In Trypanosoma brucei RNA Editing, TbMP18 (Band VII) Is Critical for Editosome Integrity and for both Insertional and Deletional Cleavages

Abstract

In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an ∼20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the ∼20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as ∼10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the ∼20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.

We thank Paul Englund and present and past members of his and our laboratories for helpful discussions, Laura Rusche for purification of the editing complex, David Reim at the Wistar Institute for sequencing the tryptic peptides, Mike Hemann for cloning the band VII gene, Ken Piller for the procyclic strain 667 cDNA library, Paul Englund for the pZJM vector and heat shock protein 70 antibody, R. Louise Krauth-Siegel for lipoamide dehydrogenase antibody, and the reviewers for helpful comments.

This work was supported by NIH grant GM34231.