Formation of guide RNA/messenger RNA chimeric molecules in vitro, the initial step of RNA editing, is dependent on an anchor sequence

Synthetic pre-edited messenger RNA (mRNA) and guide RNA (gRNA) for the 5'-edited maxicircle-encoded ND7 cryptogene from Leishmania tarentolae formed chimeric molecules upon incubation in the presence of a mitochondrial extract. These chimeric molecules consisted of the gRNAs covalently linked to the mRNAs by short oligo(U) tails at normal editing sites in most cases. Unlike the previously reported chimeras present in steady-state kinetoplast RNA, the in vitro-synthesized chimeras showed no editing of downstream editing sites. The synthesis of chimeric RNAs required ATP and was dependent on the formation of a gRNA/mRNA anchor duplex 3' of the pre-edited region, as shown by in vitro mutagenesis of the mRNA and the gRNA. mRNA sequences 3' and 5' of the pre-edited region also affected the efficiency of the chimera-forming activity. This in vitro system may accurately represent the initial step in RNA editing.