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Molecular and Cellular Biology, March 2002, p. 1567-1576, Vol. 22, No. 5
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.5.1567-1576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA Sequence and Base Pairing Effects on Insertion Editing in Trypanosoma brucei

Robert P. Igo, Jr.,1 Sobomabo D. Lawson,1 and Kenneth Stuart1,2*

Seattle Biomedical Research Institute, Seattle, Washington 98109,1 Department of Pathobiology, University of Washington, Seattle, Washington 981952

Received 16 July 2001/ Returned for modification 10 September 2001/ Accepted 29 November 2001

RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.

* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109. Phone: (206) 284-8846, ext. 316. Fax: (206) 284-0313. E-mail: kstuart{at}u.washington.edu or kstuart{at}sbri.org.

Molecular and Cellular Biology, March 2002, p. 1567-1576, Vol. 22, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.5.1567-1576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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