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Sean A. Mckenna - One of the best experts on this subject based on the ideXlab platform.
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Impact of double-stranded RNA characteristics on the activation of human 2’-5’-oligoadenylate synthetase 2 (OAS2).
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2019Co-Authors: Amit Koul, Soumya Deo, Evan P. Booy, George L. Orriss, Matthew Genung, Sean A. MckennaAbstract:Human 2'-5' oligoadenylate synthetases (OAS) are a family of interferon-inducible proteins that, upon activation by double-stranded RNA, polymerize ATP into 2'-5' linked oligoadenylates. In this study, we probed the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we developed a strategy for the expression and purification of recombinant human OAS2 from eukaryotic cells and quantified the activity of the enzyme relative to OAS1 in vitro. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1 and OAS2 in the presence of a variety of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While in this study OAS1 can be catalytically activated by dsRNA of any length greater than 19 bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35 bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs, despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.
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Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family
Journal of structural biology, 2015Co-Authors: Soumya Deo, Amit Koul, Trushar R. Patel, Edis Dzananovic, Kevin Mceleney, Grzegorz Chojnowski, Janusz M. Bujnicki, Sean A. MckennaAbstract:Abstract 2′ 5′-Oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding viral double-stranded RNA, OAS enzymes produce 2′-5′-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Truncations/mutations in the smallest human OAS isoform, OAS1, results in susceptibility to West Nile virus (WNV). We have previously demonstrated in vitro the interaction between OAS1 and the 5′-terminal region of the WNV RNA genome. Here we report that the 3′-terminal region is also able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3′-terminal region that is sufficient for activation of the enzyme. The solution conformation of the 3′-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3′-terminal region in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5′- and 3′-terminal regions, a required step for replication, is not sufficient to protect WNV from OAS1 recognition in vitro . These data provide a physical framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme.
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Activation of 2' 5'-oligoadenylate synthetase by stem loops at the 5'-end of the West Nile virus genome.
PloS one, 2014Co-Authors: Soumya Deo, Evan P. Booy, Trushar R. Patel, Edis Dzananovic, Khalid Zeid, Kevin Mceleney, Stephen E. Harding, Sean A. MckennaAbstract:West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response.
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Regulation of the Interferon‐Inducible 2′–5′-Oligoadenylate Synthetases by Adenovirus VAI RNA
Journal of molecular biology, 2012Co-Authors: Hui Meng, Soumya Deo, Edis Dzananovic, Shawn Xiong, Lynda J. Donald, Cody W. Van Dijk, Sean A. MckennaAbstract:Abstract Foreign double-stranded RNA (dsRNA) generated during the normal course of the viral life cycle serves as a key infection recognition element by proteins of the innate immune response. To circumvent this response, all adenoviruses synthesize at least one highly structured RNA (VA I ), which, after processing by the RNA silencing machinery, inhibits the innate immune response via a series of interactions with specific protein partners. Surprisingly, VA I positively regulates the activity of the interferon-induced 2′–5′-oligoadenylate synthetase (OAS) enzymes, which typically represent a key mechanism whereby host-cell protein translation is attenuated in response to foreign dsRNA. We present data investigating the regulation of the OAS1 isoform by VA I derivatives and demonstrate that a processed version of VA I lacking the terminal stem behaves as a pseudo-inhibitor of OAS1. A combination of electrophoretic mobility shift assays, dynamic light scattering, and non-denaturing mass spectrometry was used to quantitate binding affinity and characterize OAS1:VA I complex stoichiometry. Enzyme assays characterized the ability of VA I derivatives to activate OAS1. Finally, the importance of RNA 5′-end phosphorylation state is investigated, and it emphasizes its potential importance in the activation or inhibition of OAS enzymes. Taken together, these data suggest a plausible strategy whereby the virus produces a single RNA transcript capable of inhibiting a variety of members of the innate immune response.
Margo A Brinton - One of the best experts on this subject based on the ideXlab platform.
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Characteristics of Human OAS1 Isoform Proteins.
