The Two RNA Ligases of the Trypanosoma brucei RNA Editing Complex: Cloning the Essential Band IV Gene and Identifying the Band V Gene

Abstract

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified fromTrypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of ∼57 kDa (band IV) and ∼50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

ACKNOWLEDGMENTS

We gratefully acknowledge John Donelson and Najib El-Sayed for providing the cDNA library, David Reim and the Wistar protein analysis facility for protein sequencing, Larry Snyder for the HR171prr+ cells, Elisabetta Ullu and Chris Tschudi for communicating to us their unpublished results, Nina Agabian for helpful discussions, and Alevtina Zhelonkina for technical assistance with Northern blots.

L.N.R. was a Howard Hughes predoctoral fellow. This work was supported by NIH grant GM34231.

The first two authors contributed equally to this work.