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5'-Oligoadenylate Synthetase 2 (OAS2) Biochemistry and Cell Biology Impact of double-stranded RNA characteristics on the activation of human 2’-5’-oligoadenylate synthetase 2 (OAS2). Journal: Biochemistry and Cell Biology Manuscript ID bcb-2019-0060.R1 Manuscript Type: Article Date Submitted by the 29-Mar-2019 Author: Complete List of Authors: Koul, Amit; University of Manitoba Deo, Soumya; University of Lethbridge Booy, Evan; University of Manitoba Orriss, George;Draft University of Manitoba Genung, Matthew; University of Manitoba McKenna, Sean; University of Manitoba Keyword: oligoadenylate synthetase, RNA, viral RNA, enzymology Is the invited manuscript for consideration in a Special Ribowest RNA Issue? : https://mc06.manuscriptcentral.com/bcb-pubs Page 1 of 39 Biochemistry and Cell Biology Impact of double-stranded RNA characteristics on the activation of human 2’-5’- oligoadenylate synthetase 2 (OAS2). Amit, Koul1, Soumya Deo3, Evan P. Booy1, George L. Orriss1, Matthew Genung4 and Sean A. McKenna1, 2 Department of Chemistry1, Department of Biochemistry and Medical Genetics2, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, R3T2N2, Canada. Alberta RNA Research and Training Institute3, Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta T1K 3M4, Canada. Max Rady College of Medicine4, Rady Faculty of Health Sciences, University of Manitoba, 750 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W2, Canada. Corresponding Author: Dr. Sean A. McKenna [email protected], +1-204-272-1562 Draft ACKNOWLEDGEMENTS This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC). We would like to thank Emy Komatsu, Dr. Hélène Perreault, and Dr. John Sorensen for help with mass spectrometry analysis of 2-5A. We also thank and acknowledge the financial support provided by the University of Manitoba to AK through a University of Manitoba Graduate Fellowship. 1 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 2 of 39 ABSTRACT Human 2’-5’ oligoadenylate synthetases (OAS) are a family of interferon inducible proteins which, upon activation by double-stranded RNA, polymerize ATP into 2’-5’ linked oligoadenylates. In this study, we probe 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 activatedDraft by dsRNA of any length greater than 19bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35bp. 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. Key words: Oligoadenylate synthetase, oligoadenylate, viral RNA, RNA. 2 https://mc06.manuscriptcentral.com/bcb-pubs Page 3 of 39 Biochemistry and Cell Biology INTRODUCTION 2’ 5’-oligoadenylate synthetases (OAS) are a family of interferon-inducible enzymes that act as pattern recognition receptors (PRR) to bind double-stranded RNA (dsRNA) during viral infection (Hovanessian 2007). Upon interaction with dsRNA, activated OAS catalyzes the polymerization of ATP into 2’ 5’- linked oligoadenylate (2-5A) chains, which in turn bind and activate their only known target, ribonuclease L (RNase L) (Zhou et al. 1993; Dong et al. 1997). Activated RNase L degrades both viral and cellular RNA, thereby attenuating viral replication (Li et al. 2016). The OAS-RNAse L system is therefore one of the main interferon-inducible antiviral pathways that prevent viral propagation (Player et al. 1998; Han et al. 2012) The human OAS family is composed of four genes, OAS1, OAS2, OAS3 and OASL, present on chromosome 12, of which only the first three produce a catalyticallyDraft active enzyme (Hovnanian et al. 1998; Rebouillat et al. 1998). These genes encode proteins with one (OAS1), two (OAS2) and three (OAS3) OAS core domains, and are hypothesized to form tetramer, dimer and monomer respectively in their catalytic state (Chebath et al. 1987; Hovanessian et al. 1987). Through alternative splicing, these genes encode 8 different catalytically active isoforms, including five OAS1 isoforms (p42, p44, p46, p48, and p52), two OAS2 isoforms (p69 and p71), and a single OAS3 (p100) isoform, (Lin et al. 2009). A core catalytic domain corresponding to the first 346 amino acids of human OAS1 is highly conserved across different species; this conserved domain is also present in OAS2 and OAS3 in two and three homologous copies (Hovanessian et al. 2007). OAS2 domain I and domain II share 41 and 53% identity with the core catalytic domain of OAS1, whereas OAS3 domains I, II and III share 42, 47 and 60% amino acid sequence identity with the OAS1 core catalytic domain (Hovanessian et al. 2007). The core catalytic OAS domain is a polymerase beta (pol-β) nucleotidyl transferase domain that