Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures.

Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5' ends corresponding to transcription start sites (TSS), as well as 1628 processed 5' ends and 1299 3' ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5' and 3' ends, contrasting with the prevailing description of discrete 5' termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-seq was also implemented for pnp1-1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2000 termini were altered in pnp1-1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.

Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing.

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

Arabidopsis chloroplast mini-ribonuclease III participates in rRNA maturation and intron recycling.

RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3' end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3' extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.

ORRM5, an RNA recognition motif-containing protein, has a unique effect on mitochondrial RNA editing.

Plants have an RNA editing mechanism that prevents deleterious organelle mutations from resulting in impaired proteins. A typical flowering plant modifies about 40 cytidines in chloroplast transcripts and many hundreds of cytidines in mitochondrial transcripts. The plant editosome, the molecular machinery responsible for this process, contains members of several protein families, including the organelle RNA recognition motif (ORRM)-containing family. ORRM1 and ORRM6 are chloroplast editing factors, while ORRM2, ORRM3, and ORRM4 are mitochondrial editing factors. Here we report the identification of organelle RRM protein 5 (ORRM5) as a mitochondrial editing factor with a unique mode of action. Unlike other ORRM editing factors, the absence of ORRM5 in orrm5 mutant plants results in an increase of the editing extent in 14% of the mitochondrial sites surveyed. The orrm5 mutant also exhibits a reduced splicing efficiency of the first nad5 intron and slower growth and delayed flowering time. ORRM5 contains an RNA recognition motif (RRM) and a glycine-rich domain at the C terminus. The RRM provides the editing activity of ORRM5 and is able to complement the splicing but not the morphological defects.

The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part II: Bioinformatic Analyzes and Virtual RNA Blots.

Nanopore sequencing of full-length cDNAs offers unprecedented details of the plastid RNA metabolism. After the generation of the nanopore reads, several bioinformatic steps are required to analyze the data. In this chapter, we describe in a few simple command lines the processing and mapping of the reads as well as the generation of virtual Northern blots as a simple and familiar way to visualize Nanopore sequencing data.

The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part I: Library Preparation.

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.

Dissecting the molecular puzzle of the editosome core in Arabidopsis organelles.

Over the last decade, the composition of the C-to-U RNA editing complex in embryophyte organelles has turned out to be much more complex than first expected. While PPR proteins were initially thought to act alone, significant evidences have clearly depicted a sophisticated mechanism with numerous protein-protein interaction involving PPR and non-PPR proteins. Moreover, the identification of specific functional partnership between PPRs also suggests that, in addition to the highly specific PPRs directly involved in the RNA target recognition, non-RNA-specific ones are required. Although some of them, such as DYW1 and DYW2, were shown to be the catalytic domains of the editing complex, the molecular function of others, such as NUWA, remains elusive. It was suggested that they might stabilize the complex by acting as a scaffold. We here performed functional complementation of the crr28-2 mutant with truncated CRR28 proteins mimicking PPR without the catalytic domain and show that they exhibit a specific dependency to one of the catalytic proteins DYW1 or DYW2. Moreover, we also characterized the role of the PPR NUWA in the editing reaction and show that it likely acts as a scaffolding factor. NUWA is no longer required for efficient editing of the CLB19 editing sites once this RNA specific PPR is fused to the DYW catalytic domain of its partner DYW2. Altogether, our results strongly support a flexible, evolutive and resilient editing complex in which RNA binding activity, editing activity and stabilization/scaffolding function can be provided by one or more PPRs.

High-Throughput Protein-Protein Interactions Screening Using Pool-Based Liquid Yeast Two-Hybrid Pipeline.

Because of its adaptability to high-throughput approaches and a low operating cost, the yeast two-hybrid (Y2H) assay remains the most widely used one for high-throughput protein-protein interactions (PPI) mapping experiments. Here we provide a detailed protocol for a liquid culture-based high-throughput binary protein-protein Y2H screen pipeline of a pool of 50 proteins used as baits against a collection of ~12,000 Arabidopsis proteins encoded by sequence-verified open reading frames (ORFs).

DiffSegR: an RNA-seq data driven method for differential expression analysis using changepoint detection.