Viruses, 2020Co-Authors: Husni Elbahesh, Margo A BrintonAbstract:The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect splicing. The unique C-terminal sequences of the hOAS1 isoforms could differentially affect synthetase activity, protein stability, protein partner interactions and/or cellular localization. Recombinant p41, p42, p44, p46, p48, p49 and p52 hOAS1 isoform proteins expressed in bacteria were each able to synthesize trimer and higher order 2'-5' linked oligoadenylates in vitro in response to poly(I:C). The p42, p44, p46, p48 and p52 isoform proteins were each able to induce RNase-mediated rRNA cleavage in response to poly(I:C) when overexpressed in HEK293 cells. The expressed levels of the p42 and p46 isoform proteins were higher than those of the other isoforms, suggesting increased stability in mammalian cells. In a yeast two-hybrid screen, Fibrillin1 (FBN1) was identified as a binding partner for hOAS1 p42 isoform, and Supervillin (SVIL) as a binding partner for the p44 isoform. The p44-SVIL interaction was supported by co-immunoprecipitation data from mammalian cells. The data suggest that the unique C-terminal regions of hOAS1 isoforms may mediate the recruitment of different partners, alternative functional capacities and/or different cellular localization.
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variability in the 2 5 oligoadenylate synthetase gene cluster is associated with human predisposition to tick borne encephalitis virus induced disease
The Journal of Infectious Diseases, 2010Co-Authors: Andrey V Barkhash, Andrey A Perelygin, V N Babenko, Natalia G Myasnikova, Pavel I Pilipenko, A G Romaschenko, M I Voevoda, Margo A BrintonAbstract:The 2′–5′-oligoadenylate synthetase (2′–5′-OAS) family members are interferon-induced antiviral proteins. Twenty-three single nucleotide polymorphisms located within the OAS1, OAS2, OAS3, and OASL genes were analyzed in 142 patients with Russian tick-borne encephalitis. Statistically significant differences in genotype, allele, and haplotype frequencies for 3 OAS2 single nucleotide polymorphisms (rs1293762, rs15895, and rs1732778) and 2 OAS3 single nucleotide polymorphisms (rs2285932 and rs2072136) were detected between patients with central nervous system disease and both those with fever and/or meningitis and the control group. The data suggest a possible association between these 5 OAS single nucleotide polymorphisms and the outcome of tick-borne encephalitis virus infection in a Russian population.
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Characterization of the equine 2'-5' oligoadenylate synthetase 1 (OAS1) and ribonuclease L (RNASEL) innate immunity genes
BMC genomics, 2007Co-Authors: Jonathan J. Rios, Maureen T. Long, Andrey A Perelygin, Margo A Brinton, Andrey Zharkikh, Teri L. Lear, David L. AdelsonAbstract:Background The mammalian OAS/RNASEL pathway plays an important role in antiviral host defense. A premature stop-codon within the murine OAS1b gene results in the increased susceptibility of mice to a number of flaviviruses, including West Nile virus (WNV). Mutations in either the OAS1 or RNASEL genes may also modulate the outcome of WNV-induced disease or other viral infections in horses. Polymorphisms in the human OAS gene cluster have been previously utilized for case-control analysis of virus-induced disease in humans. No polymorphisms have yet been identified in either the equine OAS1 or RNASEL genes for use in similar case-control studies.
