Crystal structure of the chloroplast RNA editing factor MORF2.

RNA editing is a post-transcription process that alters the genetic information on RNA molecules. In plastids and mitochondria of flowering plants, the multiple organellar RNA editing factors (MORFs) interact with the PLS-type pentatricopeptide repeat (PPR) proteins and participate in RNA editing of cytidine-to-uridine conversion. The PPR proteins recognize cytidine targets around the editing sites, and the MORF proteins modulate the RNA-binding activity of the PPR proteins. Here, we report the structure of the Arabidopsis thaliana chloroplast MORF2 at 2.4 Å resolution. The structure, adopting typical MORF-box fold as observed in mitochondrial MORF1 and chloroplast MORF9, reveals an MORF1-like dimerization mode. The difference between the two dimerization modes can be attributed to F157 (corresponding F162 in MORF1 and W160 in MORF9), which causes a 60° shift upon dimerization. This observation, together with the PPR-MORF2 model, suggests a dimer-to-monomer transition during RNA editosome formation.

Frequent chloroplast RNA editing in early-branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors.

BACKGROUND: RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30-50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles. RESULTS: With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs. CONCLUSION: Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

Confirmation of soybean plastid rRNAs by formaldehyde denaturing agarose gel electrophoresis.

Owing to their prokaryotic origin, plastid rRNAs are mainly 23s/16s/5s rRNAs. We present a novel plant RNA isolation method in this paper. Also, not only the eukaryotic 28s (26s, 25s)/18s rRNAs but the prokaryotic 26s/23s rRNAs as well were demonstrated in a single sample for the first time by formaldehyde denaturing agarose gel electrophoresis.

Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas.

Many chloroplast transcripts are protected against exonucleolytic degradation by RNA-binding proteins. Such interactions can lead to the accumulation of short RNAs (sRNAs) that represent footprints of the protein partner. By mining existing data sets of Chlamydomonas reinhardtii small RNAs, we identify chloroplast sRNAs. Two of these correspond to the 5'-ends of the mature psbB and psbH messenger RNAs (mRNAs), which are both stabilized by the nucleus-encoded protein Mbb1, a member of the tetratricopeptide repeat family. Accordingly, we find that the two sRNAs are absent from the mbb1 mutant. Using chloroplast transformation and site-directed mutagenesis to survey the psbB 5' UTR, we identify a cis-acting element that is essential for mRNA accumulation. This sequence is also found in the 5' UTR of psbH, where it plays a role in RNA processing. The two sRNAs are centered on these cis-acting elements. Furthermore, RNA binding assays in vitro show that Mbb1 associates with the two elements specifically. Taken together, our data identify a conserved cis-acting element at the extremity of the psbH and psbB 5' UTRs that plays a role in the processing and stability of the respective mRNAs through interactions with the tetratricopeptide repeat protein Mbb1 and leads to the accumulation of protected sRNAs.

Characterization of the chloroplast genome sequence of oil palm (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis Jacq.) is an economically important crop, which is grown for oil production. To better understand the molecular basis of oil palm chloroplasts, we characterized the complete chloroplast (cp) genome sequence obtained from 454 pyrosequencing. The oil palm cp genome is 156,973 bp in length consisting of a large single-copy region of 85,192 bp flanked on each side by inverted repeats of 27,071 bp with a small single-copy region of 17,639 bp joining the repeats. The genome contains 112 unique genes: 79 protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. By aligning the cp genome sequence with oil palm cDNA sequences, we observed 18 non-silent and 10 silent RNA editing events among 19 cp protein-coding genes. Creation of an initiation codon by RNA editing in rpl2 has been reported in several monocots and was also found in the oil palm cp genome. Fifty common chloroplast protein-coding genes from 33 plant taxa were used to construct ML and MP phylogenetic trees. Their topologies are similar and strongly support for the position of E. guineensis as the sister of closely related species Phoenix dactylifera in Arecaceae (palm families) of monocot subtrees.

Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5'-processing and translational coupling.

The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 5'-UTR (905 nt). We show that it is translatable. Processing of the 5'-UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 5'-UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins.

Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts.

Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5'- and 3'-exonucleases, resulting in processed RNAs with PPR10 bound at the 5'- or 3'-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5'- or 3'-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation.

Short non-coding RNA fragments accumulating in chloroplasts: footprints of RNA binding proteins?

