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. 2003 Jun;2(3):569-77.
doi: 10.1128/EC.2.3.569-577.2003.

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes

Gonzalo J Domingo  1 , Setareh S Palazzo, Bingbing Wang, Brian Pannicucci, Reza Salavati, Kenneth D Stuart
Affiliations

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes

Gonzalo J Domingo et al. Eukaryot Cell. 2003 Jun.

Abstract

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

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Figures

FIG. 1.
FIG. 1.
Gene presence and absence in normal and mutant trypanosomes. (A) Phase-contrast (a to c) and DAPI-stained fluorescence (d to f) micrographs of T. brucei EATRO164 wild type (EATRO164wt), derived acriflavin-induced dyskinetoplastic T. brucei EATRO164 (EATRO164Dk), and T. evansi AnTat 3/3. (B) DNA products from PCR amplifications performed with whole-cell DNA, using oligonucleotide primers for maxicircle-encoded genes (lane 1, A6 [381 bp]; lane 2, ND4 [239 bp]; lane 3, ND7 [144 bp]) and nucleus-encoded RNA-editing-associated RNA ligase genes (lane 4, REL2 [1,252 bp]; lane 5, REL1 [859 bp]).
FIG. 2.
FIG. 2.
Editosomes in normal and Dk mutant trypanosomes. (A) T. brucei EATRO164wt; (B) T. brucei EATRO164Dk; (C) T. evansi AnTat 3/3. Editosome protein components were identified by Western analysis using monoclonal antibodies to proteins TbMP81, TbMP63, REL1 (TbMP52), and TbMP42 (top panels for each strain) and by autoadenylation of REL1 and REL2 (TbMP48) (bottom panels for each strain), as described in Materials and Methods. Fractions (0.5 ml) from the 10 to 30% glycerol gradients (glycerol gradient) of whole-cell lysates (bottom right), samples enriched for mitochondria (mito.), and immunoprecipitates using immobilized monoclonal antibody P1H3 to TbMP63 [I.P. (P1H3)] were examined. A marker with a sedimentation coefficient of ∼20S elutes over fractions 7 to 10 on the same glycerol gradient. IgG indicates the IgG heavy chain of P1H3, which reacts with the anti-mouse second antibody.
FIG. 3.
FIG. 3.
Precleaved deletion and insertion in vitro editing of glycerol gradient fractions. Fractions 1 to 15 (top to bottom) from whole-cell lysates of T. brucei EATRO164wt (A), T. brucei EATRO164Dk (B), and T. evansi AnTat 3/3 (C) were assayed for gRNA-directed deletion editing (left panels), and pooled fractions 7 to 11 were assayed for gRNA-directed insertion editing (right panels). The arrows indicate the radiolabeled 5′CL18 pre-mRNA substrate (S), the processed substrate from which 4U are removed (−4U) or to which 2U added (+2U), the edited product (P), and the background of the substrate ligated to the 3′ fragment without processing (∗).
FIG. 4.
FIG. 4.
Precleaved deletion (A) and insertion (B) in vitro editing of immunoprecipitates. Immunoprecipitates from whole-cell lysates of T. brucei EATRO164wt, T. brucei EATRO164Dk, and T. evansi AnTat 3/3 made with monoclonal antibody P1H3, which is specific for editosome protein TbMP63, were assayed for gRNA-directed precleaved deletion (A) and precleaved insertion (B) editing in vitro. The arrows indicate the radiolabeled 5′CL18 pre-mRNA substrate (S), the processed substrate from which 4U are removed (−4U) or to which 2U added (+2U), the edited product (P), and the background of the substrate ligated to the 3′ fragment without processing (∗).
FIG. 5.
FIG. 5.
Full-round deletion editing by editosomes from Dk mutants. The32P-labeled A6short/TAG substrate was incubated with glycerol gradient fraction 9 from T. brucei EATRO164wt, T. brucei EATRO164Dk, and T. evansi AnTat 3/3 in the presence (+g) or absence (−g) of gA6[14]USDdccc gRNA. The substrate (S) and products of specific endonuclease cleavage (Endo) and editing (P) are indicated. The right panel is an enlarged section of the full gel in the left panel to show the substrate and product region. The endonuclease doublet products reflect the addition of one and two C's during the T4 RNA ligase radiolabeling of the substrate. A control (C) lane has no added fraction 9, and RNase T1-digested substrate is used as a marker (T1).
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