RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3-terminal uridylyl transferase, 3-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an ϳ49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.Several mitochondrial RNAs are posttranscriptionally edited in kinetoplastid protozoa by the insertion and deletion of uridylates (U's) at multiple sites, to produce mature mRNAs. RNA editing creates initiation and termination codons and the likely functional open reading frames (ORFs). Indeed, translation of edited RNA has recently been directly demonstrated (11). The RNA editing appears to regulate mitochondrial respiration in different life cycle stages of Trypanosoma brucei. The insertion and deletion of U's is directed by small RNAs that are called guide RNAs (gRNAs). The editing occurs by a series of enzymatic steps. These steps include gRNA-directed cleavage of the pre-mRNA by endoribonuclease, U addition or removal at the 3Ј end of the 5Ј cleavage product by 3Ј-terminal uridylyl transferase (TUTase) or 3Ј-exouridylylase, respectively, and ligation of 5Ј and 3Ј cleavage products by RNA ligase (reviewed in references 6, 13, and 28).RNA editing occurs in association with a ribonucleoprotein complex which sediments at 20S in glycerol gradients (4, 22). Fractionation and hence partial purification of the complex by glycerol gradient and liquid chromatographic techniques have been reported (4,18,22,24). For the most part, these preparations were insufficient to identify specific proteins that are part of the editing complex. However, Rusché et al. (24) suggested that a complex of eight proteins could catalyze editing. They concluded that three of these proteins were adenylylatable and suggested that they represented the editing RNA ligase, although the role of these proteins has not yet been demonstrated. Indeed, little progress has been made on the definitive identification of proteins that are components of the editing complex. Three T. brucei mitochondrial proteins, gBP21 (15), DEAD box protein mHEL61p (19), and REAP1 (18...

RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact.RNA editing in trypanosomes posttranscriptionally inserts and deletes uridylates (U's) at multiple sites in most mitochondrial pre-mRNAs to produce mature mRNAs. U insertion and deletion are directed by guide RNAs (gRNAs) and are catalyzed by a macromolecular complex. Editing occurs by a series of enzymatic steps that include endoribonuclease, 3Ј terminal uridylyl transferase (TUTase), 3Ј exouridylylase, and RNA ligase activities (reviewed in references 3, 8, 21, and 23). Although editing can be extensive, with the insertion and deletion of numerous U's, it is also very specific. The characteristics of the enzymatic activities contribute to this specificity (7), but noncatalytic proteins may be required for editing and may contribute to the specificity.RNA editing is catalyzed by a 20S ribonucleoprotein complex (2, 15), and identification of its components and the composition of the fully functional complex is at an early stage. Initial studies estimated that a complex that can catalyze at least some of the steps of editing in vitro contains 7 to 20 polypeptides (11,14,16). Two related proteins, TbMP52 and TbMP48, were identified by mass spectrometric analysis of purified editing complexes (14), and TbMP52 was shown to be essential for RNA editing and for survival of bloodstream forms in vivo and in vitro (19). In addition, TbMP52 and TbMP48 correspond to the larger and smaller adenylatable proteins in the RNA editing complex, respectively, and were found to be RNA ligases (12,17,19). TbMP52 corresponds to T. brucei V and T. brucei p52 and TbMP48 corresponds to T. brucei IV and T. brucei p48 (12,17). The fully functional editing complex, which may consist of a catalytic core complex and accessory and regulatory factors may contain numerous proteins. Several other candidate proteins, some of which have RNA binding activities, have been described and may play a role in RNA editing (6, 9, 11, 13, 25). However, except for mHel61p (13), none of these proteins have been shown to play a direct role in editing.I...

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3 exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3 pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5 phosphate of the 3 fragment, which is not required for U removal. The ability of the 3 U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3 exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R p )␣-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.

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