TY - JOUR
T1 - Determinants for Association and Guide RNA-Directed Endonuclease Cleavage by Purified RNA Editing Complexes from Trypanosoma brucei
AU - Hernandez, Alfredo
AU - Panigrahi, Aswini
AU - Cifuentes-Rojas, Catherine
AU - Sacharidou, Anastasia
AU - Stuart, Kenneth
AU - Cruz-Reyes, Jorge
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health to J.C.-R. (GM067130) and K.S. (GM42188). The mass spectrometric studies were performed at the Seattle Biomedical Research Institute (Seattle, WA). We thank Dr. C.T. Ranjith Kumar and, from the Cruz-Reyes laboratory, Bhaskara Reddy Madina and Ambrish Kumar for their comments on the manuscript and helpful discussions. We also thank Drs. Achim Schnaufer, Ruslan Aphasizhev, and Juan D. Alfonzo for their generous advice and protocols for tandem affinity purification and transfection of trypanosomes. pLEW79TAP was a gift from Achim Schnaufer. We are also grateful to Dr. Larry Simpson for providing T. brucei genomic DNA and Dr. Andrew MacMillan for his advice on the EMSA.
PY - 2008/8/1
Y1 - 2008/8/1
N2 - U-insertion/deletion RNA editing in the single mitochondrion of kinetoplastids, an ancient lineage of eukaryotes, is a unique mRNA maturation process needed for translation. Multisubunit editing complexes recognize many pre-edited mRNA sites and modify them via cycles of three catalytic steps: guide RNA (gRNA)-directed cleavage, insertion or deletion of uridylates at the 3′-terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. This study defined relatively simple single- and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with UV photocrosslinking and functional studies. The cleaved strand required a minimal 5′ overhang of 12 nt and an ∼ 15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports in the context of purified native editing complexes show that the determinants for association, cleavage and full-round editing gradually increase in complexity as these stages progress. The native complexes in these studies contained most, if not all, known core subunits in addition to components of the MRP complex. Finally, we found that the endonuclease KREN1 in purified complexes photocrosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.
AB - U-insertion/deletion RNA editing in the single mitochondrion of kinetoplastids, an ancient lineage of eukaryotes, is a unique mRNA maturation process needed for translation. Multisubunit editing complexes recognize many pre-edited mRNA sites and modify them via cycles of three catalytic steps: guide RNA (gRNA)-directed cleavage, insertion or deletion of uridylates at the 3′-terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. This study defined relatively simple single- and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with UV photocrosslinking and functional studies. The cleaved strand required a minimal 5′ overhang of 12 nt and an ∼ 15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports in the context of purified native editing complexes show that the determinants for association, cleavage and full-round editing gradually increase in complexity as these stages progress. The native complexes in these studies contained most, if not all, known core subunits in addition to components of the MRP complex. Finally, we found that the endonuclease KREN1 in purified complexes photocrosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.
KW - RNA editing
KW - RNP
KW - U-insertion/deletion
KW - guide RNA
KW - kinetoplastids
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U2 - 10.1016/j.jmb.2008.05.003
DO - 10.1016/j.jmb.2008.05.003
M3 - Article
C2 - 18572190
AN - SCOPUS:46649094360
SN - 0022-2836
VL - 381
SP - 35
EP - 48
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -