Cross-competition in transgenic chloroplasts expressing single editing sites reveals shared cis elements

Abstract

RNA editing in organelles of angiosperm plants results in alteration of Cs to Us in transcripts. In most editing sites analyzed 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 for selection of the correct C editing target and for editing efficiency, but no consensus sequences have been identified. The effect of high-level expression of two different minigenes carrying either the rpoB-2 or the ndhF-2 editing site on editing was determined for all 31 known edited Cs in two transgenic tobacco lines. The editing efficiencies of both the corresponding rpoB and ndhF editing sites in the endogenous genes' transcripts and in several other genes' transcripts were reduced in the chloroplast transgenic plants. Conserved nucleotides could be identified in the sequences immediately 5' of each overexpressed editing site and in the sites in the additional genes that experienced a competition effect, though the conserved nucleotides differ 5' of rpoB-2 and 5' of ndhF-2. Inspection of sequences surrounding chloroplast and mitochondrial editing sites reveals that they can be grouped into clusters which carry conserved nucleotides within 30 nt 5' of the C target of editing. The data are consistent with a model in which the same trans factor recognizes several chloroplast or mitochondrial editing sites, depending on the particular sequence 5' of the edited C.

FIG. 1.

PPE assay of atpF-1. (A) PPE was performed on a site-specific RT-PCR product from a radiolabeled oligonucleotide. (A) Sequence surrounding atpF-1 and the 3′ part of atpF-1(T), the oligonucleotide used in this reaction. The primer extension is poisoned by dideoxynucleoside triphosphate incorporation, in this case a ddTTP (T*). When the site is edited, atpF-1(T) is extended only to the editing site, producing an extended oligonucleotide of 35 bases (b). When the site is not edited, the cDNA does not contain a T at the editing site, so atpF-1(T) is extended to the next T, producing a primer of 38 bases. (B) Separation on sequencing gel and phosphorimager exposure of the primer extension products. Lanes: pH, PPE from leaf extracts of wild-type tobacco; rpoB-2 and ndhF-2, PPE with transplastomic plants overexpressing rpoB-2 or ndhF-2; 0, PPE without template (this indicates the size of the radiolabeled oligonucleotide); g, PPE with a cloned unedited (genomic) PCR product; c, PPE with a cloned edited RT-PCR product.

FIG. 2.

Editing extent of 31 sites in young green leaf chloroplasts of wild-type tobacco. The editing percentage was determined for PPE products by quantifying the radioactivity associated with edited and unedited sites (ImageQuant software). The x axis represents the 31 editing sites listed in Table 2.

FIG. 3.

Alteration of editing in transplastomic tobacco, determined by PPE assay of psbL-1. (A). Sequence surrounding psbL-1 and the 3′ part of psbL-1(G), the oligonucleotide used in this PPE. PPE is performed on a cDNA amplified with primers flanking psbL-1 from radiolabeled psbL-1(G). The primer extension is poisoned in this case by ddGTP (G*) incorporation. When the site is not edited, psbL-1(G) is extended only to the editing site, producing an extended oligonucleotide of 33 bases (b). When the site is edited, the complementary strand of the cDNA contains an A at the editing site, so psbL-1(G) is extended to the next G, producing a primer of 36 bases. (B). Separation on sequencing gel and phosphorimager exposure of the primer extension products. Lanes: pH, PPE with leaf extracts of wild-type tobacco; rpoB-2 and ndhF-2, PPE with transplastomic plants overexpressing rpoB-2 or ndhF-2; 0, PPE without template (this indicates the size of the radiolabeled oligonucleotide); g, PPE with a cloned unedited (genomic) PCR product; c, PPE with a cloned edited RT-PCR product.

FIG. 4.

Competition effect. Overexpression of the rpoB-2 (A) or ndhF-2 (B) minigene induced a decrease in editing level in endogenous rpoB-2, rps14-1, psbL-1, rpoB-1, and rpoA-1 (A) and in endogenous ndhF-2, ndhB-3, and ndhD-1 (B).

FIG. 5.

Putative cis-acting elements conserved in the upstream sequence of the editing sites showing a competition effect. Sites in bold are those introduced into the tobacco chloroplast genome. Gaps were added in the alignments to show similarity. Bold letters represent conserved nucleotides. Blocks of conserved nucleotides are shaded. Large capital Cs show C targets of editing. Nt, N. tabacum; Zm, Zea mays.

FIG. 6.

Chloroplast editing sites in tobacco (Nt) (A) and in maize (Zm) (B) sharing short sequences in their upstream regions. (C) Similarities and differences in sequences upstream of edited Cs in tobacco and the corresponding C or T in Arabidopsis thaliana (At). Gaps were introduced in the alignments to show similar sequences. Bold letters represent conserved nucleotides, and blocks of conserved nucleotides are shaded. Large capital Cs show C targets of editing.

FIG. 7.

Mitochondrial editing sites with upstream regions exhibiting similarity to either rpoB-2 or ndhF-2. These editing sites are known to be edited in tobacco (N. tabacum [Nt]) or in a related species (Nicotiana sylvestris [Ns]) or genus (Petunia hybrida [Ph]). Mitochondrial editing sites are named x-C1, where x represents the codon position and 1 is the location of the C within the codon. Underlined sites are those tested for a competition effect. The asterisk in coxII-2 shows the first editing site, 148-C2, of exon 2, which must be edited to U in order to form the conserved nucleotide. Site names in bold are those introduced in tobacco chloroplast genome (Zm, Zea mays). Gaps were introduced in the alignments to illustrate similarities. Bold letters represent conserved nucleotides, and blocks of conserved nucleotides are shaded. Large capital Cs show C targets of editing.

FIG. 8.

Editing in mitochondrial sites is not altered in tobacco overexpressing rpoB-2 despite sequence similarities in their upstream sequences. A PPE assay was performed on atp9 27-C3, rpl16 13-C1, and nad3 15-C2. Lanes: pH PPE with leaf extracts of wild-type tobacco; rpoB-2, PPE with transplastomic plants overexpressing rpoB-2; 0, PPE without template showing the radiolabeled oligonucleotide (O); g, PPE with a cloned unedited (C) PCR product; c, PPE with a cloned edited (T) RT-PCR product.