Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair.
Trypanosome RNA editing utilizes a seven polypeptide complex that includes two RNA ligases, band IV and band V. We now find that band IV protein contributes to the structural stability of the editing complex, so its lethal genetic knock-out could reflect structural or catalytic requirements. To assess the catalytic role in editing, we generated cell lines which inducibly replaced band IV protein with an enzymatically inactive but structurally conserved version. This induction halts cell growth, showing that catalytic activity is essential. These induced cells have impaired in vivo editing, specifically of RNAs requiring uridylate (U) deletion; unligated RNAs cleaved at U-deletion sites accumulated. Additionally, mitochondrial extracts of cells with reduced band IV activity were deficient in catalyzing U-deletion, specifically at its ligation step, but were not deficient in U-insertion. Thus band IV ligase is needed to seal RNAs in U-deletion. U-insertion does not appear to require band IV, so it might use the other ligase of the editing complex. Furthermore, band IV ligase was also found to serve an RNA repair function, both in vitro and in vivo.
Huang
C E
CE
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Cruz-Reyes
J
J
Zhelonkina
A G
AG
O'Hearn
S
S
Wirtz
E
E
Sollner-Webb
B
B
D000818:Animals; D007700:Kinetics; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011118:Polynucleotide Ligases / Q000378:metabolism*; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D011118:Polynucleotide Ligases; C436704:band IV ligase; C436705:band V ligase