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Nucleic Acids Research 2005 33(16):5250-5261; doi:10.1093/nar/gki822
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Published online 16 September 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

Nerea Beguiristain1, Hugh D. Robertson2 and Jordi Gómez1,3,*

1Laboratorio de Medicina Interna, Hospital Vall d'Hebron Barcelona 08035, Spain 2Department of Biochemistry, Weill Medical College of Cornell University New York, NY 1002, USA 3Centro Investigación en Sanidad Animal INIA Valdeolmos, Spain

*To whom correspondence should be addressed. Tel: +34 916202300 ext. 125; Fax: +34 916202247; Email: jgomez{at}vhebron.net, castilla{at}inia.es

Received May 3, 2005. Revised July 22, 2005. Accepted August 23, 2005.

Here, we show that Escherichia coli Ribonuclease III cleaves specifically the RNA genome of hepatitis C virus (HCV) within the first 570 nt with similar efficiency within two sequences which are ~400 bases apart in the linear HCV map. Demonstrations include determination of the specificity of the cleavage sites at positions C27 and U33 in the first (5') motif and G439 in the second (3') motif, complete competition inhibition of 5' and 3' HCV RNA cleavages by added double-stranded RNA in a 1:6 to 1:8 weight ratio, respectively, 50% reverse competition inhibition of the RNase III T7 R1.1 mRNA substrate cleavage by HCV RNA at 1:1 molar ratio, and determination of the 5' phosphate and 3' hydroxyl end groups of the newly generated termini after cleavage. By comparing the activity and specificity of the commercial RNase III enzyme, used in this study, with the natural E.coli RNase III enzyme, on the natural bacteriophage T7 R1.1 mRNA substrate, we demonstrated that the HCV cuts fall into the category of specific, secondary RNase III cleavages. This reaction identifies regions of unusual RNA structure, and we further showed that blocking or deletion of one of the two RNase III-sensitive sequence motifs impeded cleavage at the other, providing direct evidence that both sequence motifs, besides being far apart in the linear RNA sequence, occur in a single RNA structural motif, which encloses the HCV internal ribosome entry site in a large RNA loop.


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