Recognition of a cluster of RNA editing sites in the atp4 mRNA

Abstract

In most editing sites analysed in vitro or in vivo, sequences within approximately 30 nucleotides (nt) 5" and 10 nt 3" of the edited C have been found to be required and sufficient for selection of the correct C editing target and for editing efficiency. All previous studies focused on the investigation of processing of singular editing sites. Here I investigate recognition and processing of several editing sites located within few nucleotides in the mitochondrial atp4 transcript. Here three editing sites are clustered in four nucleotides (CUCC at nucleotide positions 248, 250, 251). I find that a single cis-element of about 20 nucleotides serves for recognition of sites 1 and 3, while site 2 possibly requires a distinct recognition complex. Competition with this sequence element for sites 1 and 3 suppresses in vitro editing of both sites. Even RNA molecules in which all editing sites are substituted by A or G can compete in vitro editing of a wild type substrate indicating that editing site recognition can occur independently of the actual editing site. Experiments with partially pre-edited substrates confirm that the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). Cis-element duplication within the RNA template dramatically increases the in vitro editing activity. This enhancing effect is observed when the distance between the editing sites of the individual repeats is about 25 nucleotides. If this distance is increased to 40 nucleotides the enhancing effect disappears. Analysis of the editing sites status in the repeat-containing RNAs shows a random distribution of the editing events between the repeats. This observation suggests a touch-and-go mechanism of RNA editing site recognition rather than a progressive linear processivity. In vitro experiments with two different editing sites with similar surrounding sequences suggest that such similar sequences can be recognized by the same trans-factor(s).