for the induction of antiviral immunity. Here we demonstrate that the mitochondrial protein phosphoglycerate mutase family member 5 (PGAM5) is important for the antiviral cellular response. Following challenge of HeLa cells with the dsRNA-analog poly(I:C), PGAM5 oligomers and high levels of PGAM5 were found in mitochondrial aggregates. Using immunoprecipitation, a direct interaction of PGAM5 with the mitochondrial antiviral-signaling protein (MAVS) was demonstrated. In addition, PGAM5 deficient cells showed diminished expression of IFNβ and IFNβ target genes as compared to WT cells. Moreover, PGAM5 deficient mouse embryonic fibroblasts (MEFs) exhibited decreased phosphorylation levels of IRF3 and TBK1 when challenged with poly(I:C) intracellularly. Finally, PGAM5 deficient MEFs, upon infection with vesicular stomatitis virus (VSV), revealed diminished IFNβ expression and increased VSV replication. Collectively, our study highlights PGAM5 as an important regulator for IFNβ production mediated via the TBK1/IRF3 signaling pathway in response to viral infection.

scholarly journals PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Zongyi Bo ◽  
Yurun Miao ◽  
Rui Xi ◽  
Qiuping Zhong ◽  
Chenyi Bao ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Pseudorabies Virus ◽  
Nuclear Translocation ◽  
Economic Loss ◽  
Inhibitory Effect ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Creb Binding Protein ◽  
Swine Industry

Abstract Cyclic GMP-AMP (cGAMP) synthase (cGAS) is an intracellular sensor of cytoplasmic viral DNA created during virus infection, which subsequently activates the stimulator of interferon gene (STING)-dependent type I interferon response to eliminate pathogens. In contrast, viruses have developed different strategies to modulate this signalling pathway. Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujeszky’s disease (AD), a notable disease that causes substantial economic loss to the swine industry globally. Previous reports have shown that PRV infection induces cGAS-dependent IFN-β production, conversely hydrolysing cGAMP, a second messenger synthesized by cGAS, and attenuates PRV-induced IRF3 activation and IFN-β secretion. However, it is not clear whether PRV open reading frames (ORFs) modulate the cGAS–STING-IRF3 pathway. Here, 50 PRV ORFs were screened, showing that PRV UL13 serine/threonine kinase blocks the cGAS–STING-IRF3-, poly(I:C)- or VSV-mediated transcriptional activation of the IFN-β gene. Importantly, it was discovered that UL13 phosphorylates IRF3, and its kinase activity is indispensable for such an inhibitory effect. Moreover, UL13 does not affect IRF3 dimerization, nuclear translocation or association with CREB-binding protein (CBP) but attenuates the binding of IRF3 to the IRF3-responsive promoter. Consistent with this, it was discovered that UL13 inhibits the expression of multiple interferon-stimulated genes (ISGs) induced by cGAS–STING or poly(I:C). Finally, it was determined that PRV infection can activate IRF3 by recruiting it to the nucleus, and PRVΔUL13 mutants enhance the transactivation level of the IFN-β gene. Taken together, the data from the present study demonstrated that PRV UL13 inhibits cGAS–STING-mediated IFN-β production by phosphorylating IRF3.

scholarly journals Type I human complement C2 deficiency. A 28-base pair gene deletion causes skipping of exon 6 during RNA splicing.

1993 ◽  
Vol 268 (3) ◽  
pp. 2268
Author(s):  
C.A. Johnson ◽  
P. Densen ◽  
R.K. Hurford ◽  
H.R. Colten ◽  
R.A. Wetsel
Keyword(s):  
Base Pair ◽  
Rna Splicing ◽  
Gene Deletion ◽  
Type I ◽  
Human Complement ◽  
C2 Deficiency

scholarly journals Role of Alternative Splicing in Regulating Host Response to Viral Infection

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1720
Author(s):  
Kuo-Chieh Liao ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Innate Immunity ◽  
Alternative Splicing ◽  
Immune Responses ◽  
Viral Infection ◽  
Rna Splicing ◽  
Type I ◽  
Host Immune Responses ◽  
Transcriptional Regulatory Mechanisms ◽  
Host Virus Interactions

The importance of transcriptional regulation of host genes in innate immunity against viral infection has been widely recognized. More recently, post-transcriptional regulatory mechanisms have gained appreciation as an additional and important layer of regulation to fine-tune host immune responses. Here, we review the functional significance of alternative splicing in innate immune responses to viral infection. We describe how several central components of the Type I and III interferon pathways encode spliced isoforms to regulate IFN activation and function. Additionally, the functional roles of splicing factors and modulators in antiviral immunity are discussed. Lastly, we discuss how cell death pathways are regulated by alternative splicing as well as the potential role of this regulation on host immunity and viral infection. Altogether, these studies highlight the importance of RNA splicing in regulating host–virus interactions and suggest a role in downregulating antiviral innate immunity; this may be critical to prevent pathological inflammation.

scholarly journals Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wen-juan Li ◽  
Yao-hui He ◽  
Jing-jing Yang ◽  
Guo-sheng Hu ◽  
Yi-an Lin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Growth ◽  
Rna Splicing ◽  
Synergistic Effects ◽  
Arginine Methylation ◽  
Type I ◽  
Prostate Cancer Cells ◽  
Cancer Cell Growth ◽  
Cancer Mutations ◽  
Substrate Spectrum

AbstractNumerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

scholarly journals SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 530
Author(s):  
Soo Jin Oh ◽  
Ok Sarah Shin
Keyword(s):  
Nucleocapsid Protein ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Antiviral Immune Response ◽  
N Protein ◽  
Nonstructural Protein 1 ◽  
Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

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