In Trypanosoma brucei RNA Editing, Band II Enables Recognition Specifically at Each Step of the U Insertion Cycle

Abstract

Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.

ACKNOWLEDGMENTS

We thank Zefeng Wang, Jim Morris, Mark Drew, and Paul Englund for the pZJM vector; Alevtina Zhelonkina for expert technical assistance in verifying cleavage specificity; R. Luise Krauth-Siegel for the lipoamide dehydrogenase antibody; Jorge Cruz-Reyes for the use of I47G prior to its complete publication; Paul Englund, Michelle Klingbeil, Kevin Hoff, and Alevtina Zhelonkina for suggestions on the manuscript; and members of our laboratory for helpful discussions.

This work was supported by NIH grant GM34231.