Cis-acting Elements Stimulating Kinetoplastid Guide RNA-directed Editing

Lisa M. Oppegard, Matthew Hillestad, Ryan T. McCarthy, Raj D. Pai, Gregory J. Connell
2003 Journal of Biological Chemistry  
The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in
more » ... quirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A ؉ U sequence element within the 5-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction. The kinetoplastid uridylate insertional and deletional editing reactions have been proposed to be initiated by an endonuclease cleavage at an editing site that produces a 5Ј mRNA fragment with a 3Ј-hydroxyl and a 3Ј fragment with a 5Ј
doi:10.1074/jbc.m307997200 pmid:14532294 fatcat:aksdvdxslvfkfc5bmh35js6lai