In vitro guide RNA/mRNA chimaera formation in Trypanosoma bruceiRNA editing

Abstract

THE post-transcriptional processing of various mitochondrial transcripts in kinetoplastids, kRNA editing, adds and removes uridines, producing mature messenger RNAs1,2. This editing seems to be directed by 'guide' RNAs (gRNAs) which are complementary to portions of the mature message3. The editing mechanism has been proposed to entail transesterification4,5. Detection of chimaeric gRNA-mRNA molecules, intermediates predicted by transesterification, support this model4. We report here the in vitro formation of such chimaeras where endogenous gRNAs are covalently linked to added synthetic mRNA. Addition of gel-purified gRNAs to the standard reaction mix increases chimaera formation. This increase is not observed when the gRNA 3′-hydroxyl group is chemically modified, identifying this terminal hydroxyl as the reactive group. These results provide the first experimental evidence for an in vitroRNA editing event and support the involvement of transesterification as a chemical mechanism.