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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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