title: Guide RNA-directed uridine insertion RNA editing in vitro.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, UCLA School of Medicine, 90095-1662, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: Byrne
authors.forename: G J
authors.initials: GJ
authors.lastname: Connell
authors.forename: L
authors.initials: L
authors.lastname: Simpson
mesh_terms: D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D006493:Heparin / Q000494:pharmacology; D007891:Leishmania / Q000235:genetics / Q000502:physiology*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D017393:RNA Editing; D017394:RNA, Guide, Kinetoplastida / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000378:metabolism; D013379:Substrate Specificity; D014529:Uridine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D012313:RNA; D006493:Heparin; D014529:Uridine; D017394:RNA, Guide, Kinetoplastida