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the mammalian 2 5 oligoadenylate synthetase gene family evidence for concerted evolution of paralogous OAS1 genes in rodentia and artiodactyla
Journal of Molecular Evolution, 2006Co-Authors: Andrey A Perelygin, Andrey Zharkikh, Svetlana V Scherbik, Margo A BrintonAbstract:Multiple 2′-5′ oligoadenylate (2-5A) synthetases are important components of innate immunity in mammals. Gene families encoding these proteins have previously been studied mainly in humans and mice. To reconstruct the evolution of this gene family in mammals, a search for additional 2-5A synthetase genes was performed in rat, cattle, pig, and dog. Twelve 2′-5′ oligoadenylate synthetase (Oas) genes were identified in the rat genome, including eight OAS1 genes, two OAS1 pseudogenes, single copies of Oas2 and Oas3, and two Oas-like genes, Oasl1 and Oasl2. Four OAS genes were detected in the pig genome and five OAS genes were found in both the cattle and dog genomes. An OAS3 gene was not found in either the cattle or the pig genome. While two tandemly duplicated OAS-like (OASL) genes were identified in the dog genome, only a single OASL orthologue was found in both the cattle and the pig genomes. The bovine and porcine OASL genes contain premature stop codons and encode truncated proteins, which lack the typical C-terminal double ubiquitin domains. The cDNA sequences of the rat, cattle, pig, and dog OAS genes were amplified, sequenced and compared with each other and with those in the human, mouse, horse, and chicken genomes. Evidence of concerted evolution of paralogous 2′-5′ oligoadenylate synthetase 1 genes was obtained in rodents (Rodentia) and even-toed ungulates (Artiodactyla). Calculations using the nonparametric Kolmogorov-Smirnov test suggested that the homogenization of paralogous OAS1 sequences was due to gene conversion rather than stabilizing selection.
Jaideep Chaudhary - One of the best experts on this subject based on the ideXlab platform.
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Abstract C57: The expression of 2′-5′ oligoadenylate synthetase in prostate cancer and its effect on prostate cancer cell cycle
Cancer Research, 2012Co-Authors: Sanjay Mandal, Jaideep ChaudharyAbstract:Abstract 2-5 oligoadenylate synthetase (OAS) is an IFN inducible enzyme that is part of innate immune response. During viral infections 2′-5′ OAS is activated by viral dsRNA or cellular RNA with significant double strand structure to synthesize 2-5 oligoadenylates (2-5A) from ATP; which in turn activates RNaseL, the latent ribonuclease. RNAseL degrades infectious RNA which imparts antiviral protection to the cell. OAS-RNaseL system also degrades cellular RNA and inhibits protein synthesis. The role of RNAseL as a tumor suppressor has been studied in prostate cancer cell lines and has been linked to hereditary prostate cancer (HPC). However HPC accounts for less than 9% of the overall prostate cancer incidence but the role of RNAseL in sporadic prostate cancer has not been established. Since OAS1 is the rate limiting enzyme in RNaseL activation, we therefore hypothesized that OAS could potentially act as tumor suppressor in the context of sporadic prostate cancer. Methods: Immunohistochemistry was performed on human prostate cancer and normal prostate tissue to determine the expression of OAS1. RT-PCR and western blot was performed to detect the expression of OAS1 in LNCaP, DU145 and PC3 cell lines. Two alternative approaches were then used to investigate the effect of OAS1 on cell survival and its potential mechanism of action: OAS1 was over-expressed in LNCaP whereas it was silenced in DU145 cells. Cell cycle and apoptosis was quantitated by FACS analysis and the expression of key regulatory genes was determined by qRT-PCR and western blot analysis. Results: Immunohistochemistry demonstrated that OAS1 expression is high in the normal prostate but decreased progressively with stage in prostate cancer. OAS1 was not expressed in LNCaP cells but its expression was high in DU145 and PC3 prostate cancer lines. Overexpression of OAS1 in LNCaP cells induced apoptosis and G1 arrest that was associated with increased p53, BAX and p21 expression. Alternatively, silencing of OAS in DU145 cells led to an increase in S phase and G2 cell population with a concomitant decrease in apoptosis. Conclusion: Our prostate cancer cell line and prostate cancer tissue data strongly suggest that OAS1 is a candidate tumor suppressor that could be used as a potential biomarker for prostate cancer. Moreover, our previous study had shown a link between OAS1 polymorphism and prostate cancer incidence. Collectively data suggest the role of immune/inflammatory pathway in prostate cancer progression. Citation Format: Sanjay Kumar Mandal, Jaideep Chaudhary. The expression of 2′-5′ oligoadenylate synthetase in prostate cancer and its effect on prostate cancer cell cycle [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C57.
