Determinants for Association and Guide RNA-Directed Endonuclease Cleavage by Purified RNA Editing Complexes from Trypanosoma brucei

Alfredo Hernandez, Aswini Panigrahi, Catherine Cifuentes-Rojas, Anastasia Sacharidou, Kenneth Stuart, Jorge Cruz-Reyes

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)35-48
Number of pages14
JournalJournal of Molecular Biology
Volume381
Issue number1
DOIs
StatePublished - Aug 1 2008
Externally publishedYes

Keywords

  • guide RNA
  • kinetoplastids
  • RNA editing
  • RNP
  • U-insertion/deletion

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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