Chloroplast RNA metabolism is controlled and excecuted by hundreds of nuclear-encoded, chloroplast-localized RNA binding proteins. Contrary to the nucleo-cytosolic compartment or bacteria, there is little evidence for non-coding RNAs that play a role as riboregulators of chloroplasts. We mined deep-sequencing datasets to identify short (16-28 nt) RNAs in the chloroplast genome and found 50 abundant small RNAs (sRNAs) represented by multiple, in some cases, thousands of sequencing reads, whereas reads are in general absent from the surrounding sequence space. Other than sRNAs representing the most highly abundant mRNAs, tRNAs and rRNAs, most sRNAs are located in non-coding regions and many are found a short distance upstream of start codons. By transcript end mapping we show that the 5' and 3' termini of chloroplast RNAs coincide with the ends of sRNAs. Sequences of sRNAs identified in Arabidopsis are conserved between different angiosperm species and in several cases, we identified putative orthologs in rice deep sequencing datasets. Recently, it was suggested that small chloroplast RNA fragments could result from the protective action of pentatricopeptide repeat (PPR) proteins against exonucleases, i.e. footprints of RNA binding proteins. Our data support this scenario on a transcriptome-wide level and suggest that a large number of sRNAs are in fact remnants of PPR protein targets.

Studying the structure and processing of chloroplast transcripts.

Most chloroplast genes in land plants are represented by multiple transcript isoforms that arise via differential splicing, endo- and exo-nucleolytic processing, and/or RNA editing. Exploration of the functional significance and mechanisms of these processing events is an active area of current research. This chapter focuses on methods that can be used to define the termini of chloroplast RNAs, quantify the relative levels of alternative processed RNA isoforms, and identify the binding sites of proteins that mediate chloroplast RNA processing. Various approaches for defining the sequence specificity of chloroplast RNA binding proteins are discussed, as are the parameters to consider in designing in vitro assays for RNA binding activities. A protocol is provided for a poisoned-primer extension assay for quantifying different splice isoforms.

CURE-Chloroplast: a chloroplast C-to-U RNA editing predictor for seed plants.

BACKGROUND: RNA editing is a type of post-transcriptional modification of RNA and belongs to the class of mechanisms that contribute to the complexity of transcriptomes. C-to-U RNA editing is commonly observed in plant mitochondria and chloroplasts. The in vivo mechanism of recognizing C-to-U RNA editing sites is still unknown. In recent years, many efforts have been made to computationally predict C-to-U RNA editing sites in the mitochondria of seed plants, but there is still no algorithm available for C-to-U RNA editing site prediction in the chloroplasts of seed plants. RESULTS: In this paper, we extend our algorithm CURE, which can accurately predict the C-to-U RNA editing sites in mitochondria, to predict C-to-U RNA editing sites in the chloroplasts of seed plants. The algorithm achieves over 80% sensitivity and over 99% specificity. We implement the algorithm as an online service called CURE-Chloroplast http://bioinfo.au.tsinghua.edu.cn/pure. CONCLUSION: CURE-Chloroplast is an online service for predicting the C-to-U RNA editing sites in the chloroplasts of seed plants. The online service allows the processing of entire chloroplast genome sequences. Since CURE-Chloroplast performs very well, it could be a helpful tool in the study of C-to-U RNA editing in the chloroplasts of seed plants.

GOBASE: an organelle genome database.

The organelle genome database GOBASE, now in its 21st release (June 2008), contains all published mitochondrion-encoded sequences (approximately 913,000) and chloroplast-encoded sequences (approximately 250,000) from a wide range of eukaryotic taxa. For all sequences, information on related genes, exons, introns, gene products and taxonomy is available, as well as selected genome maps and RNA secondary structures. Recent major enhancements to database functionality include: (i) addition of an interface for RNA editing data, with substitutions, insertions and deletions displayed using multiple alignments; (ii) addition of medically relevant information, such as haplotypes, SNPs and associated disease states, to human mitochondrial sequence data; (iii) addition of fully reannotated genome sequences for Escherichia coli and Nostoc sp., for reference and comparison; and (iv) a number of interface enhancements, such as the availability of both genomic and gene-coding sequence downloads, and a more sophisticated literature reference search functionality with links to PubMed where available. Future projects include the transfer of GOBASE features to NCBI/GenBank, allowing long-term preservation of accumulated expert information. The GOBASE database can be found at http://gobase.bcm.umontreal.ca/. Queries about custom and large-scale data retrievals should be addressed to gobase@bch.umontreal.ca.

Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae): unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer.