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2′‐5′ oligoadenylate synthetase 1 polymorphism is associated with prostate cancer
Cancer, 2011Co-Authors: Sanjay Mandal, Fisseha Abebe, Jaideep ChaudharyAbstract:BACKGROUND: The antiviral, proapoptotic, antiproliferative gene 2′-5′ oligoadenylate synthetase (2-5OAS1) converts adenosine triphosphate into a series of 2′-5′ oligoadenylates (2-5A). In turn, 2-5A activates latent ribonuclease (RNaseL), a candidate hereditary prostate cancer gene. OAS1 polymorphism (reference single nucleotide polymorphism [SNP] 2660 [rs2660]) has been associated with increased susceptibility to infections and various diseases. In general, the low-enzyme-activity adenine-adenine (AA) genotype promotes susceptibility, whereas the high-enzyme-activity guanosine-guanosine (GG) genotype confers protection. In this study, the authors investigated the association of this functional OAS1 polymorphism (rs2660) with prostate cancer. METHODS: Sample size and power were calculated using a power calculation software program for case-control genetic association analyses. Genomic DNA samples from a control group (n = 140) and from a case group of patients with prostate cancer (n = 164) were used for genotyping SNPs rs2660, rs1131454, and rs34137742 in all samples. Statistical analyses were performed using a logistic regression model. RESULTS: A significant association was observed between the rs2660 genotype (A/G) and prostate cancer. Genotype AA increased the risk, whereas genotype GG decreased the risk of prostate cancer. The GG genotype was not observed in the African American samples. The AA genotype also increased the risk of prostate cancer with age. CONCLUSIONS: The OAS1 SNP rs2660 AA genotype was associated significantly with prostate cancer, whereas the GG genotype protected against prostate cancer. OAS1 rs2660 may be a prostate cancer susceptibility polymorphism, which is a significant observation, especially in a context of the OAS1-RNaseL pathway. Thus, a functional defect in OAS1 because of the rs2660 SNP not only can attenuate RNaseL function but also can alter cell growth and apoptosis independent of RNaseL. Cancer 2011;. © 2011 American Cancer Society.
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Abstract 2772: 2’-5’oligoadenylate synthetase polymorphism is associated with prostate cancer: Effect modified by age and race
Epidemiology, 2011Co-Authors: Sanjay Mandal, Fisseha Abebe, Jaideep ChaudharyAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: 2’ -5’ -oligoadenylate synthetase (2-5 OAS1) is an antiviral gene that converts ATP to a series of (2-5A). 2-5A promotes dimerization of latent ribonuclease (RNASE L) to form catalytically active RNASEL, a candidate hereditary prostate cancer gene. Thus a defect in OAS1 can not only attenuate RNASEL function, but can also alter cell growth and apoptosis independent of RNASEL. OAS1 polymorphism (rs2660) has been associated with increased disease susceptibility and viral infection like, HCV, West Nile virus infection, SARS, diabetes and MS. The AA genotype, known to have lower enzymatic activity promotes disease susceptibility whereas GG genotype with higher enzyme activity confers protection. In the present study we investigated this functional polymorphism (rs2660) in OAS1 in a large prostate cohort. Methods: Genomic DNA from Buffy coat samples was used from age matched controls (n=140) and prostate cancer patients (n=164) who had undergone prostatectomy for genotyping. Information regarding age, race, and cancer stage were available for all samples. PCR-RFLP, followed by direct sequencing was used for genotyping. SNPs rs2660, rs1131454 and rs34137742 were genotyped on all samples. Statistical analysis was performed using chi2 test and logistic regression model with NCSS software. Results: The rs2660 genotype distribution is summarized in [table 1][1]. Genotype AA increases the risk of prostate cancer by 61%. Prostate cancer incidence increases with age, however when combined with rs2660 genotype AA the odds of prostate cancer incidence are higher.SNPs rs1131454 and rs34137742 were not associated with prostate cancer. Conclusion: OAS1 SNP rs2660 AA genotype is significantly associated with prostate cancer whereas GG genotype protects against prostate cancer. The effect of this genotype distribution was more profound in African American population as compared to Caucasians. ![Figure][2] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2772. doi:10.1158/1538-7445.AM2011-2772 [1]: #F1 [2]: pending:yes
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2’, 5’-Oligoadenylate Synthetase 1 (OAS1) expression and function in prostate cancer
