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Molecular and Cellular Biology, February 2001, p. 884-892, Vol. 21, No. 3
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.
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
and
*
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.
Present address: Department of Molecular and Cell Biology,
University of California at Berkeley, Berkeley, CA 94720.
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