Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae

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Abstract
The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection–amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5′ of the editing sites and guanosines immediately 3′ of guiding nucleotides. U insertions are directed both 5′ and 3′ of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.