BACKGROUND: To gain insight into the branching order of the five main lineages currently recognized in the green algal class Chlorophyceae and to expand our understanding of chloroplast genome evolution, we have undertaken the sequencing of chloroplast DNA (cpDNA) from representative taxa. The complete cpDNA sequences previously reported for Chlamydomonas (Chlamydomonadales), Scenedesmus (Sphaeropleales), and Stigeoclonium (Chaetophorales) revealed tremendous variability in their architecture, the retention of only few ancestral gene clusters, and derived clusters shared by Chlamydomonas and Scenedesmus. Unexpectedly, our recent phylogenies inferred from these cpDNAs and the partial sequences of three other chlorophycean cpDNAs disclosed two major clades, one uniting the Chlamydomonadales and Sphaeropleales (CS clade) and the other uniting the Oedogoniales, Chaetophorales and Chaetopeltidales (OCC clade). Although molecular signatures provided strong support for this dichotomy and for the branching of the Oedogoniales as the earliest-diverging lineage of the OCC clade, more data are required to validate these phylogenies. We describe here the complete cpDNA sequence of Oedogonium cardiacum (Oedogoniales). RESULTS: Like its three chlorophycean homologues, the 196,547-bp Oedogonium chloroplast genome displays a distinctive architecture. This genome is one of the most compact among photosynthetic chlorophytes. It has an atypical quadripartite structure, is intron-rich (17 group I and 4 group II introns), and displays 99 different conserved genes and four long open reading frames (ORFs), three of which are clustered in the spacious inverted repeat of 35,493 bp. Intriguingly, two of these ORFs (int and dpoB) revealed high similarities to genes not usually found in cpDNA. At the gene content and gene order levels, the Oedogonium genome most closely resembles its Stigeoclonium counterpart. Characters shared by these chlorophyceans but missing in members of the CS clade include the retention of psaM, rpl32 and trnL(caa), the loss of petA, the disruption of three ancestral clusters and the presence of five derived gene clusters. CONCLUSION: The Oedogonium chloroplast genome disclosed additional characters that bolster the evidence for a close alliance between the Oedogoniales and Chaetophorales. Our unprecedented finding of int and dpoB in this cpDNA provides a clear example that novel genes were acquired by the chloroplast genome through horizontal transfers, possibly from a mitochondrial genome donor.

Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts.

The chloroplast genome of higher plants contains 20-40 C-to-U RNA editing sites, whose number and locations are diversified among plant species. Biochemical analyses using in vitro RNA editing systems with chloroplast extracts have suggested that there is one-to-one recognition between proteinous site recognition factors and their respective RNA editing sites, but their rigidness and generality are still unsettled. In this study, we addressed this question with the aid of an in vitro RNA editing system from tobacco chloroplast extracts and with UV-crosslinking experiments. We found that the ndhB-9 and ndhF-1 editing sites of tobacco chloroplast transcripts are both bound by the site-specific trans-acting factors of 95 kDa. Cross-competition experiments between ndhB-9 and ndhF-1 RNAs demonstrated that the 95 kDa proteins specifically binding to the ndhB-9 and ndhF-1 sites are the identical protein. The binding regions of the 95 kDa protein on the ndhB-9 and ndhF-1 transcripts showed 60% identity in nucleotide sequence. This is the first biochemical demonstration that a site recognition factor of chloroplast RNA editing recognizes plural sites. On the basis of this finding, we discuss how plant organellar RNA editing sites have diverged during evolution.

A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons.

We describe a rapid, high-throughput method to scan for new RNA editing sites. This method is adapted from high-resolution melting (HRM) analysis of amplicons, a technique used in clinical research to detect mutations in genomes. The assay was validated by the discovery of six new editing sites in different chloroplast transcripts of Arabidopsis thaliana. A screen of a collection of mutants uncovered a mutant defective for editing of one of the newly discovered sites. We successfully adapted the technique to quantify editing of partially edited sites in different individuals or different tissues. This new method will be easily applicable to RNA from any organism and should greatly accelerate the study of the role of RNA editing in physiological processes as diverse as plant development or human health.

Sequence elements critical for efficient RNA editing of a tobacco chloroplast transcript in vivo and in vitro.

In tobacco chloroplast transcripts 34 nt are efficiently edited to U. No common consensus region is present around all editing sites; however, sites can be grouped in clusters that share short common sequences. Transgene transcripts carrying either the wild-type -31/+22 or -31/+60 sequence near NTrpoB C473, an editing site within tobacco rpoB transcripts, or three different mutated sequences, were all highly edited in vivo. Endogenous transcripts of rpoB, psbL and rps14, all of which contain common sequences S1, S2 and S3 5' to NTrpoB C473, NTpsbL C2 and NTrps14 C80, were less edited in transgenic plants that over-express transcripts from NTrpoB C473 transgenes. Extent of reduction of endogenous editing differed between transgenic lines expressing mutated -31/+22 regions, depending on the abundance of the transgene transcripts. The -20/-5 sequence contains critical 5' sequence elements. Synthetic RNA templates with alterations within this 5' region were less efficiently edited in vitro than wild-type templates, by either tobacco or maize chloroplast extracts. The tobacco chloroplast extract supports both RNA editing and processing of 3' transcript termini. We conclude that within the -20/-5 region, sequences common to editing sites in the transcripts of rpoB, psbL and rps14 are critical for efficient NTrpoB C473 editing.

