An in vitro RNA editing system from cauliflower mitochondria: Editing site recognition parameters can vary in different plant species

Abstract

Most of the 400 RNA editing sites in flowering plant mitochondria are found in mRNAs. Consequently, the sequence vicinities of homologous sites are highly conserved between different species and are presumably recognized by likewise conserved transfactors. To investigate the evolutionary adaptation to sequence variation, we have now analyzed the recognition elements of an editing site with divergent upstream sequences in the two species pea and cauliflower. This variation is tolerated at the site selected, because the upstream cis-elements reach into the 5 0 -UTR of the mRNA. To compare cis-recognition features in pea and cauliflower mitochondria, we developed a new in vitro RNA editing system for cauliflower. In vitro editing assays with deleted and mutated template RNAs show that the major recognition elements for both species are located within the conserved sequence. In cauliflower, however, the essential upstream nucleotides extend further upstream than they do in pea. In-depth analysis of single-nucleotide mutations reveals critical spacing of the editing site and the specific recognition elements, and shows that the +1 nucleotide identity is important in cauliflower, but not in pea.

A Study of New Arabidopsis Chloroplast RNA Editing Mutants Reveals General Features of Editing Factors and Their Target Sites

“…In vivo experiments using transgenic chloroplasts have shown that mRNA editing sites are recognized via cis-acting elements that are generally located within ;30 nucleotides of the editing site (Chaudhuri et al, 1995;Bock et al, 1996Chaudhuri and Maliga, 1996;Reed et al, 2001). This has been confirmed in vitro using synthetic RNA substrates to explore editing specificity in both chloroplast (Miyamoto et al, 2002;Hegeman et al, 2005;Hayes et al, 2006) and mitochondrial extracts (Neuwirt et al, 2005;Takenaka et al, 2004Takenaka et al, , 2008Verbitskiy et al, 2008). In other systems with such pervasive RNA editing, target sites are recognized by guide RNAs complementary to the RNA strand to be edited (Simpson and Emeson, 1996).…”

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

“…The cis-acting elements have been delineated to be about 30 nt surrounding the editing site for both organelles (33,34). Plant site-specific editing factors belong to the PLS subfamily of the PPR protein family (14).…”

RNA editing sites in plant mitochondria can share cis‐elements

“…The interdependence of two sites in the atp9 mRNA separated by only 30 nucleotides was analyzed in an in vitro system from cauliflower mitochondria [17].…”