PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of plantsigLink to Publisher's site
 
Plant Signal Behav. 2010 June; 5(6): 727–729.
PMCID: PMC3001572

The moss Physcomitrella patens, a model plant for the study of RNA editing in plant organelles

Abstract

RNA editing is an enigmatic phenomenon in which specific cytidines (C) in the transcripts are changed to uridines (U). In flowering plants, over 500 editing sites have been identified in the mitochondria and plastids. By contrast, in a moss Physcomitrella patens, only 12 editing sites are found in both organelles. Recent extensive genetics studies have revealed involvement of the pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain (PPR-DYW) in site-specific RNA editing events. Flowering plants have ~100 PPR-DYW genes while P. patens has only 10 PPR-DYW genes. Thus, the number of PPR-DYW genes is somewhat related to the number of RNA editing sites. The P. patens gametophyte, the haploid phase of the life cycle, is dominant, making it possible to study the phenotype of knockouts directly after transformation for gene-targeting. This makes it easier to identify the PPR-DYW proteins required for RNA editing. Recently, we have shown that one of 10 PPR-DYW proteins, PpPPR_71, was responsible for RNA editing in the mitochondrial ccmFc mRNA. In this study, we propose a working model of PpPPR_71 function on RNA editing.

Key words: DYW domain, mitochondria, Physcomitrella patens, PPR protein, RNA editing

C-to-U RNA editing frequently occurs in many transcripts of most plant organelles.1,2 For example, Arabidopsis thaliana has 508 RNA editing sites in the mitochondria3 and 34 sites in the plastids.4 In P. patens, RNA editing in the translated regions is a rarely occurring event as only one site in the plastids5 and 11 sites in the mitochondria.6,7 Since RNA editing was discovered in plant organelles 20 year ago,8,9 the molecular mechanism of RNA editing was unknown for a long time. In 2005, a nucleus-encoded and plastid-targeted PPR protein was first identified as an RNA editing factor for plastid ndhD transcript from A. thaliana.10 Last year several PPR-DYW proteins were found to act as site recognition factors for RNA editing.1118 Among 450 A. thaliana PPR genes, 87 encode PPR-DYW proteins.19,20 The C-terminal DYW domain (95 amino acids) contains invariant residues that match the active site of cytidine deaminases from bacteria, plants, animals and yeast.21 Interestingly, neither RNA editing nor DYW domains could be identified in algae and one clade of liverworts. There is a correlation between the presence of nuclear DYW genes and organelle RNA editing among embryophytes.21,22 Therefore, the DYW domains are proposed to be responsible for RNA editing in plant organelles and catalyze RNA editing. The moss P. patens has only 10 PPR-DYW genes,20 which raises the question whether PPR-DYW proteins are responsible for RNA editing in mosses.

A Moss PPR-DYW Protein is Required for RNA Editing at One Specific Site Among 11 Mitochondrial Editing Sites

One of the 10 PPR-DYW proteins, PpPPR_71, is a mitochondrial-targeted protein that consists of 833 amino acids with 17 PPR motifs.20 Targeted-disruption of the PpPPR_71 gene resulted in an RNA editing defect for the ccmF-2 site of the ccmFc transcript while the 10 editing sites, including the ccmF-1 site, were not affected.7 The PpPPR_71 disruptants displayed severe growth retardation of the moss protonemata. RNA editing at this site (the 122nd nucleotide from the AUG of ccmFc mRNA) results in a serine (uCu) to phenylalanine (uUu) change at the 41st residue of CcmFc. This suggests that phenylalanine at this position is critical for the functioning of CcmFc.23

The ccmF-1 and ccmF-2 editing sites are separated only by 18 nt and therefore RNA editing at both sites could be influenced by each other. To investigate this possibility the RNA editing status of ccmFc transcripts was examined. We cloned the RT-PCR products from the wild type protonemata RNA sample and randomly sequenced 108 independent cDNA clones. Among them, 89 cDNAs (82.4%) were fully edited and 10 (9.3%) were unedited for both sites, and 9 were edited at the ccmF-1 site but not at the ccmF-2 site (Fig. 1). However, only cDNA edited at ccmF-2 could not be isolated. This strongly suggests that RNA editing for ccmF-2 depends on RNA editing of ccmF-1.

