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Nucleic Acids Research Advance Access originally published online on February 3, 2008
Nucleic Acids Research 2008 36(5):1645-1653; doi:10.1093/nar/gkn020
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Nucleic Acids Research, 2008, Vol. 36, No. 5 1645-1653
© 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.

RNA

Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications

Veronica K. Coventry1 and Graeme L. Conn1,2,*

1Faculty of Life Sciences and 2Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, M1 7DN, UK

*To whom correspondence should be addressed. Tel: +44 161 3064218; Fax: +44 161 2365201; Email: graeme.l.conn{at}manchester.ac.uk

Received November 13, 2007. Revised January 14, 2008. Accepted January 14, 2008.

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.

Present address: Veronica K. Coventry, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.


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