Stéphane Bentolila

Interactions of Mitochondrial and Nuclear Genes That Affect Male Gametophyte Development

Apart from their agronomic importance in hybrid seed production, mutations that encode cytoplasmic male sterility (CMS) provide a means to probe the role of the mitochondrion in reproductive development. Fertility restorers are examples of nuclear genes that affect cytoplasmic gene expression, and

A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants

Known in over 150 species, cytoplasmic male sterility is encoded by aberrant mitochondrial genes that prevent pollen development. The RNA- or protein-level expression of most of the mitochondrial genes encoding cytoplasmic male sterility is altered in the presence of one or more nuclear genes called restorers of fertility that suppress the male-sterile phenotype. Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have previously been cloned. Here we report the isolation of a gene directly involved in the control of the expression of a cytoplasmic male sterility-encoding gene. The Petunia restorer of fertility gene product is a mitochondrially targeted protein that is almost entirely composed of 14 repeats of the 35-aa pentatricopeptide repeat motif. In a nonrestoring genotype we identified a homologous gene that exhibits a deletion in the promoter region and is expressed in roots but not in floral buds.

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing

Transcripts of plant organelle genes are modified by cytidine-to-uridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virus-induced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.

Comprehensive High-Resolution Analysis of the Role of an Arabidopsis Gene Family in RNA Editing

In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.

Genetic Architecture of Mitochondrial Editing in Arabidopsis thaliana

We have analyzed the mitochondrial editing behavior of two Arabidopsis thaliana accessions, Landsberg erecta (Ler) and Columbia (Col). A survey of 362 C-to-U editing sites in 33 mitochondrial genes was conducted on RNA extracted from rosette leaves. We detected 67 new editing events in A. thaliana rosette leaves that had not been observed in a prior study of mitochondrial editing in suspension cultures. Furthermore, 37 of the 441 C-to-U editing events reported in A. thaliana suspension cultures were not observed in rosette leaves. Forty editing sites that are polymorphic in extent of editing were detected between Col and Ler. Silent editing sites, which do not change the encoded amino acid, were found in a large excess compared to nonsilent sites among the editing events that differed between accessions and between tissue types. Dominance relationships were assessed for 15 of the most polymorphic sites by evaluating the editing values of the reciprocal hybrids. Dominance is more common in nonsilent sites than in silent sites, while additivity was observed only in silent sites. A maternal effect was detected for 8 sites. QTL mapping with recombinant inbred lines detected 12 major QTL for 11 of the 13 editing traits analyzed, demonstrating that efficiency of editing of individual mitochondrial C targets is generally governed by a major factor.

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

Significance Transcripts in plant organelles are altered by conversion of cytidines to uridines in a process termed RNA editing. Members of two protein families have been identified in the plant editosome, but its complete composition is unknown. Now a unique protein that contains an RNA recognition motif has been found to be essential for editing of multiple plastid transcripts in both Arabidopsis and maize. Phylogenetic analysis indicates that this protein belongs to a sub-family of RNA recognition-motif proteins predominantly predicted to be targeted to organelles and that are thus likely to play roles in organelle RNA metabolism. Plant RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of both mitochondria and plastids. Specific targeting of particular Cs is achieved by pentatricopeptide proteins that recognize cis elements upstream of the C that is edited. Members of the RNA-editing factor interacting protein (RIP) family in Arabidopsis have recently been shown to be essential components of the plant editosome. We have identified a gene that contains a pair of truncated RIP domains (RIP-RIP). Unlike any previously described RIP family member, the encoded protein carries an RNA recognition motif (RRM) at its C terminus and has therefore been named Organelle RRM protein 1 (ORRM1). ORRM1 is an essential plastid editing factor; in Arabidopsis and maize mutants, RNA editing is impaired at particular sites, with an almost complete loss of editing for 12 sites in Arabidopsis and 9 sites in maize. Transfection of Arabidopsis orrm1 mutant protoplasts with constructs encoding a region encompassing the RIP-RIP domain or a region spanning the RRM domain of ORRM1 demonstrated that the RRM domain is sufficient for the editing function of ORRM1 in vitro. According to a yeast two-hybrid assay, ORRM1 interacts selectively with pentatricopeptide transfactors via its RIP-RIP domain. Phylogenetic analysis reveals that the RRM in ORRM1 clusters with a clade of RRM proteins that are targeted to organelles. Taken together, these results suggest that other members of the ORRM family may likewise function in RNA editing.

