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Molecular and Cellular Biology, October 1998, p. 6014-6022, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Association of Guide RNA Binding Protein gBP21 with Active RNA Editing Complexes in Trypanosoma brucei

Thomas E. Allen,1,2 Stefan Heidmann,3 RoseMary Reed,1,2 Peter J. Myler,1,2 H. Ulrich Göringer,4 and Kenneth D. Stuart1,2,*

Seattle Biomedical Research Institute, Seattle, Washington, 98109-1651,1 and Department of Pathobiology, University of Washington Seattle, Washington 98195,2 and Department of Genetics, University of Bayreuth, 95440 Bayreuth,3 and Laboratorium für Molekulare Biologie, Genzentrum der LMU München am MPI für Biochemie, 82152 Martinsried,4 Germany

Received 6 April 1998/Returned for modification 3 June 1998/Accepted 19 June 1998

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

* Corresponding author. Mailing address: Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109-1651. Phone: (206) 284-8846, ext. 316. Fax: (206) 284-0313. E-mail: kstuart{at}u.washington.edu.

Molecular and Cellular Biology, October 1998, p. 6014-6022, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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