TRYPANOSOMA-BRUCEI MITOCHONDRIAL GUIDE RNA-MESSENGER-RNA CHIMERA-FORMING ACTIVITY COFRACTIONATES WITH AN EDITING-DOMAIN-SPECIFIC ENDONUCLEASE AND RNA LIGASE AND IS MIMICKED BY HETEROLOGOUS NUCLEASE AND RNA LIGASE

RNA editing in trypanosomes has been proposed to occur through transes terification or endonuclease cleavage and RNA ligation reactions, Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresp onding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondri al extracts had been reported to completely separate the gRNA-mRNA chi mera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to r ule out the endonuclease-RNA ligase mechanism. However, we show that a n editing-domain-specific endonuclease activity does cosediment with t he chimera-forming activity, as does the RNA ligase activity, but dete ction of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming a ctivity is mimicked by mung bean nuclease and T4 RNA ligase. Using cyt ochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that the se heterologous enzymes specifically generate CYb gRNA-mRNA chimeras a nalogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of c himera formation.