Figure 1
RNA editing status of the ccmFc transcripts and RNA-binding affinities of PpPPr_71 and its DYW domain to the RNA probes. Four different cDNAs can be cloned; unedited (-C-C-), partially edited (-U-C- or -C-U-), or fully edited (-U-U-) cDNAs. Numbers of ...

PpPPR_71 Preferably Binds to the Edited ccmF-1 Transcript

To investigate the RNA binding properties of PpPPR_71, we performed an electrophoretic mobility shift assay (EMSA) using the recombinant (r) PPR-DYW or rDYW proteins and 46-nt RNA probes, RNA1 to RNA3.7 The sequences of the three RNA probes differed by the C or U residue at the ccmF-1 and ccmF-2 editing sites (Fig. 1). This assay indicated that PpPPR_71 binds strongly to the RNA2 probe, which carries edited U at ccmF-1 and unedited C at ccmF-2. In contrast, PpPPR_71 binds weakly to RNA1 (unedited) and RNA3 (fully edited). Thus, the binding activity of PpPPR_71 likely depends on the RNA editing status of the target RNA. It is interesting to observe that the rDYW domain also binds to the same extent to the three RNA probes but its binding affinity is much weaker than that of rPPR-DYW.

Furthermore, EMSA with excess amounts of cold RNA1 to 3 probes as a competitor indicated that rDYW binds somewhat stronger to RNA1 and RNA2, which carry unedited C at ccmF-2, than to RNA3 carrying an edited ccmF-2 site. This suggests that the DYW domain itself recognizes and binds to the surrounding unedited ccmF-2 site of ccmFc RNA. Taken together with the in vivo RNA editing status of ccmFc transcripts, we propose a model of how PpPPR_71 protein works for RNA editing of the ccmFc transcript (Fig. 2). When RNA editing at the ccmF-1 site is completed, then PpPPR_71 recognizes and binds to the cis-acting element carrying U at ccmF-1 and acts as a trans-acting factor for ccmF-2 RNA editing. PPR motifs and the DYW domain cooperatively bind to the cis-acting element for RNA editing, then recruit unidentified RNA editing enzyme. After the ccmF-2 site is edited, PpPPR_71 dissociates from the target RNA.

Figure 2
A model of RNA editing event in the ccmFc transcript. When RNA editing at the ccmF-1 site is completed by an unidentified trans-acting factor (probably a PPR-DYW protein) or RNA editing enzyme, then PpPPR_71 specifically binds to the cis-acting element ...

Alternatively, the result of cDNA sequence analysis can be explained as follows. Editing at the ccmF-1 site is simply faster or more efficient than editing at the ccmF-2 site.2 If a defect of the ccmF-1 recognition factor also abolishes or strongly inhibits the editing of ccmF-2, our model will be supported. To answer this question, trans-acting factors for ccmF-1 RNA editing remain to be identified. One of the remaining 9 PPR-DYW proteins of P. patens is possibly involved in RNA editing at the ccmF-1 site. We are currently screening the knockout mutants of other PPR-DYW genes and have identified five PPR-DYW proteins required for RNA editing in the mitochondrial transcripts (Ohtani S, Tasaki E, Koumura Y, Aoki Y and Sugita M, unpublished results).

Notes

Addendum to: Tasaki E, Hattori M, Sugita M. The moss pentatricopeptide repeat protein with a DYW domain is responsible for RNA editing of mitochondrial ccmFc transcriptPlant J2010 doi: 10.1111/j.1365-313X.2010.04175.x.