Cancer Research, 2008Co-Authors: Roblena Walker, Ashley Evans, Tandeih A. Ghogomu, Jaideep ChaudharyAbstract:4562 Our recent studies have suggested that androgens primarily regulate the immune inflammatory pathways in normal prostate epithelial cells. In general androgens up-regulate the anti-tumor acute inflammatory pathway but down-regulate the pro-tumor chronic inflammatory pathways in normal prostate epithelial cells. In prostate cancer, this regulatory role of androgens is switched in which there is a general loss or decrease in expression of acute inflammatory response genes (e.g. IFN) and a concomitant increase in chronic inflammatory response genes (e.g. IL6). One of the key androgen regulated acute inflammatory gene is 2’, 5’-oligoadenylate synthetase 1 (OAS1). OAS1 is also an IFN regulated gene that is expressed as part of the innate immune response to viruses. The induction of OAS1 leads to the polymerization of ATP to 2’-5’ -linked adenosine oligomers or 2-5A (pppA(2’p5’A)n) in the presence of dsRNA. 2-5A in turn activates RNASEL, which degrades viral dsRNA. The expression and function of OAS1 is therefore critical for functional RNaseL. RNASEL gene inactivation has also been linked to hereditary prostate cancer. Recently, RNASEL has been reported to have tumor suppressor activities in vitro. However, in sporadic prostate cancer no RNASEL mutations or a significant change in its expression is observed. These observations led us to hypothesize that upstream regulators of RNASEL, such as OAS1 may be disrupted leading to loss of RNASEL function and prostate cancer. In order to test this hypothesis, we performed OAS1 reverse transcription (RT) polymerase chain reactions (PCR) on prostate cancer cell lines PC3, DU145, and LNCaP. Our observations indicate that OAS1 is highly expressed in PC3, but not in DU145 or LNCaP cells. Interestingly, RNASEL is only expressed in DU145 but not in PC3 or LNCaP cells. These results suggest that the OAS1-RNASEL pathway is non-functional in prostate cancer cells. Attenuation of the pathway through genetic approach is currently being used to address the significance of OAS-RNaseL in prostate cancer cell survival and viral response and will be presented.
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2 5 oligoadenylate synthetase 1 OAS1 expression and function in prostate cancer
Cancer Research, 2008Co-Authors: Roblena Walker, Ashley Evans, Tandeih A. Ghogomu, Jaideep ChaudharyAbstract:4562 Our recent studies have suggested that androgens primarily regulate the immune inflammatory pathways in normal prostate epithelial cells. In general androgens up-regulate the anti-tumor acute inflammatory pathway but down-regulate the pro-tumor chronic inflammatory pathways in normal prostate epithelial cells. In prostate cancer, this regulatory role of androgens is switched in which there is a general loss or decrease in expression of acute inflammatory response genes (e.g. IFN) and a concomitant increase in chronic inflammatory response genes (e.g. IL6). One of the key androgen regulated acute inflammatory gene is 2’, 5’-oligoadenylate synthetase 1 (OAS1). OAS1 is also an IFN regulated gene that is expressed as part of the innate immune response to viruses. The induction of OAS1 leads to the polymerization of ATP to 2’-5’ -linked adenosine oligomers or 2-5A (pppA(2’p5’A)n) in the presence of dsRNA. 2-5A in turn activates RNASEL, which degrades viral dsRNA. The expression and function of OAS1 is therefore critical for functional RNaseL. RNASEL gene inactivation has also been linked to hereditary prostate cancer. Recently, RNASEL has been reported to have tumor suppressor activities in vitro. However, in sporadic prostate cancer no RNASEL mutations or a significant change in its expression is observed. These observations led us to hypothesize that upstream regulators of RNASEL, such as OAS1 may be disrupted leading to loss of RNASEL function and prostate cancer. In order to test this hypothesis, we performed OAS1 reverse transcription (RT) polymerase chain reactions (PCR) on prostate cancer cell lines PC3, DU145, and LNCaP. Our observations indicate that OAS1 is highly expressed in PC3, but not in DU145 or LNCaP cells. Interestingly, RNASEL is only expressed in DU145 but not in PC3 or LNCaP cells. These results suggest that the OAS1-RNASEL pathway is non-functional in prostate cancer cells. Attenuation of the pathway through genetic approach is currently being used to address the significance of OAS-RNaseL in prostate cancer cell survival and viral response and will be presented.
