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

Alevtina G. Zhelonkina, Sean F. O'Hearn, Julie A. Law, Jorge Cruz-Reyes, Catherine E. Huang, Vadim S. Alatortsev, Barbara Sollner-Webb

Research output: Contribution to journalArticle

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. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish (US)
Pages (from-to)476-487
Number of pages12
JournalRNA
Volume12
Issue number3
DOIs
Publication statusPublished - Mar 2006

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Keywords

  • Editing
  • RNA
  • Terminal U transferase
  • Trypanosome
  • TUTase

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Zhelonkina, A. G., O'Hearn, S. F., Law, J. A., Cruz-Reyes, J., Huang, C. E., Alatortsev, V. S., & Sollner-Webb, B. (2006). T. brucei RNA editing: Action of the U-insertional TUTase within a U-deletion cycle. RNA, 12(3), 476-487. https://doi.org/10.1261/rna.2243206