The two RNA ligases of the Trypanosoma brucei RNA editing complex: Cloning the essential band IV gene and identifying the band V gene

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. ​(Fig.1A)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 3′ end of the upstream cleavage product by a terminal-U-transferase or 3′-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. FIG. 1 Process of RNA editing and mechanism of RNA ligase action. (A) RNA editing has been found to involve the indicated enzymatic steps, as described in the introduction. TUTase, terminal-U-transferase. (B) Mechanism of many RNA and DNA ligases, including ... 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 3′ hydroxyl and 5′ phosphate termini, evidently by a common mechanism (30, 31; Fig. ​Fig.1B).1B). First, the ligase autoadenylylates, using ATP to form a covalent protein-AMP intermediate while releasing pyrophosphate (PPi). This reaction occurs in the absence of RNA and reverses with high concentrations of PPi. The AMP is then transferred to the 5′ phosphate of a donor RNA, generating a 5′-5′ linkage, and the 3′ hydroxyl of the acceptor RNA finally displaces this 5′ AMP, forming the new phosphodiester bond. T. brucei mitochondrial extract contains adenylylatable proteins of ∼57 and ∼50 kDa that deadenylylate when incubated with PPi or ligatable RNA but not when incubated with nonligatable RNA or ligatable DNA (29–31), indicating that they are RNA ligases. While some T. brucei lines show one ∼57-kDa polypeptide (8, 31), the TREU 667 line our laboratory works with has two closely migrating ∼57-kDa forms, as well as the ∼50-kDa species (29). These three polypeptides are all constituents of the minimal editing complex (29), indicating their roles in RNA editing. To facilitate characterization of the enzymology and biological requirement for the ∼57-kDa RNA ligase, we cloned its cDNA, expressed it in Escherichia coli and trypanosomes, and performed genetic knockout analysis. These studies show that we have cloned the gene for a mitochondrially targeted RNA ligase that is essential in procyclic trypanosomes, regulated in abundance, and highly related to another predicted protein, evidently the ∼50-kDa ligase of the RNA editing complex.