Specific roles of 5' RNA secondary structures in stabilizing transcripts in chloroplasts.

RNA secondary structures, e.g. stem-loops that are often found at the 5' and 3' ends of mRNAs, are in many cases known to be crucial for transcript stability but their role in prolonging the lifetime of transcripts remains elusive. In this study we show for an essential RNA-stabilizing stem-loop at the 5' end of rbcL gene transcripts in Chlamydomonas that it neither prevents ribonucleases from binding to the RNA nor impedes their movement along the RNA strand. The stem-loop has a formative function in that it mediates folding of a short sequence around its base into a specific RNA conformation, consisting of a helical and single-stranded region, i.e. the real structure required for longevity of rbcL transcripts in chloroplasts. Disturbing this structure renders transcripts completely unstable, even if the sequence of this element is not altered. The requirement of a specific 5' sequence and structure for RNA longevity suggests an interaction of this element with a trans-acting factor that protects transcripts from rapid degradation in chloroplasts.

Molecular evolution of the trnTUGU-trnFGAA region in Bryophytes.

Structure, variability, and molecular evolution of the trnT-F region in the Bryophyta (mosses and liverworts) is analyzed based on about 200 sequences of the trnT-L spacer and trnL 5' exon, 1000 sequences of the trnL intron, and 800 sequences of the trnL 3' exon and trnL-F spacer, including comparisons of lengths, GC contents, sequence similarities, and functional elements. Mutations occurring in the trnL 5' and 3' exons, including compensatory base pair changes, and a transition in the trnL anticodon in Takakia lepidozioides, are discussed. All three non-coding regions display a mosaic structure of highly variable elements (V1 - V3 in the trnT-L spacer, V4/V5 corresponding to stem-loop regions P6/P8 in the trnL intron, and V6/V7 in the trnL-F spacer) and more conserved elements. In the trnL intron this structure is a consequence of the defined secondary structure necessary for correct splicing, whereas in both spacers conserved regions are restricted to promoter elements. At least the highly variable regions in the trnT-L spacer and stem-loop region P8 of the trnL intron seem to evolve independently in the major bryophyte lineages and are therefore not suitable for high taxonomic level phylogenetic reconstructions. In mosses, a trend of length reduction towards the more derived lineages is observed in all three non-coding regions. GC contents are mostly linked to sequence variability, with the conserved regions being more GC rich and the more variable AT rich. The lowest GC values (< 10 %) are found in the trnT-L spacer of mosses. In addition to two putative sigma (70)-type promoters in the trnT-L spacer, a third putative promoter is present in the trnL-F spacer, although trnL and trnF are assumed to be co-transcribed. Consensus sequences are provided for the -35 and -10 sequences of the major bryophyte lineages. The third promoter is part of a hairpin secondary structure, whose loop region is highly homoplastic in mosses due to an inversion occurring independently in non-related taxa, even at the intraspecific level.

Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction.

BACKGROUND: A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1. RESULTS: The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases. CONCLUSION: Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.

A structural and functional study of plastid RNAs homologous to catalytic bacterial RNase P RNA.

Ribonuclease P (RNase P), the ubiquitous enzyme required for 5' maturation of transfer RNA, is a ribonucleoprotein containing an essential RNA subunit. This RNA (P RNA) is the catalytic component of RNase P in Bacteria and some Archaea. A putative P RNA is encoded in the chloroplast genome of three algae: Cyanophora paradoxa, Porphyra purpurea and Nephroselmis olivacea. In no case, the P RNAs from the plastids were active in vitro in conditions where bacterial and some archaeal P RNAs are functional. By using lead-ion-induced hydrolysis, we conclude that the catalytic deficiency is most likely due to the perturbation of the global structure of the plastid P RNAs compared to the bacterial counterpart. As a consequence, the plastid P RNAs are unable to bind to the precursor tRNA substrates. We discuss these results in the context of plastid and RNase P evolution.

Analysis of RNA motifs.

RNA motifs are directed and ordered stacked arrays of non-Watson-Crick base pairs forming distinctive foldings of the phosphodiester backbones of the interacting RNA strands. They correspond to the 'loops' - hairpin, internal and junction - that intersperse the Watson-Crick two-dimensional helices as seen in two-dimensional representations of RNA structure. RNA motifs mediate the specific interactions that induce the compact folding of complex RNAs. RNA motifs also constitute specific protein or ligand binding sites. A given motif is characterized by all the sequences that fold into essentially identical three-dimensional structures with the same ordered array of isosteric non-Watson-Crick base pairs. It is therefore crucial, when analyzing a three-dimensional RNA structure in order to identify and compare motifs, to first classify its non-Watson-Crick base pairs geometrically.