Guide RNA-directed uridine insertion RNA editing in vitro

Elaine M. Byrne, Gregory J. Connell, Larry Simpson

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66 Scopus citations


Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa, The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and α-β bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to ~13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.

Original languageEnglish (US)
Pages (from-to)6758-6765
Number of pages8
JournalEMBO Journal
Issue number23
StatePublished - Dec 2 1996


  • Guide RNA
  • Kinetoplast DNA
  • ND7
  • RNA editing
  • Uridine insertion


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