Publications by authors named "Bernard Gutmann"

14 Publications

  • Page 1 of 1

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria.

Commun Biol 2021 May 10;4(1):545. Epub 2021 May 10.

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia.

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.
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http://dx.doi.org/10.1038/s42003-021-02062-9DOI Listing
May 2021

The Pentatricopeptide Repeat Protein MEF100 Is Required for the Editing of Four Mitochondrial Editing Sites in .

Cells 2021 Feb 22;10(2). Epub 2021 Feb 22.

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia.

In there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides. A combined approach of RNA-Seq, mutant complementation, electrophoresis of mitochondrial protein complexes and Western blotting allowed us to show that MEF100, a PPR protein identified in a genetic screen for mutants resistant to an inhibitor of γ -glutamylcysteine synthetase, is required for the editing of -493, -403, -698 and -356 sites in Arabidopsis mitochondria. The absence of editing in leads to a decrease in mitochondrial Complex I activity, which probably explains the physiological phenotype. Some plants have lost the requirement for MEF100 at one or more of these sites through mutations in the mitochondrial genome. We show that loss of the requirement for MEF100 editing leads to divergence in the MEF100 binding site.
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http://dx.doi.org/10.3390/cells10020468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926422PMC
February 2021

The Expansion and Diversification of Pentatricopeptide Repeat RNA-Editing Factors in Plants.

Mol Plant 2020 02 21;13(2):215-230. Epub 2019 Nov 21.

Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, WA, Australia; School of Molecular Sciences, The University of Western Australia, Crawley, Perth 6009, WA, Australia. Electronic address:

The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, indicating massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well defined due to the sparse sequence data available from early-diverging land plant lineages. In this study, we exploit the comprehensive OneKP datasets of over 1000 transcriptomes from diverse plants and algae toward establishing a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterize over 2 250 000 PPR motifs in over 400 000 proteins. In lycophytes, polypod ferns, and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variations that imply functional differences between PPR proteins in seed plants versus non-seed plants and variations we propose to be linked to seed-plant-specific editing co-factors. Finally, using the sequence variations across the datasets, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA-editing factors, given their phylogenetic distribution and sequence characteristics.
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http://dx.doi.org/10.1016/j.molp.2019.11.002DOI Listing
February 2020

Towards a plant model for enigmatic U-to-C RNA editing: the organelle genomes, transcriptomes, editomes and candidate RNA editing factors in the hornwort Anthoceros agrestis.

New Phytol 2020 03 14;225(5):1974-1992. Epub 2019 Dec 14.

Institut für Zelluläre und Molekulare Botanik (IZMB), University of Bonn, Kirschallee 1, 53115, Bonn, Germany.

Hornworts are crucial to understand the phylogeny of early land plants. The emergence of 'reverse' U-to-C RNA editing accompanying the widespread C-to-U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort-tracheophyte clade. C-to-U RNA editing is well understood after identification of many editing factors in models like Arabidopsis thaliana and Physcomitrella patens, but there is no plant model yet to investigate U-to-C RNA editing. The hornwort Anthoceros agrestis is now emerging as such a model system. We report on the assembly and analyses of the A. agrestis chloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors. Both organelles in A. agrestis feature high amounts of RNA editing, with altogether > 1100 sites of C-to-U and 1300 sites of U-to-C editing. The nuclear genome reveals > 1400 genes for PPR proteins with variable carboxyterminal DYW domains. We observe significant variants of the 'classic' DYW domain, in the meantime confirmed as the cytidine deaminase for C-to-U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U-to-C editing targets according to the PPR-RNA binding code.
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http://dx.doi.org/10.1111/nph.16297DOI Listing
March 2020

The E domain of CRR2 participates in sequence-specific recognition of RNA in plastids.

New Phytol 2019 04 6;222(1):218-229. Epub 2018 Dec 6.

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, WA, Australia.

Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids. We identified a surprisingly large number of small RNAs that represent in vivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both in vitro and in vivo experiments to reveal the role of the E domain of CRR2. The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR-RNA interaction. The 'code' of the E domain PPR motifs is different from that of P- and S-motifs. The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs in vivo.
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http://dx.doi.org/10.1111/nph.15578DOI Listing
April 2019

Editing of Chloroplast by PPR Editing Factor EMB2261 Is Essential for Development.

Front Plant Sci 2018 20;9:841. Epub 2018 Jun 20.

Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia.

RNA editing in plastids is known to be required for embryogenesis, but no single editing event had been shown to be essential. We show that the mutation is lethal through a failure to express an editing factor that specifically recognizes the site. EMB2261 was predicted to bind the -element upstream of the site and genetic complementation with promoters of different strength followed by RNA-seq analysis was conducted to test the correlation between editing and expression. is the only editing event in chloroplasts that correlates with expression. Sequence divergence between the -element and the EMB2261 protein sequence in plants where editing is not required adds support to the association between them. We conclude that EMB2261 is the specificity factor for editing. This editing event converts P51 in Rps14 to L51, which is conserved among species lacking RNA editing, implying the importance of the editing event to Rps14 function. Rps14 is an essential ribosomal subunit for plastid translation, which, in turn, is essential for embryogenesis.
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http://dx.doi.org/10.3389/fpls.2018.00841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019781PMC
June 2018

Protein Complexes Implicated in RNA Editing in Plant Organelles.

Mol Plant 2017 10 25;10(10):1255-1257. Epub 2017 Sep 25.

ARC Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.molp.2017.09.011DOI Listing
October 2017

Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants.

Plant J 2016 Feb;85(4):532-47

Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, 6009, Australia.

The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30-40 amino acid motifs that form an extended binding surface capable of sequence-specific recognition of RNA strands. Almost all of them are post-translationally targeted to plastids and mitochondria, where they play important roles in post-transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide-binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C-terminus of many RNA-editing factors. We show that the super-helical RNA-binding surface of RNA-editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre-requisite for accurate large-scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community.
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http://dx.doi.org/10.1111/tpj.13121DOI Listing
February 2016

Distribution of Ribonucleoprotein and Protein-Only RNase P in Eukarya.

Mol Biol Evol 2015 Dec 3;32(12):3186-93. Epub 2015 Sep 3.

Institut de Biologie Moléculaire des Plantes du CNRS, Strasbourg, France

RNase P is the endonuclease that removes 5' leader sequences from tRNA precursors. In Eukarya, separate RNase P activities exist in the nucleus and mitochondria/plastids. Although all RNase P enzymes catalyze the same reaction, the different architectures found in Eukarya range from ribonucleoprotein (RNP) enzymes with a catalytic RNA and up to 10 protein subunits to single-subunit protein-only RNase P (PRORP) enzymes. Here, analysis of the phylogenetic distribution of RNP and PRORP enzymes in Eukarya revealed 1) a wealth of novel P RNAs in previously unexplored phylogenetic branches and 2) that PRORP enzymes are more widespread than previously appreciated, found in four of the five eukaryal supergroups, in the nuclei and/or organelles. Intriguingly, the occurrence of RNP RNase P and PRORP seems mutually exclusive in genetic compartments of modern Eukarya. Our comparative analysis provides a global picture of the evolution and diversification of RNase P throughout Eukarya.
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http://dx.doi.org/10.1093/molbev/msv187DOI Listing
December 2015

PPR proteins shed a new light on RNase P biology.

RNA Biol 2013 19;10(9):1457-68. Epub 2013 Jun 19.

Institut de Biologie Moléculaire des Plantes du CNRS; Université de Strasbourg; Strasbourg, France.

A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5' leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process.
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http://dx.doi.org/10.4161/rna.25273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858429PMC
May 2015

Structural insights into protein-only RNase P complexed with tRNA.

Nat Commun 2013 ;4:1353

Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.

