T. bruceiRNA editing: Action of the U-insertional TUTase within a U-deletion cycle

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Abstract
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. When physiological UTP levels are provided, it adds U’s to the upstream cleavage fragment after U-deletional endonuclease and 3′-U-exo action, but before rejoining by the U-deletional ligase, generating partial U-deletion products. TUTase activity in U-deletion was not previously appreciated since its detection requires UTP, which is not normally added to in vitro U-deletion reactions. Fractionation and RNAi analyses show this U-addition in U-deletion requires TbMP57 TUTase be present and competent for U-insertion; such U-addition does not occur with another mitochondrial TUTase that is separate from the basic editing complex. Efficient TbMP57 action in both U-insertion and U-deletion suggests these two editing forms may be less separate than generally envisioned. Should such promiscuous TUTase action also occur in vivo, it could explain why editing utilizes substantially fewer U-deletional than U-insertional events and why partial editing appears preferential in U-deletion.