In vitro analysis of cis-elements and trans-factors for the RNA editing of tobacco chloroplast psbE and petB mRNAs

Abstract

RNA editing is one of the posttranscriptional processes in higher plant chloroplasts. Editing is found in many cases in internal protein-coding regions, resulting in amino acid substitutions. Therefore, it is important to understand editing events to use the chloroplast as a factory for protein production. The number of editing sites so far observed is 31 in tobacco and these editing events are C- to -U conversions. To analyze the molecular mechanism, we have developed an in vitro RNA editing system from tobacco green chloroplasts (1). Extracts were prepared from intact chloroplasts isolated from 5-10 cm tobacco leaves. Pre-mRNA portions in which the C residue to be edited was specifically labelled with 32P were used as in vitro substrates. Editing activity was assayed by detecting 32P-labelled U mononucleotides on cellulose TLC after digestion of substrates with nuclease P1. Using the in vitro system, cis-acting elements for editing were defined in tobacco chloroplast mRNAs from psbE and petB. In vitro competition assays indicated that trans-acting factors interact with these cis-elements and that the trans-factor is site-specific. UV cross-linking assays showed the existence of site-specific trans-factors for these mRNA editing. (1) T. Hirose and M. Sugiura, Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system. EMBO J. 20: 1144-1152 (2001)