Impact of double-stranded RNA characteristics on the activation of human 2′–5′-oligoadenylate synthetase 2 (OAS2)

Author:

Koul Amit1,Deo Soumya2,Booy Evan P.1,Orriss George L.1,Genung Matthew3,McKenna Sean A.14

Affiliation:

1. Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.

2. Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.

3. Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, R3E 0W2, Canada.

4. Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.

Abstract

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.

Publisher

Canadian Science Publishing

Subject

Cell Biology,Molecular Biology,Biochemistry

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