Trypanosoma brucei RNA editing: coupled cycles of U deletion reveal processive activity of the editing complex.

RNA editing in Trypanosoma brucei is posttranscriptional uridylate removal/addition, generally at vast numbers of pre-mRNA sites, but to date, only single editing cycles have been examined in vitro. We here demonstrate achieving sequential cycles of U deletion in vitro, with editing products confirmed by sequence analysis. Notably, the subsequent editing cycle is much more efficient and occurs far more rapidly than single editing cycles; plus, it has different recognition requirements.

T. brucei RNA editing: action of the U-insertional TUTase within a U-deletion cycle.

Trypanosome RNA editing is massive post-transcriptional U-insertion and U-deletion, which generates mature mRNA coding regions through cycles of endonuclease, terminal U transferase (TUTase) or 3'-U-exo, and ligase action. Both types of editing are thought to be catalyzed by distinct sets of proteins of a multiprotein complex, and no enzymatic activity of wild-type editing complex had been shown to function in both forms of editing. By examining the individual steps of the U-deletion cycle using purified editing complex, traditional mitochondrial extract, and rapidly prepared cell lysate, we here demonstrate that TbMP57 TUTase of U-insertion can act efficiently within a U-deletion cycle.

Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins.

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII.

Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair.

Trypanosome RNA editing utilizes a seven polypeptide complex that includes two RNA ligases, band IV and band V. We now find that band IV protein contributes to the structural stability of the editing complex, so its lethal genetic knock-out could reflect structural or catalytic requirements. To assess the catalytic role in editing, we generated cell lines which inducibly replaced band IV protein with an enzymatically inactive but structurally conserved version.