A Reevaluation of Rice Mitochondrial Evolution Based on the Complete Sequence of Male-Fertile and Male-Sterile Mitochondrial Genomes

Plant mitochondrial genomes have features that distinguish them radically from their animal counterparts: a high rate of rearrangement, of uptake and loss of DNA sequences, and an extremely low point mutation rate. Perhaps the most unique structural feature of plant mitochondrial DNAs is the presence of large repeated sequences involved in intramolecular and intermolecular recombination. In addition, rare recombination events can occur across shorter repeats, creating rearrangements that result in aberrant phenotypes, including pollen abortion, which is known as cytoplasmic male sterility (CMS). Using next-generation sequencing, we pyrosequenced two rice (Oryza sativa) mitochondrial genomes that belong to the indica subspecies. One genome is normal, while the other carries the wild abortive-CMS. We find that numerous rearrangements in the rice mitochondrial genome occur even between close cytotypes during rice evolution. Unlike maize (Zea mays), a closely related species also belonging to the grass family, integration of plastid sequences did not play a role in the sequence divergence between rice cytotypes. This study also uncovered an excellent candidate for the wild abortive-CMS-encoding gene; like most of the CMS-associated open reading frames that are known in other species, this candidate was created via a rearrangement, is chimeric in structure, possesses predicted transmembrane domains, and coopted the promoter of a genuine mitochondrial gene. Our data give new insights into rice mitochondrial evolution, correcting previous reports.

Natural Variation in Arabidopsis Leads to the Identification of REME1, a Pentatricopeptide Repeat-DYW Protein Controlling the Editing of Mitochondrial Transcripts

In vascular plants, organelle RNAs are edited by C-to-U base modification. Hundreds of mitochondrial C residues are targeted for editing in flowering plants. In this study, we exploited naturally occurring variation in editing extent to identify Required for Efficiency of Mitochondrial Editing1 (REME1), an Arabidopsis (Arabidopsis thaliana) pentatricopeptide repeat protein-encoding gene belonging to the DYW subclass that promotes editing of at least two C residues on different mitochondrial transcripts. Positional cloning identified REME1 unambiguously as the gene controlling editing of nad2-558. Virus-induced gene silencing of REME1 confirmed its role in editing of nad2-558 and allowed us to identify orfX-552 as a second C whose editing is positively controlled by REME1. An unexpected outcome of REME1 silencing was the finding of a number of mitochondrial C targets whose editing extent exhibits a significant and reproducible increase in silenced tissues. That increase was shown to be partly due to the virus inoculation and partly to REME1-specific silencing. Analysis of an insertional T-DNA mutant within the REME1 coding sequence confirmed the findings of the virus-induced gene silencing experiments: decrease in editing extent of nad2-558 and orfX-552 and increase in editing extent of two sites, matR-1771 and rpl5-92. Transgenic complementation of the low-edited accession (Landsberg erecta) restored the editing of nad2-558 and orfX-552 to high-edited accession (Columbia)-type levels or to even higher levels than Columbia. There was no effect of the transgene on editing extent of matR-1771 and rpl5-92. The strategy and tools used in this report can be applied to identify additional genes that affect editing extent in plant mitochondria.

The Unexpected Diversity of Plant Organelle RNA Editosomes

Flowering plants convert many hundreds of organelle cytidines (Cs) to uridines (Us) during post-transcriptional RNA editing. Pentatricopeptide repeat (PPR) proteins dictate specificity by recognizing RNA sequences near C targets. However, the complete mechanism of the editing machinery is not yet understood. Recently, non-PPR editing factors [RNA editing factor interacting proteins (RIPs)/multiple organellar RNA editing factors (MORFs), organelle RNA recognition motif (ORRM) proteins, organelle zinc-finger (OZ) proteins, and protoporphyrinogen oxidase 1 (PPO1)] have been identified as components of the plant RNA editosome, which is a small RNA-protein complex. Surprisingly, plant editosomes are highly diverse not only with regard to the PPR proteins they contain but also in the non-PPR components that are present. Here we review the most recent progress in the field and discuss the implications of the diversity of plant editosomes for the evolution of RNA editing and for possible future applications.

A zinc finger motif-containing protein is essential for chloroplast RNA editing.

C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.