Just Justesen - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial localization of the OAS1 p46 isoform associated with a common single nucleotide polymorphism.
BMC cell biology, 2014Co-Authors: Karina Hansen Kjaer, Just Justesen, Jesper Buchhave Poulsen, Jytte Pahus, Mariann Fagernæs Hansen, Erik Ilsø Christensen, Pia M. MartensenAbstract:The expression of 2′-5′-Oligoadenylate synthetases (OASs) is induced by type 1 Interferons (IFNs) in response to viral infection. The OAS proteins have a unique ability to produce 2′-5′ Oligoadenylates, which bind and activate the ribonuclease RNase L. The RNase L degrades cellular RNAs which in turn inhibits protein translation and induces apoptosis. Several single nucleotide polymorphisms (SNPs) in the OAS1 gene have been associated with disease. We have investigated the functional effect of two common SNPs in the OAS1 gene. The SNP rs10774671 affects splicing to one of the exons in the OAS1 gene giving rise to differential expression of the OAS1 isoforms, and the SNP rs1131454 (former rs3741981) resides in exon 3 giving rise to OAS1 isoforms with either a Glycine or a Serine at position 162 in the core OAS unit. We have used three human cell lines with different genotypes in the OAS1 SNP rs10774671, HeLa cells with the AA genotype, HT1080 cells with AG, and Daudi cells with GG. The main OAS1 isoform expressed in Daudi and HT1080 cells was p46, and the main OAS1 isoform expressed in HeLa cells was p42. In addition, low levels of the OAS1 p52 mRNA was detected in HeLa cells and p48 mRNA in Daudi cells, and trace amounts of p44a mRNA were detected in the three cell lines treated with type 1 interferon. We show that the OAS1 p46 isoform was localized in the mitochondria in Daudi cells, whereas the OAS1 isoforms in HeLa cells were primarily localized in cytoplasmic vacuoles/lysosomes. By using recombinantly expressed OAS1 mutant proteins, we found that the OAS1 SNP rs1131454 (former rs3741981) did not affect the enzymatic OAS1 activity. The SNP rs10774671 determines differential expression of the OAS1 isoforms. In Daudi and HT1080 cells the p46 isoform is the most abundantly expressed isoform associated with the G allele, whereas in HeLa cells the most abundantly expressed isoform is p42 associated with the A allele. The SNP rs1131454 (former rs3741981) does not interfere with OAS1 enzyme activity. The OAS1 p46 isoform localizes to the mitochondria, therefore a full 2-5A system can now be found in the mitochondria.
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Evolution of the 2′-5′-Oligoadenylate Synthetase Family in Eukaryotes and Bacteria
Journal of Molecular Evolution, 2009Co-Authors: Karina Hansen Kjaer, Jesper Buchhave Poulsen, Tõnu Reintamm, Emilie Saby, Pia Moeller Martensen, Merike Kelve, Just JustesenAbstract:The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins in bacteria that contains the five OAS-specific motifs. This indicates a specific relationship to OAS. The wide distribution of the OAS genes has made it possible to suggest how the OAS1 gene could have evolved from a common ancestor to choanoflagellates and metazoans. Furthermore, we suggest that the OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS may differ from its function in the interferon system, and that this original function of OAS is preserved even in OAS genes that code for proteins, which do not have 2′-5′-oligoadenylate synthetase activity.
