Arabidopsis chloroplast quantitative editotype

AbstractChloroplast C-to-U RNA editing is an essential post-transcriptional process. Here we analyzed RNA editing in Arabidopsis thaliana using strand-specific deep sequencing datasets from the wild-type and a mutant defective in RNA 3′ end maturation. We demonstrate that editing at all sites is partial, with an average of 5–6% of RNAs remaining unedited. Furthermore, we identified nine novel sites with a low extent of editing. Of these, three sites are absent from the WT transcriptome because they are removed by 3′ end RNA processing, but these regions accumulate, and are edited, in a mutant lacking polynucleotide phosphorylase

Intron RNA editing is essential for splicing in plant mitochondria

Most plant mitochondria messenger RNAs (mRNAs) undergo editing through C-to-U conversions located mainly in exon sequences. However, some RNA editing events are found in non-coding regions at critical positions in the predicted secondary and tertiary structures of introns, suggesting that RNA editing could be important for splicing. Here, we studied the relationships between editing and splicing of the mRNA encoding the ribosomal protein S10 (rps10), which has a group II intron and five editing sites. Two of them, C2 and C3, predicted to stabilize the folded structure of the intron necessary for splicing, were studied by using rps10 mutants introduced into isolated potato mitochondria by electroporation. While mutations of C2 involved in EBS2/IBS2 interactions did not affect splicing, probably by the presence of an alternative EBS2′ region in domain I of the intron, the edition of site C3 turned out to be critical for rps10 mRNA splicing; only the edited (U) form of the transcript was processed. Interestingly, RNA editing was strongly reduced in transcripts from two different intronless genes, rps10 from potato and cox2 from wheat, suggesting that efficient RNA processing may require a close interaction of factors engaged in different maturation processes. This is the first report linking editing and splicing in conditions close to the in vivo situation

The RNA Editing Pattern of cox2 mRNA Is Affected by Point Mutations in Plant Mitochondria

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

Salt stress affects mRNA editing in soybean chloroplasts

Abstract Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress

ChloroSeq, an Optimized Chloroplast RNA-Seq Bioinformatic Pipeline, Reveals Remodeling of the Organellar Transcriptome Under Heat Stress

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsis thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators