Analysis of cis-elements for RNA editing at two sites in the atp9 mRNA

Abstract

To analyse the cis-requirements for RNA editing sites a newly developed in-vitro RNA editing system was employed. The cis-requirements for editing of atp9 site 1 in pea were investigated by templates deleted, sequence exchanged templates, and competition reactions. In the pea system, deleted templates in steps of 10 nucleotides between -40 to -20 edit correctly but with decreased efficiency, while deletions with less than 20 upstream nucleotides do not support editing. 3’ deletions have little effect in pea, suggesting little influence on recognition. These results suggest that 20 nucleotides upstream are necessary and sufficient for recognition of the editing site. Stepwise mutated RNAs as templates or competitors reveal distinct substructures of the cis-elements. In pea a sequence element situated -40 to -35 enhances editing. The essential core region for recognition is restricted to the 10 nucleotides between -15 to -5. Experiments show that the enhancing effect of the region -40 to -35 can be titrated. This suggests, that either an abundant trans-factor is participating in recognition or an in-vitro artificial sequence effect is observed. The trans-factors interacting with the core region are present in restricted amounts, since they are sensitive to competition. To investigate the evolutionary adaptation of sequence variations 5’ of an editing site in another plant, atp9 (1) was investigated in cauliflower. Like in pea, 20 upstream nucleotides are essential and sufficient for editing. In cauliflower 3’ deletions affect editing up to 50 %, suggesting a (sterical) influence of the +1 nucleotide. This inference is experimentally supported by the effect of +1 exchange mutants. Relative to pea, the core recognition region extends further upstream, suggesting adaptation of the trans-element(s). All recognition elements are located within the conserved area.