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Molecular and Cellular Biology, February 2001, p. 884-892, Vol. 21, No. 3
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.3.884-892.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold

Jorge Cruz-Reyes, Alevtina Zhelonkina, Laura Rusche,dagger and Barbara Sollner-Webb*

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 21 July 2000/Returned for modification 13 September 2000/Accepted 7 November 2000

Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3' OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.

* Corresponding author. Mailing address: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Phone: (410) 955-6278. Fax: (410) 955-0192. E-mail: bsw{at}jhmi.edu.

dagger Present address: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720.

Molecular and Cellular Biology, February 2001, p. 884-892, Vol. 21, No. 3
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.3.884-892.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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