Guide RNA‐directed uridine insertion RNA editing in vitro.

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 alpha‐beta bond hydrolysis of ATP were required, and the activity was inhibited above 50–100 mM KCl. A gRNA‐independent insertion of up to approximately 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.