RNase P is the essential activity removing 5'-leader sequences from transfer RNA precursors. RNase P was always associated with ribonucleoprotein complexes before the discovery of protein-only RNase P enzymes called PRORPs (PROteinaceous RNase P) in eukaryotes. Here we provide biophysical and functional data to understand the mode of action of PRORP enzymes. Activity assays and footprinting experiments show that the anticodon domain of transfer RNA is dispensable, whereas individual residues in D and TψC loops are essential for PRORP function. PRORP proteins are characterized in solution and a molecular envelope is derived from small-angle X-ray scattering. Conserved residues are shown to be involved in the binding of one zinc atom to PRORP. These results facilitate the elaboration of a model of the PRORP/transfer RNA interaction. The comparison with the ribonucleoprotein RNase P/transfer RNA complex suggests that transfer RNA recognition by PRORP proteins is similar to that by ribonucleoprotein RNase P.
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http://dx.doi.org/10.1038/ncomms2358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562450PMC
June 2013

PlantRNA, a database for tRNAs of photosynthetic eukaryotes.

Nucleic Acids Res 2013 Jan 12;41(Database issue):D273-9. Epub 2012 Oct 12.

Institut de Biologie Moléculaire des Plantes, UPR 2357-CNRS, Université de Strasbourg, 12 rue du Général Zimmer, F-67084 Strasbourg Cedex, France.

PlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA) gene sequences retrieved from fully annotated plant nuclear, plastidial and mitochondrial genomes. The set of annotated tRNA gene sequences has been manually curated for maximum quality and confidence. The novelty of this database resides in the inclusion of biological information relevant to the function of all the tRNAs entered in the library. This includes 5'- and 3'-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and enzymes responsible for tRNA maturation and modification. Finally, data on mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the respective tRNAs and tRNA-binding proteins are also included. The current annotation concerns complete genomes from 11 organisms: five flowering plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and Brachypodium distachyon), a moss (Physcomitrella patens), two green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom (Phaeodactylum tricornutum). The database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on tRNA biology to the research community.
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http://dx.doi.org/10.1093/nar/gks935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531208PMC
January 2013

PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis.

Genes Dev 2012 May 1;26(10):1022-7. Epub 2012 May 1.

Institut de Biologie moléculaire des plantes du CNRS, University of Strasbourg, 67084 Strasbourg, France.

RNase P is an essential enzyme that cleaves the 5' leader sequence of tRNA precursors. RNase Ps were believed until now to occur universally as ribonucleoproteins in organisms performing RNase P activity. Here we find that protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) support RNase P activity in vivo in both organelles and the nucleus in Arabidopsis. Beyond tRNA, PRORP proteins are involved in the maturation of small nucleolar RNA (snoRNA) and mRNA. Finally, ribonucleoprotein RNase MRP is not involved in tRNA maturation in plants. Altogether, our results indicate that ribonucleoprotein enzymes have been entirely replaced by proteins for RNase P activity in plants.
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http://dx.doi.org/10.1101/gad.189514.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360558PMC
May 2012

A single Arabidopsis organellar protein has RNase P activity.

Nat Struct Mol Biol 2010 Jun 16;17(6):740-4. Epub 2010 May 16.

Institut de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique, Strasbourg, France.

The ubiquitous endonuclease RNase P is responsible for the 5' maturation of tRNA precursors. Until the discovery of human mitochondrial RNase P, these enzymes had typically been found to be ribonucleoproteins, the catalytic activity of which is associated with the RNA component. Here we show that, in Arabidopsis thaliana mitochondria and plastids, a single protein called 'proteinaceous RNase P' (PRORP1) can perform the endonucleolytic maturation of tRNA precursors that defines RNase P activity. In addition, PRORP1 is able to cleave tRNA-like structures involved in the maturation of plant mitochondrial mRNAs. Finally, we show that Arabidopsis PRORP1 can replace the bacterial ribonucleoprotein RNase P in Escherichia coli cells. PRORP2 and PRORP3, two paralogs of PRORP1, are both localized in the nucleus.
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http://dx.doi.org/10.1038/nsmb.1812DOI Listing
June 2010