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 trans-factors. 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'-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.

FIGURE 1.

Comparison of the RNA editing lysates prepared from cauliflower and pea mitochondria. (A) Sample gel images of in vitro editing assays in the pea and cauliflower lysates. Numbers give the sizes of the full-length (207 nucleotides) and edited (78 nucleotides) RT-PCR-TDG products. (B) RNA editing activities in the two lysates as determined toward the pea atp9 −40/+49 template. The average of three experiments is given with the respective standard error shown for the cauliflower activity (cf) relative to the pea, the latter taken as 100% in each set of experiments.

FIGURE 2.

Sequence comparison between pea (Pisum sativum) and cauliflower (Brassica oleracea) mitochondria surrounding the first RNA editing site in the atp9 coding region. Numbering is centered around this editing site. The native cauliflower and pea sequences deviate upstream of nucleotide −23, which is located in the 5′-untranslated leader region. This sequence variation between the two species allows a cross-wise comparison of the nucleotides necessary for site recognition. The AUG of the translational start in pea is boxed, the edited C is in large type, and identical nucleotides are indicated by the dashes between the two sequences. In cauliflower another AUG occurs in frame further upstream, which could theoretically be used (data not shown). The sequence between nucleotides +10 and +45 is not shown but is identical in the two plants. The complete sequence of the atp9 gene in cauliflower is deposited in the databases (accession no. DQ102391).

FIGURE 3.

Pea and cauliflower templates are processed with similar efficiency in the cauliflower lysate. This observation suggests that in both plant species, the cis-elements for recognition of this editing site reside within the conserved 23 nucleotides (and possibly the few positions conserved further upstream). The cauliflower lysate activity appears to be slightly higher toward its cognate sequence, but the differences between the two templates are within the experimental variation. The average of four independent assays is shown with the activity toward the pea template taken as 100% in each assay. On the right, gel images are shown for representative assays of pea and cauliflower (cf) templates. Different cloning sites result in different respective TDG product sizes in pea (78) and cauliflower (69).

FIGURE 4.

Delineation of the cis-recognition region by deletion clones in the cauliflower mitochondrial lysate. The sequences upstream and downstream of the monitored editing site were deleted in steps of 10 nucleotides; that is, they were in effect substituted by bacterial vector sequences. (A) Deletion of the 5′-sequence up to 20 upstream nucleotides (−20) still allows editing. Further deletion closer than −20 nucleotides from the editing site completely abolishes recognition of this editing site. Control was the pea template −40/+10; the deletions were all done+with the pea template to allow a direct comparison of the cis-elements. (B) Deleting the 3′-region has little effect up to +10 nucleotides, but the complete substitution up to the edited nucleotide (−30/0) shows substantial inhibition of the in vitro editing activity. The longest 3′-extension was used as standard, and the relative activities with the 3′-deleted templates are shown.

FIGURE 5.

The effects of scanning mutations around the first atp9 editing site as substrates and as competitors on in vitro RNA editing in pea and cauliflower mitochondrial lysates are compared. (A) The respective nucleotide quintet altered to its complementary sequence in each set of experiments for maximum effect and to maintain the G+C content is shown, and its designation is given beneath the mutated sequences. (B) The mutated pea templates are tested for their effectiveness in cauliflower (dark bars on the right) and pea (light bars on the left). Notable differences between the two species are observed toward mutants M5 and M9. (C) The wild-type pea template is competed with 1500-fold excess of the mutants M1–M10 from part A in the cauliflower lysate (dark bars; cf) and 1000-fold in the pea lysate assays, respectively (light bars; pea). Control template is the pea −40/+49 wild-type sequence without competitor. Vector sequences compete little, but the wild-type competitor suppresses recognition of the template completely in the pea lysate. Please note that this suppression is not complete in the more active cauliflower lysate, even though a 50% higher excess of competitor was used. The most striking difference between the lysates from these two plant species is seen with competitor M5. Further details are discussed in the text.

FIGURE 6.

Importance of individual nucleotide alterations around the editing site. The influence of the identity of the first nucleotide downstream of the edited nucleotide was determined by mutating this position through all three alternative nucleotides. Any of these changes results in the loss of ~70% of the editing activity. All mutants were tested in four separate experiments, and the mean percentages of the wild-type editing activity were determined. The resulting standard error is indicated for each mutant. The tolerance of the in vitro editing activity for distance alterations between the upstream cis-recognition element and the edited nucleotide was investigated by deleting or inserting an adenosine nucleotide within the run of four As in the wild-type sequence. No activity was observed with either template, showing that the distance from the cis-element is crucial. Please note that in the deletion template a concomitant A-to-G change at the +1 position has occurred.