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The RNA editing pattern of cox2 mRNA is affected by point mutations in plant mitochondria.

Castandet B, Araya A - PLoS ONE (2011)

Bottom Line: It has been reported that a short region surrounding the target C forms the cis-recognition elements, but individual residues on it do not play similar roles for the different editing sites.We found that four different recognition patterns can be distinguished: (a) +1 dependency, (b) -1 dependency, (c) +1/-1 dependency, and (d) no dependency on nearest neighbor residues.Our data suggest that the complex response observed for cox2 mRNA may be a consequence of the fate of the transcript during mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, MCMP- UMR5234, Centre National de la Recherche Scientifique and Université Bordeaux Segalen. Bordeaux, France.

ABSTRACT
The mitochondrial transcriptome from land plants undergoes hundreds of specific C-to-U changes by RNA editing. These events are important since most of them occur in the coding region of mRNAs. One challenging question is to understand the mechanism of recognition of a selected C residue (editing sites) on the transcript. It has been reported that a short region surrounding the target C forms the cis-recognition elements, but individual residues on it do not play similar roles for the different editing sites. Here, we studied the role of the -1 and +1 nucleotide in wheat cox2 editing site recognition using an in organello approach. We found that four different recognition patterns can be distinguished: (a) +1 dependency, (b) -1 dependency, (c) +1/-1 dependency, and (d) no dependency on nearest neighbor residues. A striking observation was that whereas a 23 nt cis region is necessary for editing, some mutants affect the editing efficiency of unmodified distant sites. As a rule, mutations or pre-edited variants of the transcript have an impact on the complete set of editing targets. When some Cs were changed into Us, the remaining editing sites presented a higher efficiency of C-to-U conversion than in wild type mRNA. Our data suggest that the complex response observed for cox2 mRNA may be a consequence of the fate of the transcript during mitochondrial gene expression.

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Editing of mutants of overlapping C167 and C169 sites.(A) Sequence encompassing sites C167 and C169. ×1 and ×2 represent the −1 and the +1 mutations of the site C167 respectively. ×2 is also the −1 mutant of site C169. ×3 represents the +1 mutation of the site C169. (B) Chromatopherograms of the sequences surrounding the sites C167 and C169 from different constructs. Arrows indicate the C167 (left) and C169 (right) editing sites. The editing efficiency in wild type and mutant transcripts is indicated below the chromatopherogram. C167T and C169T indicate the presence of a residue T at the place of the editing target C167 and C169 respectively. At least 16 independent RT-PCR clones were sequenced for each construct.
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pone-0020867-g004: Editing of mutants of overlapping C167 and C169 sites.(A) Sequence encompassing sites C167 and C169. ×1 and ×2 represent the −1 and the +1 mutations of the site C167 respectively. ×2 is also the −1 mutant of site C169. ×3 represents the +1 mutation of the site C169. (B) Chromatopherograms of the sequences surrounding the sites C167 and C169 from different constructs. Arrows indicate the C167 (left) and C169 (right) editing sites. The editing efficiency in wild type and mutant transcripts is indicated below the chromatopherogram. C167T and C169T indicate the presence of a residue T at the place of the editing target C167 and C169 respectively. At least 16 independent RT-PCR clones were sequenced for each construct.

Mentions: In the case of contiguous editing sites, the C targets are also part of the cis-recognition sequence of the neighboring one. The contiguous C167 and C169 editing sites allow the reciprocal influence between editing sites to be studied. For this purpose, different constructs containing either −1 or +1 mutations were expressed in isolated mitochondria. Fig. 4A depicts the different mutations used. As shown above, editing of C167 was strongly affected by −1 or +1 mutation (×1 and ×2). It was reduced about 50% by the mutation at position +3 (×3). In contrast, editing of C169 was not affected by the −1 mutation (×2) but was severely reduced in the mutant + 1 (×3) (Fig. 4B). It should be noted that ×2 represents a +1 modification for C167, but is a −1 mutant with regard to C169. The same mutations were introduced into pre-edited C167T and C169T constructs. Both C167T and C169T presented slight modifications on the editing efficiency of the contiguous residue. The C169T mutant did not affect the editing efficiency of C167 when combined with the ×2 and ×3 mutations compared to the wild type construct. On the other hand, the pre-edited C167T form increased the inhibitory effect of ×1 mutations (Fig. 4B, line C167T). The C169T mutant did not affect the editing efficiency of C167. Similarly, ×1 and ×2 mutations had an effect on C167 editing whatever the C169 status. However, the pre-edited C169T alleviated the editing inhibition when combined with the ×1 mutation (Fig. 4B, line C169T).


