Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of ϳ57 kDa (band IV) and ϳ50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.In kinetoplastid protozoans, many mitochondrial transcripts undergo RNA editing, a specific insertion and deletion of U residues at multiple sites, directed by guide RNAs (reviewed in references 2, 13, 15, 38, and 39). Both U deletional and U insertional editing cycles have been reproduced in vitro and shown to involve three enzymatic steps ( Fig. 1A) (9,20,34,35; see also reference 7). First, the mRNA is cleaved by a guide RNA (gRNA)-directed endonuclease, U residues are then added to or removed from the 3Ј end of the upstream cleavage product by a terminal-U-transferase or 3Ј-U-exonuclease, and the mRNA is then rejoined by RNA ligase. A complex consisting of seven different polypeptides that contains all these activities and catalyzes both U-deletional and U-insertional editing rounds has been purified from Trypanosoma brucei mitochondria (10, 29). We have undertaken the cloning and characterization of the polypeptides that make up this complex, beginning with one identified as an RNA ligase.RNA ligases are used by many cells in tRNA splicing (e.g., see references 5, 14, 45, and 49) and by bacteriophage T4 in tRNA repair (reviewed in reference 41), and they are also present in trypanosome mitochondria (3,17,46). These enzymes join RNA 3Ј hydroxyl and 5Ј phosphate termini, evidently by a common mechanism (30, 31; Fig. 1B). First, the ligase autoadenylylates, using ATP to form a covalent protein-AMP intermediate while releasing pyrophosphate (PP i ). This reaction occurs in the absence of RNA and reverses with high concentrations of PP i . The AMP is then transferred to the 5Ј phosphate of a donor RNA, generating a 5Ј-5Ј linkage, and the 3Ј hydroxyl of the acceptor RNA finally displaces this 5Ј AMP, forming the new phosphodiester bond. T. brucei mitochondrial extract ...

In the currently envisioned mechanism of trypanosome mitochondrial RNA editing, U-insertion and U-deletion cycles begin with a common kind of gRNA-directed cleavage. However, natural, altered, and mutationally interconverted editing sites reveal that U-deletional cleavage is inefficient without and activated by ATP and ADP, while U-insertional cleavage shows completely reverse nucleotide effects. The adenosine nucleotides' effects appear to be allosteric and determined solely by sequences immediately adjacent to the anchor duplex. Both U-deletional and U-insertional cleavages are reasonably active at physiological mitochondrial ATP concentration. Notably, ATP and ADP markedly stimulate complete U-deletion and inhibit U-insertion reactions, reflecting their effects on cleavage. These plus previous results suggest that U deletion and U insertion are remarkably distinct.