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Interaction between the 2′−5′ oligoadenylate synthetase‐like protein p59 OASL and the transcriptional repressor methyl CpG‐binding protein 1
European journal of biochemistry, 2004Co-Authors: Jesper B. Andersen, Dorthe Strandbygård, Rune Hartmann, Just JustesenAbstract:The human 2′−5′ oligoadenylate synthetases (OAS) form a conserved family of interferon-induced proteins consisting of four genes: OAS1, OAS2, OAS3 and the 2′−5′ oligoadenylate synthetase-like gene (OASL). When activated by double-stranded RNA, OAS1–3 polymerize ATP into 2′−5′-linked oligoadenylates; 2′−5′-linked oligoadenylates, in turn, activate a latent endoribonuclease that degrades viral and cellular RNAs. In contrast, while the p59 OASL protein is highly homologous to the OAS family (45% identity), its 350 amino acid N-terminal domain lacks 2′−5′ oligoadenylate synthetase activity. A C-terminal 164 amino acid domain, which is 30% homologous to a tandem repeat of ubiquitin, further distinguishes the p59 OASL protein and suggests that it serves a biological role which is distinct from other OAS family members. To dissect the function of p59 OASL, we utilized the yeast two-hybrid system to identify interacting proteins. Methyl CpG-binding protein 1 (MBD1), which functions as a transcriptional repressor, was identified as a strong p59 OASL interactor. Interestingly, like p59 OASL, transcription of the MBD1 gene was induced by interferon, indicating that these genes are co-ordinately regulated. The interaction was confirmed in vitro and in vivo and was mapped to the ubiquitin-like domain of p59 OASL. The p59 OASL–MBD1 interaction was specific, because p59 OASL did not interact with any of the other MBD family members and MBD1 did not interact with OAS1. These findings link the p59 OASL with MBD1 transcriptional control in the context of an interferon-stimulated cell, and provide the basis for future studies to examine the functional role of this interaction.
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Gene structure of the murine 2'-5'-oligoadenylate synthetase family.
Cellular and molecular life sciences : CMLS, 2002Co-Authors: S. Eskildsen, R. Hartmann, N. O. Kjeldgaard, Just JustesenAbstract:The 2′-5′-oligoadenylate synthetases (OASs) are members of a family of interferon-induced proteins playing an important role in the antiviral effect of interferons as well as being involved in apoptosis and control of cellular growth. Based on sequence data from the murine BAC clone (RP23-39M18), and a number of EST and IMAGE clones and the Celera Mouse database, we identified twelve Oas genes in the mouse genome, all localized to the chromosome 5F region. In contrast to the single OAS1 gene found in humans, we identified eight closely linked OAS1 genes in the murine genome, together with the genes of Oas2 and Oas3. Compared to the single OASL gene found in humans, two genes of OAS-like proteins, Oasl1 and Oasl2, were identified. All the putative genes seem to be transcribed.¶The exon/intron structures of the murine Oas genes were found to be identical to those of the human genes.
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Inhibition of 2′‐5′ oligoadenylate synthetase by divalent metal ions
FEBS letters, 2001Co-Authors: Rune Hartmann, Gernot Walko, Just JustesenAbstract:Abstract OAS1 is the small form and OAS2 is the medium form of the human interferon-induced 2′-5′ oligoadenylate synthetases. The p42 isoform of OAS1 and the p69 isoform of OAS2 have been expressed in insect cells and purified to give pure, highly active 2′-5′ oligoadenylate synthetase. The catalysis of 2′-5′ oligoadenylate synthesis is strictly dependent on double-stranded RNA and magnesium ions. We have examined the effect of a series of divalent metal ions: copper, iron and zinc ions strongly inhibited the enzymatic activity, cobalt and nickel ions were partly inhibitory whereas calcium and manganese ions were without effect. However, manganese ions can replace magnesium ions as activator. The inhibitory effect of zinc ions was characterised in detail. The inhibitory constants of Zn2+ were estimated to be 0.10 mM for OAS1p42 and to 0.02 mM for OAS2p69. Cross-linking experiments showed that zinc ions can control the oligomerisation by enhancing the formation of tetrameric forms of OAS1p42
Soumya Deo - One of the best experts on this subject based on the ideXlab platform.
