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Molecular and Cellular Biology, February 2001, p. 979-989, Vol. 21, No. 4
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.4.979-989.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Laura N. Rusché,1,dagger Catherine E. Huang,1 Kenneth J. Piller,1,Dagger Michael Hemann,1 Elizabeth Wirtz,2 and Barbara Sollner-Webb1,*

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,1 and Laboratory of Molecular Parasitology, The Rockefeller University, New York, New York 100212

Received 25 August 2000/Returned for modification 20 October 2000/Accepted 20 November 2000

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 from Trypanosoma 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.

* Corresponding author. Mailing address: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-6278. Fax: (410) 955-0192. E-mail: bsw{at}jhmi.edu.

dagger Present address: Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720.

Dagger Present address: Pharmacia-Monsanto Laboratories, St. Louis, MO 63198.

Molecular and Cellular Biology, February 2001, p. 979-989, Vol. 21, No. 4
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.4.979-989.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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