To fully understand gene regulation, it is necessary to have a thorough understanding of both the transcriptome and the enzymatic and RNA-binding activities that shape it. While many RNA-Seq-based tools have been developed to analyze the transcriptome, most only consider the abundance of sequencing reads along annotated patterns (such as genes). These annotations are typically incomplete, leading to errors in the differential expression analysis. To address this issue, we present DiffSegR - an R package that enables the discovery of transcriptome-wide expression differences between two biological conditions using RNA-Seq data. DiffSegR does not require prior annotation and uses a multiple changepoints detection algorithm to identify the boundaries of differentially expressed regions in the per-base log2 fold change. In a few minutes of computation, DiffSegR could rightfully predict the role of chloroplast ribonuclease Mini-III in rRNA maturation and chloroplast ribonuclease PNPase in (3'/5')-degradation of rRNA, mRNA and tRNA precursors as well as intron accumulation. We believe DiffSegR will benefit biologists working on transcriptomics as it allows access to information from a layer of the transcriptome overlooked by the classical differential expression analysis pipelines widely used today. DiffSegR is available at https://aliehrmann.github.io/DiffSegR/index.html.

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that "mitochondrial translation factors" mTRAN1 and mTRAN2 are land plant-specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)-like RNA binding proteins of the mitoribosomal "small" subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5' regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

The nucleocytoplasmic conflict, a driving force for the emergence of plant organellar RNA editing.

RNA editing challenges the central dogma of molecular biology by changing the genetic information at the transcript level. In plant organelles, RNAs are modified by deamination of some specific cytosine residues, but the origin of this process remains puzzling. Different from the generally accepted neutral model to explain the emergence of RNA editing in plant organelles, we propose a new hypothesis based on the nucleocytoplasmic conflict theory. We assume that mutations in organellar genomes arose first and spread into the population provided they increased the transmission of their own maternally inherited genome. RNA editing appeared subsequently as a nuclear-encoded correction mechanism to restore the transmission of the nuclear genome. In plants, a well-known consequence of the nucleocytoplasmic conflict is cytoplasmic male sterility (CMS) which is counteracted by the emergence of fertility restorer genes (Rf) belonging to the pentatricopeptide repeat (PPR) protein family. Interestingly, RNA-editing deficiency can lead to CMS, and it now clearly appears that PPR proteins are major players in RNA editing. This striking similarity between the mechanisms of fertility restoration and RNA editing can be explained if both reactions are the consequence of the same driving force, the nucleocytoplasmic conflict. Similarly, the prevalence of RNA editing in eukaryotic organellar genomes could also be a consequence of the genetic antagonism between organellar and nuclear genomes.

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.

Analyzing Multifactorial RNA-Seq Experiments with DicoExpress.

The proper use of statistical modeling in NGS data analysis requires an advanced level of expertise. There has recently been a growing consensus on using generalized linear models for differential analysis of RNA-Seq data and the advantage of mixture models to perform co-expression analysis. To offer a managed setting to use these modeling approaches, we developed DiCoExpress that provides a standardized R pipeline to perform an RNA-Seq analysis. Without any particular knowledge in statistics or R programming, beginners can perform a complete RNA-Seq analysis from quality controls to co-expression through differential analysis based on contrasts inside a generalized linear model. An enrichment analysis is proposed both on the lists of differentially expressed genes, and the co-expressed gene clusters. This video tutorial is conceived as a step-by-step protocol to help users take full advantage of DiCoExpress and its potential in empowering the biological interpretation of an RNA-Seq experiment.

RNA editing restores critical domains of a group I intron in fern mitochondria.

In the leptosporangiate fern Osmunda regalis, cox1 gene is disrupted by a 1071-nucleotide-long group I intron that is homologous to the Marchantia polymorpha cox1 intron 4 (cox1i395g1). This intron, which shares 89% sequence identity with its bryophyte counterpart, lost the capacity to encode for a maturase due to insertion/deletion mutations. The cox1 coding region is interrupted by a stop codon in both exons. The cox1 transcript undergoes 58 C-to-U and 13 U-to-C conversions, including the suppression of two stop codons that result in the recovery of a functional cox1 ORF. Interestingly, 4 C-to-U conversions found in mRNA precursors showed that the O. regalis cox1i395g1 intron is efficiently edited. These modifications improved the sequence identity with the Marchantia cox1i395 intron. In particular, the RNA editing events affect regions involved in secondary and tertiary structures of the intron, restoring three base pairing in the structural P5a and P9 helices, and correcting a highly conserved U in the P7 helix that contributes to the catalytic core. Moreover, cox1 intron orthologous from three different fern species were found to be edited by both C-to-U and U-to-C conversions in P7 and P9. Thus, RNA editing helps to correct the conserved domains of group I introns in "true ferns", suggesting a possible link between editing and splicing. We present here the first experimental evidence of RNA editing concerning a group I intron in plant organelles.

