Date Published: December 4, 2013
Publisher: Public Library of Science
Author(s): Stefanie Grüttner, Christina Hopf, Abhishek Kumar, Frank Kempken, Stefan Maas.
As previously demonstrated, the maize cox2 RNA is fully edited in cauliflower mitochondria. Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites.
Mitochondrial RNA editing in higher plants has long been described [1–3], and is discussed in recent reviews [4–8]. In short, RNA editing is characterized mostly by C-to-U changes, with rare U-to-C editing, targeting hundreds of RNA editing sites in plant mitochondrial transcripts, and to a lesser degree also in higher plant plastids. Sequences close to the editing sites are required for RNA editing. Employing a wheat in organello approach, a region of 16 nucleotides upstream and six nucleotides downstream was shown to be required for recognition of two cox2 editing sites [9,10]. Using an in vitro RNA editing system isolated from pea mitochondria a 20-nucleotide region upstream of the first atp9 editing site was shown to be essential. However, for efficient editing, an upstream sequence of 40 nucleotides was required . A cauliflower in vitro assay not only confirmed this result, but identified one nucleotide downstream of the atp9 editing site as essential .
A growing number of PPR proteins have been implicated in editing site recognition. However, their target motifs appear to be little defined . In previous work, we found limited evidence for involvement of the secondary or tertiary structure of an mRNA in the editing process by comparison of editing site recognition of cox2 mRNA from mono- and dicot origin . One way to further test this possibility is to introduce deletions in the mRNA sequences. If RNA editing is solely due to site-specific binding of PPR proteins or PPR protein networks such deletions should have an effect at editing sites directly adjacent to the deletion at best. Any observed long-range effect provides direct evidence for an involvement of the secondary or tertiary structure of the mRNA on editing site recognition.
Several studies employing either in vitro or in organello RNA editing systems have provided important evidence on the process of mitochondrial and plastid RNA editing [23,26–28]. Even more relevant, several pentatricopeptide repeat proteins of the PLS subfamily are essential for editing of specific RNA editing sites [4–8]. Furthermore, the MORF family provides additional components of the RNA editing machinery . Some PPR editing factors recognize different editing sites with divergent surrounding nucleotide sequences thus raising the question of how specificity of editing site recognition is managed [17,19]. It is noteworthy that even contiguous editing sites may be recognized by two different PPR proteins . Hence the existence of a network of PPR trans-factors was suggested which bind to RNA sequences at a low level of specificity . This may explain the discrepancy of how 140 potential PPRs may bind to 400 editing sites. However, the data present here demonstrate that deletions in the exons or removal of the intron do not influence editing sites adjacent to the deletion. Instead, in the cauliflower in organelle system, they drastically reduce RNA editing at all sites of the maize cox2 mRNA. The size of these deletions varied from 27 to 99 base pairs in either exon. All but one abolished editing and splicing completely. In contrast, some partial edited sites remained upon removal of the cox2 intron. A similar observation was made using a wheat in organello system and employing potato rps10 and wheat cox2 mRNA, where removal of the respective introns led to loss or partial loss of RNA editing in the transcripts . The study also confirmed earlier observations [32,33] that editing of certain sites in or close by an intron is a prerequisite of splicing, although in some cases editing may occur after splicing . This data were interpreted as an indication of a close linkage of editing and splicing factors during RNA processing . In the study current, it was not possible to reestablish RNA splicing by in vitro mutation of editing sites close-by or in the intron. However, it is possible that we missed additional editing sites in the intron sequence, although not previously reported from other cox2 mRNAs. An alternative explanation is provided by changes in the RNA secondary structures of pCH737 (exon1∆75 bp), pCH765 (exon2∆52 bp), and pCH768 (exon2∆33 bp) (see Figure S2), which have additional bulges and interior loops in the proximity of the intron binding sites, which may explain loss of splicing, as the proper RNA secondary structure is required for splicing [29,35].