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Molecular and Cellular Biology, April 2008, p. 2437-2445, Vol. 28, No. 7
0270-7306/08/$08.00+0     doi:10.1128/MCB.01886-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Trypanosoma brucei RNA Editing: Coupled Cycles of U Deletion Reveal Processive Activity of the Editing Complex{triangledown}

Vadim S. Alatortsev ,#,{dagger} Jorge Cruz-Reyes,#,{ddagger} Alevtina G. Zhelonkina,# and Barbara Sollner-Webb*

Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205

Received 18 October 2007/ Returned for modification 12 December 2007/ Accepted 18 January 2008

RNA editing in Trypanosoma brucei is posttranscriptional uridylate removal/addition, generally at vast numbers of pre-mRNA sites, but to date, only single editing cycles have been examined in vitro. We here demonstrate achieving sequential cycles of U deletion in vitro, with editing products confirmed by sequence analysis. Notably, the subsequent editing cycle is much more efficient and occurs far more rapidly than single editing cycles; plus, it has different recognition requirements. This indicates that the editing complex acts in a concerted manner and does not dissociate from the RNA substrate between these cycles. Furthermore, the multicycle substrate exhibits editing that is unexpected from a strictly 3'-to-5' progression, reminiscent of the unexpected editing that has been shown to occur frequently in T. brucei mRNAs edited in vivo. This unexpected editing is most likely due to alternate mRNA:guide RNA (gRNA) alignment forming a hyphenated anchor; its having only a 2-bp proximal duplex helps explain the prevalence of unexpected editing in vivo. Such unexpected editing was not previously reported in vitro, presumably because the common use of artificially tight mRNA:gRNA base pairing precludes alternate alignments. The multicycle editing and unexpected editing presented in this paper bring in vitro reactions closer to reproducing the in vivo editing process.

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

{triangledown} Published ahead of print on 28 January 2008.

# V.S.A., J.C.-R., and A.G.Z. contributed equally to this work and are listed alphabetically.

{dagger} Present address: Division of Metabolism, Departments of Pediatrics and Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287.

{ddagger} Present address: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843.

Molecular and Cellular Biology, April 2008, p. 2437-2445, Vol. 28, No. 7
0270-7306/08/$08.00+0     doi:10.1128/MCB.01886-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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