A Guide to the Chloroplast Transcriptome Analysis Using RNA-Seq.

Since its first use in plants in 2007, high-throughput RNA sequencing (RNA-Seq) has generated a vast amount of data for both model and nonmodel species. Organellar transcriptomes, however, are virtually always overlooked at the data analysis step. We therefore developed ChloroSeq, a bioinformatic pipeline aimed at facilitating the systematic analysis of chloroplast RNA metabolism, and we provide here a step-by-step user's manual. Following the alignment of quality-controlled data to the genome of interest, ChloroSeq measures genome expression level along with splicing and RNA editing efficiencies. When used in combination with the Tuxedo suite (TopHat and Cufflinks), ChloroSeq allows the simultaneous analysis of organellar and nuclear transcriptomes, opening the way to a better understanding of nucleus-organelle cross talk. We also describe the use of R commands to produce publication-quality figures based on ChloroSeq outputs. The effectiveness of the pipeline is illustrated through analysis of an RNA-Seq dataset covering the transition from growth to maturation to senescence of Arabidopsis thaliana leaves.

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.

Strand-specific RNA sequencing uncovers chloroplast ribonuclease functions.

Organellar gene expression incorporates ribonucleases as indispensable participants. Here, we explored the capacity of strand-specific RNA sequencing (RNA-Seq) as a tool to analyze chloroplast ribonuclease functions using the 3'→5' exoribonuclease polynucleotide phosphorylase (PNPase) as a proof of concept. The role of PNPase in transcript 3' end maturation was easily monitored, and additional targeted mRNAs were discovered. Moreover, a role in tRNA precursor degradation was predicted and validated. These results, together with previously published data, suggest that RNA-Seq represents a unique opportunity to decipher the roles of organellar ribonucleases and deepen our understanding of chloroplast gene expression.

Arabidopsis chloroplast quantitative editotype.

Chloroplast 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.

A molecular contribution to the assessment of the Tricholoma equestre species complex.

In recent years, interest in the Tricholoma equestre species complex has increased because of several cases of severe and sometimes fatal rhabdomyolysis reported in France and Poland. These occurred after repeated consumption of large portions of T. equestre sporophores during consecutive meals, despite the fact that this species is renowned as a tasty edible wild mushroom. The T. equestre species complex includes three ectomycorrhizal species Tricholoma flavovirens (Pers.) S. Lundell, Tricholoma auratum (Paulet) Gillet, and T. equestre (L.) P. Kummer. All these species produce sporophores with intense yellow gills but are difficult to distinguish by morphological analyses at both the macroscopic and microscopic levels. In T. equestre, two additional varieties are recognized: T. equestre var. populinum (Christensen & Noordeloos) associated with Populus sp. and/or Betula sp. trees and sometimes recognized as Tricholoma frondosae (Kalamees & Shchukin) and T. equestre var. pallidifolia characterized by pale to white gills, frequently recognized as Tricholoma joachimii (Bon & Riva). To explore the taxonomic (species delimitation), ecological, and geographical extent and limits of the T. equestre species complex, we have carried out a molecular comparison of worldwide strains belonging to this complex by using sequences of two molecular markers: the internal transcript spacer (ITS)1/5.8S/ITS2 region of the nuclear ribosomal unit and the 5' part of the mitochondrial cox1 gene. Phylogenetic analyses support the placement of European T. equestre, T. flavovirens, and T. auratum strains as representatives of a single species. This species appears associated with various conifers trees, depending on the geographic origin (Pinus pinaster for T. auratum, Pinus sylvestris or Abies alba for T. equestre and T. flavovirens). However, in the context of a single T. equestre species, the geographical location could lead to the characterization of sub-species or varieties, as suggested by the gathering of the four Asian (Japanese) T. auratum strains in a strongly supported distinct phylogenetic clade. Moreover, our analysis strongly argues for considering T. joachimii and the synonymised T. equestre var. pallidifolia as two representatives of a different species not belonging to the T. equestre group. This species would be phylogenetically related to the Tricholoma columbetta species with which they share white gills. Similarly, the phylogenetic analysis of the molecular data and the lack of gene flow between the strains associated with broad-leaved trees and those of the T. equestre complex, rather argues for two distinct species depending on the ecological niche: T. frondosae under broad-leaved trees and T. equestre under conifers.