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Molecular and Cellular Biology, November 2000, p. 8447-8457, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Uridylate Addition and RNA Ligation Contribute to the Specificity of Kinetoplastid Insertion RNA Editing

Robert P. Igo Jr., Setareh S. Palazzo, Moffett L. K. Burgess, Aswini K. Panigrahi, and Kenneth Stuart*

Seattle Biomedical Research Institute, Seattle, Washington 98109

Received 10 May 2000/Returned for modification 21 June 2000/Accepted 24 August 2000

RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5' fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5' fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.

* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109. Phone: (206) 284-8846 x316. Fax: (206) 284-0313. E-mail: kstuart{at}u.washington.edu.

Molecular and Cellular Biology, November 2000, p. 8447-8457, Vol. 20, No. 22
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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