The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created only after the addition or deletion of specific uridines. Although in vitro systems have been valuable in characterizing the editing mechanism, only a limited number of mRNAs are accurately edited in vitro. We demonstrate here that in vitro editing of cytochrome b mRNA is inhibited by an A-U sequence present on both the 5-untranslated sequence and on a cytochrome b guide RNA. Mutation of the sequence on the guide RNA stimulates directed editing and results in the loss of binding to at least one component within the editing extract. Mutation of the sequence on the mRNA increases the accuracy of the editing. Evidence is provided that suggests the A-U sequence interacts with the editing machinery both in vitro and in vivo.
Several mitochondrial mRNAs of the kinetoplastid protozoa do not encode a functional open reading frame until they have been edited through the addition or deletion of U nucleotides at specific sites. Genetic information specifying the location and extent of editing is present on guide RNAs (gRNAs). The sequence adjacent to most mRNA editing sites has a high purine content which previously has been proposed to facilitate the editing reaction through base-pairing to a poly(U) tail at the 3' end of the gRNA. We demonstrate here that gRNA binding alone is insufficient to create an editing site and that the mRNA sequence near an editing site is an additional determinant affecting the efficiency of the reaction.
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