belongs to the superfamily that includes poly-A polymerase (PAP1) (Hartmann et al. 2003) and 3 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 4 of 39 CCA adding enzymes (Torralba et al. 2008). Human OAS1 consists of a single pol-β like domain, whereas OAS2 and OAS3 consist of two and three such domains respectively, likely acquired by gene duplication during evolution (Justesen et al. 2000). However, OAS enzymes are distinct in this superfamily as they require a cofactor (dsRNA) for catalytic activity, in contrast to CCA and PAP1 that are constitutively active (Hovanessian et al. 2007). The core OAS1 domain adopts a bilobal structure with the catalytic aspartic acid triad located at the intersection of the N- and C- terminal lobes (Hartmann et al. 2003). On the opposite face from the active site, two dsRNA binding regions (separated by approximately 30 Å) enable recognition of two adjacent minor grooves encompassing approximately 17 base pairs (bp) (Donovan et al. 2013). The dsRNA-protein interaction is largely mediated through recognition of ribose 2’-OH in the minor groove and electrostatic interactions via basic aminoDraft acids in the dsRNA binding regions (Hartmann et al. 2003). Interaction with dsRNA requires reorientation of helix N3 in OAS1, leading to compression of active site aspartic acid residues (D75, D77, D148) into close proximity, enabling coordination of two magnesium ions required for substrate (ATP) binding during catalysis (Justesen et al. 2000; Donovan et al. 2013). The substrate specificities of the three OAS isozymes have begun to be investigated. OAS1, comprising a single domain, will bind dsRNA of any length provided that it exceeds 17 bp (Donovan et al. 2013). OAS1 also shows enhanced affinity for dsRNA with 5’ di/triphosphate ends as opposed to 5’ -OH (Meng et al. 2012; Donovan et al. 2015). A 3’ single-stranded pyrimidine overhang has been shown to enhance the catalytic activity of both perfect dsRNA and highly structured RNA containing sufficient stretches of dsRNA (Vachon et al. 2015). dsRNA with a single 3’ uridine overhang appears to enable optimal OAS1 activation (Vachon et al. 2015). In OAS3, the first two core domains (DI & DII) lack the aspartic acids required for catalytic activity but retain dsRNA binding, while the third domain (DIII) is catalytically active (Donovan et al. 4 https://mc06.manuscriptcentral.com/bcb-pubs Page 5 of 39 Biochemistry and Cell Biology 2015). The low-resolution solution structure of OAS3 suggests that the three domains are in an extended linear arrangement (Ibsen et al. 2014). A strong preference of OAS3 for dsRNA longer than 50 bp has been observed, and is suggested to require interaction with all three domains for optimal catalytic activity (Donovan et al. 2015). This length dependence is logical as a single OAS domain requires approximately 17 bp. Comparatively, little is known about OAS2, which contains an N-terminal catalytically inactive domain (DI) lacking the aspartic acid triad and a C-terminal catalytic domain (DII) (Sarkar et al. 1999b). OAS enzymes are differentially expressed in specific cell types, and their presence in different sub-cellular fractions such as mitochondria, nuclear, and rough/smooth microsomal fractions suggest that these proteins may have distinct functional roles (Chebath et al. 1987; Hovanessian et al. 1987). A comprehensiveDraft study of different OAS enzymes in dengue viral (DEN) infection demonstrated that OAS1 and OAS3, but not OAS2, were able to show anti-DEN activity (Lin et al. 2009). In contrast, two single nucleotide polymorphism (SNPs) in the OAS2 gene have been suggested to increase the susceptibility against DEN infection and no such association was found with SNPs of OAS1 and OAS3 (Thamizhmani et al. 2014). Murine cells expressing OAS2 isoforms show resistance against encephalomyocarditis virus (ECMV) infection but not towards the vesicular stomatitis virus (Marié et al. 1999). Porcine OAS2 has been shown to provide resistance against the porcine reproductive and respiratory syndrome virus (PRRSV) (Zhao et al. 2017) whereas porcine OAS1 has been shown to inhibit Japanese encephalitis virus (JEV) replication better than porcine OAS2 (Zheng et al. 2015). SNPs in OAS2 have also demonstrated an association with a predisposition to chronic hepatitis C (HCV) infection in the Russian population (Barkhash et al. 2014). In cases of severe chronic obstructive pulmonary disease, exposure to cigarette smoke can lead to more severe outcomes from influenza infection (Zhou et al.
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