Association of guide RNA binding protein gBP21 with active RNA editing complexes in Trypanosoma brucei.
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.
Allen
T E
TE
Seattle Biomedical Research Institute, Seattle, Washington, 98109-1651, USA.
Heidmann
S
S
Reed
R
R
Myler
P J
PJ
Göringer
H U
HU
Stuart
K D
KD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000913:Antibodies, Protozoan / Q000096:biosynthesis / Q000276:immunology; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D015800:Protozoan Proteins / Q000276:immunology / Q000378:metabolism*; D012313:RNA; D017393:RNA Editing; D017394:RNA, Guide, Kinetoplastida / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D015800:Protozoan Proteins; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; C104816:gBP21 protein, Trypanosoma brucei; D012313:RNA; D017394:RNA, Guide, Kinetoplastida