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Impact of double-stranded RNA characteristics on the activation of human 2’-5’-oligoadenylate synthetase 2 (OAS2).
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2019Co-Authors: Amit Koul, Soumya Deo, Evan P. Booy, George L. Orriss, Matthew Genung, Sean A. MckennaAbstract:Human 2'-5' oligoadenylate synthetases (OAS) are a family of interferon-inducible proteins that, upon activation by double-stranded RNA, polymerize ATP into 2'-5' linked oligoadenylates. In this study, we probed the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we developed a strategy for the expression and purification of recombinant human OAS2 from eukaryotic cells and quantified the activity of the enzyme relative to OAS1 in vitro. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1 and OAS2 in the presence of a variety of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While in this study OAS1 can be catalytically activated by dsRNA of any length greater than 19 bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35 bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3'-overhangs, despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.
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Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family
Journal of structural biology, 2015Co-Authors: Soumya Deo, Amit Koul, Trushar R. Patel, Edis Dzananovic, Kevin Mceleney, Grzegorz Chojnowski, Janusz M. Bujnicki, Sean A. MckennaAbstract:Abstract 2′ 5′-Oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding viral double-stranded RNA, OAS enzymes produce 2′-5′-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Truncations/mutations in the smallest human OAS isoform, OAS1, results in susceptibility to West Nile virus (WNV). We have previously demonstrated in vitro the interaction between OAS1 and the 5′-terminal region of the WNV RNA genome. Here we report that the 3′-terminal region is also able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3′-terminal region that is sufficient for activation of the enzyme. The solution conformation of the 3′-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3′-terminal region in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5′- and 3′-terminal regions, a required step for replication, is not sufficient to protect WNV from OAS1 recognition in vitro . These data provide a physical framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme.
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Activation of 2' 5'-oligoadenylate synthetase by stem loops at the 5'-end of the West Nile virus genome.
PloS one, 2014Co-Authors: Soumya Deo, Evan P. Booy, Trushar R. Patel, Edis Dzananovic, Khalid Zeid, Kevin Mceleney, Stephen E. Harding, Sean A. MckennaAbstract:West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response.
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Regulation of the Interferon‐Inducible 2′–5′-Oligoadenylate Synthetases by Adenovirus VAI RNA
Journal of molecular biology, 2012Co-Authors: Hui Meng, Soumya Deo, Edis Dzananovic, Shawn Xiong, Lynda J. Donald, Cody W. Van Dijk, Sean A. MckennaAbstract:Abstract Foreign double-stranded RNA (dsRNA) generated during the normal course of the viral life cycle serves as a key infection recognition element by proteins of the innate immune response. To circumvent this response, all adenoviruses synthesize at least one highly structured RNA (VA I ), which, after processing by the RNA silencing machinery, inhibits the innate immune response via a series of interactions with specific protein partners. Surprisingly, VA I positively regulates the activity of the interferon-induced 2′–5′-oligoadenylate synthetase (OAS) enzymes, which typically represent a key mechanism whereby host-cell protein translation is attenuated in response to foreign dsRNA. We present data investigating the regulation of the OAS1 isoform by VA I derivatives and demonstrate that a processed version of VA I lacking the terminal stem behaves as a pseudo-inhibitor of OAS1. A combination of electrophoretic mobility shift assays, dynamic light scattering, and non-denaturing mass spectrometry was used to quantitate binding affinity and characterize OAS1:VA I complex stoichiometry. Enzyme assays characterized the ability of VA I derivatives to activate OAS1. Finally, the importance of RNA 5′-end phosphorylation state is investigated, and it emphasizes its potential importance in the activation or inhibition of OAS enzymes. Taken together, these data suggest a plausible strategy whereby the virus produces a single RNA transcript capable of inhibiting a variety of members of the innate immune response.