Uridine insertion into preedited mRNA by a mitochondrial extract from Leishmania tarentolae: stereochemical evidence for the enzyme cascade model.
An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L Simpson, and A. M. Simpson, EMBO J. 14:178-187, 1995) and a 3'-terminal U addition activity (N. Bakalara, A. M. Simpson, and L. Simpson, J. Biol. Chem. 264:18679-18686, 1989) have been previously described by using a mitochondrial extract from Leishmania tarentolae. Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of these nucleotidyl transfer reactions. The extract utilizes (SP)-alpha-S-UTP for both 3' and internal U incorporation into substrate RNA. The internal as well as the 3' incorporation of (SP)-alpha-S-UTP proceeds via inversion of the stereoconfiguration. Furthermore, internal U incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. Our results are compatible with an enzyme cascade model for this in vitro U insertion activity involving sequential endonuclease and uridylyl transferase directly from UTP and RNA ligase steps and are incompatible with models involving the transfer of U residues from the 3' ends of guide RNAs.
Frech
G C
GC
Howard Hughes Medical Institute, University of California, Los Angeles 90095-1662, USA.
Simpson
L
L
D000818:Animals; D007891:Leishmania / Q000235:genetics*; D008928:Mitochondria / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D017393:RNA Editing; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000378:metabolism*; D013237:Stereoisomerism; D013873:Thionucleotides / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D013873:Thionucleotides; D012313:RNA; D011130:RNA Ligase (ATP); D014529:Uridine