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 cleav age-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertio ns into a specific editing site of a pre-edited mRNA which was incubat ed in a mitochondrial extract from Leishmania tarentolae. The predomin ant number of U insertions was determined by the number of guiding nuc leotides in the added gRNA, and the formation of a gRNA-mRNA anchor du plex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCI , A gRNA-independent insertion of up to similar to 13 Us occurred in t he absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. He parin inhibited the gRNA-independent U insertion activity and had no e ffect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA h ad little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are deri ved directly from UTP.