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

ABSTRACT

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.

ACKNOWLEDGMENTS

This work was supported by NIH grant GM42188 to K.D.S., who is also a Burroughs Wellcome Scholar of Molecular Parasitology. T.E.A. was supported by NIH postdoctoral fellowship 1F32AI09206-01; S.H. and H.U.G. were supported by funding from the Deutsche Forschungsgemeinschaft.

We thank Reza Salavati and Rob Igo for critical reading of the manuscript and members of the Stuart laboratory for many helpful discussions.