Uridine insertion/deletion RNA editing in trypanosomatid mitochondria: In search of the editosome.
The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a approximately 1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze-thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or "editosome."
Osato
Daren
D
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA.
Rogers
Kestrel
K
Guo
Qiang
Q
Li
Feng
F
Richmond
Greg
G
Klug
Felix
F
Simpson
Larry
L
D000818:Animals; D019729:Carbon-Oxygen Ligases / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014529:Uridine / Q000235:genetics* / Q000378:metabolism
D024101:Mitochondrial Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; C497156:gBP25 protein, Trypanosoma brucei; D012313:RNA; D019729:Carbon-Oxygen Ligases; C476580:REL1 RNA ligase, Trypanosoma brucei; D014529:Uridine