RNA



REPORT

Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei


MICHAEL T.  McMANUS a1, MASAKO  SHIMAMURA a2, JAYLEEN  GRAMS a1 and STEPHEN L.  HAJDUK a1c1
a1 Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
a2 Department of Pediatrics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

Abstract

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3′ uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

(Received October 12 2000)
(Revised October 27 2000)
(Accepted November 6 2000)


Key Words: epitope tag; nucleotidyl transferase; PSI-BLAST; trypanosome.

Correspondence:
c1 Reprint requests to: Stephen Hajduk, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA; e-mail: shajduk@uab.edu.