The RNA editing pattern of cox2 mRNA is affected by point mutations in plant mitochondria.

Castandet B, Araya A - PLoS ONE (2011)

Editing of mutants of overlapping C167 and C169 sites.(A) Sequence encompassing sites C167 and C169. ×1 and ×2 represent the −1 and the +1 mutations of the site C167 respectively. ×2 is also the −1 mutant of site C169. ×3 represents the +1 mutation of the site C169. (B) Chromatopherograms of the sequences surrounding the sites C167 and C169 from different constructs. Arrows indicate the C167 (left) and C169 (right) editing sites. The editing efficiency in wild type and mutant transcripts is indicated below the chromatopherogram. C167T and C169T indicate the presence of a residue T at the place of the editing target C167 and C169 respectively. At least 16 independent RT-PCR clones were sequenced for each construct.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113845&req=5

pone-0020867-g004: Editing of mutants of overlapping C167 and C169 sites.(A) Sequence encompassing sites C167 and C169. ×1 and ×2 represent the −1 and the +1 mutations of the site C167 respectively. ×2 is also the −1 mutant of site C169. ×3 represents the +1 mutation of the site C169. (B) Chromatopherograms of the sequences surrounding the sites C167 and C169 from different constructs. Arrows indicate the C167 (left) and C169 (right) editing sites. The editing efficiency in wild type and mutant transcripts is indicated below the chromatopherogram. C167T and C169T indicate the presence of a residue T at the place of the editing target C167 and C169 respectively. At least 16 independent RT-PCR clones were sequenced for each construct.
Mentions: In the case of contiguous editing sites, the C targets are also part of the cis-recognition sequence of the neighboring one. The contiguous C167 and C169 editing sites allow the reciprocal influence between editing sites to be studied. For this purpose, different constructs containing either −1 or +1 mutations were expressed in isolated mitochondria. Fig. 4A depicts the different mutations used. As shown above, editing of C167 was strongly affected by −1 or +1 mutation (×1 and ×2). It was reduced about 50% by the mutation at position +3 (×3). In contrast, editing of C169 was not affected by the −1 mutation (×2) but was severely reduced in the mutant + 1 (×3) (Fig. 4B). It should be noted that ×2 represents a +1 modification for C167, but is a −1 mutant with regard to C169. The same mutations were introduced into pre-edited C167T and C169T constructs. Both C167T and C169T presented slight modifications on the editing efficiency of the contiguous residue. The C169T mutant did not affect the editing efficiency of C167 when combined with the ×2 and ×3 mutations compared to the wild type construct. On the other hand, the pre-edited C167T form increased the inhibitory effect of ×1 mutations (Fig. 4B, line C167T). The C169T mutant did not affect the editing efficiency of C167. Similarly, ×1 and ×2 mutations had an effect on C167 editing whatever the C169 status. However, the pre-edited C169T alleviated the editing inhibition when combined with the ×1 mutation (Fig. 4B, line C169T).

Bottom Line: It has been reported that a short region surrounding the target C forms the cis-recognition elements, but individual residues on it do not play similar roles for the different editing sites.We found that four different recognition patterns can be distinguished: (a) +1 dependency, (b) -1 dependency, (c) +1/-1 dependency, and (d) no dependency on nearest neighbor residues.Our data suggest that the complex response observed for cox2 mRNA may be a consequence of the fate of the transcript during mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, MCMP- UMR5234, Centre National de la Recherche Scientifique and Université Bordeaux Segalen. Bordeaux, France.

ABSTRACT
The mitochondrial transcriptome from land plants undergoes hundreds of specific C-to-U changes by RNA editing. These events are important since most of them occur in the coding region of mRNAs. One challenging question is to understand the mechanism of recognition of a selected C residue (editing sites) on the transcript. It has been reported that a short region surrounding the target C forms the cis-recognition elements, but individual residues on it do not play similar roles for the different editing sites. Here, we studied the role of the -1 and +1 nucleotide in wheat cox2 editing site recognition using an in organello approach. We found that four different recognition patterns can be distinguished: (a) +1 dependency, (b) -1 dependency, (c) +1/-1 dependency, and (d) no dependency on nearest neighbor residues. A striking observation was that whereas a 23 nt cis region is necessary for editing, some mutants affect the editing efficiency of unmodified distant sites. As a rule, mutations or pre-edited variants of the transcript have an impact on the complete set of editing targets. When some Cs were changed into Us, the remaining editing sites presented a higher efficiency of C-to-U conversion than in wild type mRNA. Our data suggest that the complex response observed for cox2 mRNA may be a consequence of the fate of the transcript during mitochondrial gene expression.

Show MeSH