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 contr ibutes to the structural stability of the editing complex, so its lethal ge netic knock-out could reflect structural or catalytic requirements. To asse ss the catalytic role in editing, we generated cell lines which inducibly r eplaced band IV protein with an enzymatically inactive but structurally con served version. This induction halts cell growth, showing that catalytic ac tivity is essential. These induced cells have impaired in vivo editing, spe cifically of RNAs requiring uridylate (U) deletion; unligated RNAs cleaved at U-deletion sites accumulated. Additionally, mitochondrial extracts of ce lls with reduced band IV activity were deficient in catalyzing U-deletion, specifically at its ligation step, but were not deficient in U-insertion. T hus band IV ligase is needed to seal RNAs in U-deletion. U-insertion does n ot appear to require band IV, so it might use the other ligase of the editi ng complex. Furthermore, band IV ligase was also found to serve an RNA repa ir function, both in vitro and in vivo.