GUIDE RNA REQUIREMENT FOR EDITING-SITE-SPECIFIC ENDONUCLEOLYTIC CLEAVAGE OF PREEDITED MESSENGER-RNA BY MITOCHONDRIAL RIBONUCLEOPROTEIN-PARTICLES IN TRYPANOSOMA-BRUCEI

RNA editing in trypanosome mitochondria entails the posttranscriptiona l internal addition and occasional deletion of uridines from precursor mRNAs. Ample evidence exists to show that the information specifying the site and number of uridines added or deleted comes from small, mit ochondrially encoded guide RNAs (gRNAs), More recent work indicates th at the process involves an enzymatic cascade, initiating with an endon ucleolytic cleavage of the pre-mRNA at an editing site, The cleaved ed iting site can undergo uridine (U) addition to or deletion from the 3' end of the 5' fragment via a mitochondrial terminal uridylyl transfer ase (TUTase) or terminal uridylyl exonuclease, respectively. Mitochond rial RNA ligase subsequently rejoins the mRNA. Activities to carry out these processes have been found in trypanosome mitochondria, includin g an editing-site-specific endonuclease activity which cleaves preedit ed but not edited mRNAs. We have found that this enzymatic activity co sediments with the same 19S ribonucleoprotein particle previously show n to contain TUTase, RNA ligase, and gRNAs and remains stable after sa lt treatment, Depletion of endogenous cytochrome b gRNAs by the additi on of complementary oligonucleotides in vitro completely inhibits edit ing-site cleavage of synthetic preedited cytochrome b mRNA. The additi on of synthetic cognate gRNA for cytochrome b but not unrelated small RNA restores editing-site cleavage, These studies show that in additio n to specifying the site and number of uridines added or deleted, gRNA s provide the necessary information for cleavage by the editing-site s pecific endonuclease.