Footnotes

References

1. Shikanai T. RNA editing in plant organelles: machinery, physiological function and evolution. Cell Mol Life Sci. 2006;63:698–708. [PubMed]
2. Takenaka M, Verbitskly D, van der Merwe JA, Zehrmann A, Brennicke A. The process of RNA editing in plant mitochondria. Mitochondrion. 2008;8:35–46. [PubMed]
3. Bentolila S, Elliott LE, Hanson MR. Genetic architecture of mitochondrial editing in Arabidopsis thaliana. Genetics. 2008;178:1693–1708. [PubMed]
4. Chateigner-Boutin AL, Small I. A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons. Nucl Acids Res. 2007;35:114. [PMC free article] [PubMed]
5. Miyata Y, Sugita M. Tissue- and stage-specific RNA editing of rps14 transcripts in moss (Physcomitrella patens) chloroplasts. J Plant Physiol. 2004;161:113–115. [PubMed]
6. Rüdinger M, Funk HT, Rensing SA, Maier UG, Knoop V. RNA editing: only eleven sites are present in the Physcomitrella patens miotochondrial transcriptome and a universal nomenclature proposal. Mol Genet Genom. 2009;281:473–481. [PubMed]
7. Tasaki E, Hattori M, Sugita M. The moss pentatricopeptide repeat protein with a DYW domain is responsible for RNA editing of mitochondrial ccmFc transcript. Plant J. 2010 doi: 10.1111/j.1365-313-X.2010.04175.x. [PubMed] [Cross Ref]
8. Gualberto JM, Lamattina L, Bonnard G, Weil JH, Grienenberger JM. RNA editing in wheat mitochondria results in the conversion of protein sequences. Nature. 1989;341:660–662. [PubMed]
9. Hoch B, Maier RM, Appel K, Igloi GJ, Kössel H. Editing of a chloroplast mRNA by creation of an initiation codon. Nature. 1991;353:178–180. [PubMed]
10. Kotera E, Tasaka M, Shikanai T. A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature. 2005;433:326–330. [PubMed]
11. Okuda K, Chateigner-Boutin AL, Nakamura T, Delannoy E, Sugita M, Myouga F, et al. Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts. Plant Cell. 2009;21:146–156. [PubMed]
12. Zehrmann A, Verbitskiy D, van der Merwe JA, Brennicke A, Takenaka M. A DYW domain-containing pentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell. 2009;21:558–567. [PubMed]
13. Cai W, Ji D, Peng L, Guo J, Ma J, Zou M, et al. LPA66 is required for editing psbF chloroplast transcripts in Arabidopsis. Plant Physiol. 2009;150:1260–1271. [PubMed]
14. Zhou W, Cheng Y, Yap A, Chateigner-Boutin AL, Delannoy E, Hammani K, et al. The Arabidopsis gene YS1 encoding a DYW protein is required for editing of rpoB transcripts and the rapid development of chloroplasts during early growth. Plant J. 2009;58:82–96. [PubMed]
15. Robbins JC, Heller WP, Hanson MR. A comparative genomics approach identifies a PPR-DYW protein that is essential for C-to-U editing of the Arabidopsis chloroplast accD transcript. RNA. 2009;15:1142–1153. [PubMed]
16. Kim SR, Yang JI, Moon S, Ryu CH, An K, Kim KM, et al. Rice OGR1 encodes a pentatricopeptide repeat-DYW protein and is essential for RNA editing in mitochondria. Plant J. 2009;59:738–739. [PubMed]
17. Yu QB, Jiang Y, Chong K, Yang ZN. AtECB2, a pentatricopeptide repeat protein, is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana. Plant J. 2009;59:1011–1023. [PubMed]
18. Hammani K, Okuda K, Tanz SK, Chateigner-Boutin AL, Shikanai T, Small I. A study of new Arabidopsis chloroplast RNA editing mutants reveals general features of editing factors and their target sites. Plant Cell. 2009;21:3686–3699. [PubMed]
19. Lurin C, Andrés C, Aubourg S, Bellaui M, Bitton F, Bruyíre C, et al. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell. 2004;16:2089–2103. [PubMed]
20. O'Toole N, Hattori M, Andrés C, Iida K, Lurin C, Schmitz-Linneweber C, et al. On the expansion of the pentatricopeptide repeat gene family in plants. Mol Biol Evol. 2008;25:1120–1128. [PubMed]
21. Salone V, Rüdinger M, Polsakiewicz M, Hoffmann B, Groth-Malonek M, Szurek B, et al. A hypothesis on the identification of the editing enzyme in plant organelles. FEBS Lett. 2007;581:4132–4138. [PubMed]
22. Rüdinger M, Polsakiewicz M, Knoop V. Organellar RNA editing and plant-specific extensions of pentatricopeptide repeat proteins in jungermanniid but not in marchantiid liverworts. Mol Biol Evol. 2008;25:1405–1414. [PubMed]
23. Giegé P, Rayapuram N, Meyer EH, Grienenberger JM, Bonnard G. CcmFc involved in cytochrome c maturation is present in a large sized complex in wheat mitochondria. FEBS Lett. 2004;562:165–169. [PubMed]

Articles from Plant Signaling & Behavior are provided here courtesy of Taylor & Francis