Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.
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.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 35294, USA.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D017393:RNA Editing / Q000235:genetics*; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000302:isolation & purification*; D017394:RNA, Guide, Kinetoplastida / Q000502:physiology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000255:Adenosine Triphosphate; D011130:RNA Ligase (ATP); D017394:RNA, Guide, Kinetoplastida