Analysis of adenovirus VA RNA(I) structure and stability using compensatory base pair modifications

• Type of Material: Article
• Authors: Veronica K. Coventry, University of Manchester; Graeme Conn, Emory University
• Language: English
• Date: 2008-03-01
• Publisher: Oxford University Press (OUP): Policy C - Option B
• Publication Version: Final Published Version
• Copyright Statement: © 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gkn020
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 36
• Issue: 5
• Start Page: 1645
• End Page: 1653
• Grant/Funding Information: This work was supported by a Wellcome Trust Research Career Development Fellowship (ref. 061444) to G.L.C. and Medical Research Council PhD studentship to V.K.C.; Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.
• Keywords: PKR; BIOCHEMISTRY & MOLECULAR BIOLOGY; Science & Technology; INTERFERON; CENTRAL DOMAIN; Biochemistry & Molecular Biology; PURIFICATION; Life Sciences & Biomedicine; ACTIVATION; PROTEIN-KINASE DAI; SECONDARY; VIRUS-ASSOCIATED RNA; BINDING; STRANDED-RNA
• Research Categories: Chemistry, Biochemistry; Health Sciences, Pathology

Abstract

Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNAI were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNAI was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent Tm of ∼60°C. In contrast, the Apical Stem unfolds independently and with much higher apparent Tm of ∼83°C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNAI Central Domain structure essential for PKR inhibitory activity was identified.