RNA LIGASE AND ITS INVOLVEMENT IN GUIDE RNA MRNA CHIMERA FORMATION - EVIDENCE FOR A CLEAVAGE-LIGATION MECHANISM OF TRYPANOSOMA-BRUCEI MESSENGER-RNA EDITING/

RNA editing in Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. Editing is th ought to be directed by guide RNAs and may proceed via a chimeric guid e RNA/mRNA intermediate. We have previously shown that chimera-forming activity sediments with 19 S and 35-40 S mitochondrial ribonucleoprot ein particles (RNPs). In this report we examine the involvement of RNA ligase in the production of chimeric molecules in vitro. Two adenylyl ated proteins of 50 and 57 kDa cosediment on glycerol gradients with R NA ligase activity as components of the ribonucleoprotein particles. T he two adenylylated proteins differ in sequence and contain AMP linked via a phosphoamide bond. Both proteins are deadenylylated by the addi tion of ligatable RNA substrate with the concomitant release of AMP an d by the addition of pyrophosphate to yield ATP. Incubation with nonli gatable RNA substrate results in an accumulation of the adenylylated R NA intermediate. These experiments identify the adenylylated proteins as RNA ligases. AMP release from the mitochondrial RNA ligase is also concomitant with chimera formation. Inhibition by nonhydrolyzable anal ogs indicates that both RNA ligase and chimera-forming activities requ ire alpha-beta bond hydrolysis of ATP. Deadenylylation of the ligase i nhibits chimera formation. These results strongly suggest the involvem ent of RNA ligase in in vitro chimera formation and support the cleava ge-ligation mechanism for kinetoplastid RNA editing.