Distinct Functions of Two RNA Ligases in Active Trypanosoma brucei RNA Editing Complexes

Abstract

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

We thank Laura Rusché for providing the purified editing complex, Sean O'Hearn for letting us mention provocative unpublished results, Michele Klingbeil, Paul Englund, Pierre Coulombe, Randy Bryant, and members of our lab for helpful discussions, Marilyn Parsons, Al Mildvan, and Pete Peterson for advice on nomenclature, and Vadim S. Alatortsev for computer and software expertise in the preparation of figures.

We thank the NIH (grant GM34231) for support.

The first two authors contributed equally to this work.