RESUMO
Pathogenic bacteria employ complex systems to cope with metal ion shortage conditions and propagate in the host. IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum Staphylococcus aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here, we report that IsrR regulates the tricarboxylic acid (TCA) cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop, underscoring the tight control required to prevent aconitase expression. Beyond its canonical enzymatic role, aconitase becomes an RNA-binding protein with regulatory activity in iron-deprived conditions, a feature that is conserved in S. aureus. Aconitase not only negatively regulates its own expression, but also impacts the enzymes involved in both its substrate supply and product utilization. This moonlighting activity concurrently upregulates pyruvate carboxylase expression, allowing it to compensate for the TCA cycle deficiency associated with iron scarcity. These results highlight the cascade of complex posttranscriptional regulations controlling S. aureus central metabolism in response to iron deficiency.
Assuntos
Aconitato Hidratase , Proteínas de Bactérias , Ciclo do Ácido Cítrico , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus , Aconitato Hidratase/metabolismo , Aconitato Hidratase/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/enzimologia , Ciclo do Ácido Cítrico/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ferro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Pequeno RNA não Traduzido/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Deficiências de FerroRESUMO
Bone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR+ ) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype.
Assuntos
Medula Óssea , Células-Tronco Mesenquimais , Medula Óssea/metabolismo , Plasmócitos , Células Estromais , Moléculas de Adesão Celular/metabolismo , Células da Medula ÓsseaRESUMO
Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.
Assuntos
Clostridioides difficile/crescimento & desenvolvimento , Clostridioides/fisiologia , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , Esporos Bacterianos/fisiologia , Virulência , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Genoma Bacteriano , Fator Proteico 1 do Hospedeiro/genética , Humanos , RNA Bacteriano/genéticaRESUMO
In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working with Escherichia coli as a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , RNA Bacteriano , RNA de Transferência/genética , Biologia Computacional/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , RNA de Transferência/química , Análise de Sequência de RNARESUMO
Rfam is a database of RNA families where each of the 3444 families is represented by a multiple sequence alignment of known RNA sequences and a covariance model that can be used to search for additional members of the family. Recent developments have involved expert collaborations to improve the quality and coverage of Rfam data, focusing on microRNAs, viral and bacterial RNAs. We have completed the first phase of synchronising microRNA families in Rfam and miRBase, creating 356 new Rfam families and updating 40. We established a procedure for comprehensive annotation of viral RNA families starting with Flavivirus and Coronaviridae RNAs. We have also increased the coverage of bacterial and metagenome-based RNA families from the ZWD database. These developments have enabled a significant growth of the database, with the addition of 759 new families in Rfam 14. To facilitate further community contribution to Rfam, expert users are now able to build and submit new families using the newly developed Rfam Cloud family curation system. New Rfam website features include a new sequence similarity search powered by RNAcentral, as well as search and visualisation of families with pseudoknots. Rfam is freely available at https://rfam.org.
Assuntos
Bases de Dados de Ácidos Nucleicos , Metagenoma , MicroRNAs/genética , RNA Bacteriano/genética , RNA não Traduzido/genética , RNA Viral/genética , Bactérias/genética , Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Humanos , Internet , MicroRNAs/classificação , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/classificação , RNA Bacteriano/metabolismo , RNA não Traduzido/classificação , RNA não Traduzido/metabolismo , RNA Viral/classificação , RNA Viral/metabolismo , Alinhamento de Sequência , Análise de Sequência de RNA , Software , Vírus/genética , Vírus/metabolismoRESUMO
In Archaea and Bacteria, the arrays called CRISPRs for 'clustered regularly interspaced short palindromic repeats' and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR-Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR-Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.
Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bases de Dados Genéticas , Genoma Arqueal , Genoma Bacteriano , Software , Archaea/classificação , Archaea/enzimologia , Archaea/genética , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , FilogeniaRESUMO
RsaE is a regulatory RNA highly conserved amongst Firmicutes that lowers the amount of mRNAs associated with the TCA cycle and folate metabolism. A search for new RsaE targets in Staphylococcus aureus revealed that in addition to previously described substrates, RsaE down-regulates several genes associated with arginine catabolism. In particular, RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. The faster growth rate of ΔrsaE compared to its parental strain in media containing amino acids as sole carbon source points to an underlying role for RsaE in amino acid catabolism. Collectively, the data support a model in which RsaE acts as a global regulator of functions associated with metabolic adaptation.
Assuntos
Arginina/metabolismo , RNA Bacteriano/fisiologia , Sequências Reguladoras de Ácido Ribonucleico , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Sequência Conservada , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Organismos Geneticamente Modificados , Sequências Reguladoras de Ácido Ribonucleico/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.
Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Software , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , InternetRESUMO
Bacterial regulatory RNAs have been extensively studied for over a decade, and are progressively being integrated into the complex genetic regulatory network. Transcriptomic arrays, recent deep-sequencing data and bioinformatics suggest that bacterial genomes produce hundreds of regulatory RNAs. However, while some have been authenticated, the existence of the others varies according to strains and growth conditions, and their detection fluctuates with the methodologies used for data acquisition and interpretation. For example, several small RNA (sRNA) candidates are now known to be parts of UTR transcripts. Accurate annotation of regulatory RNAs is a complex task essential for molecular and functional studies. We defined bona fide sRNAs as those that (i) likely act in trans and (ii) are not expressed from the opposite strand of a coding gene. Using published data and our own RNA-seq data, we reviewed hundreds of Staphylococcus aureus putative regulatory RNAs using the DETR'PROK computational pipeline and visual inspection of expression data, addressing the question of which transcriptional signals correspond to sRNAs. We conclude that the model strain HG003, a NCTC8325 derivative commonly used for S. aureus genetic regulation studies, has only about 50 bona fide sRNAs, indicating that these RNAs are less numerous than commonly stated. Among them, about half are associated to the S. aureus sp. core genome and a quarter are possibly expressed in other Staphylococci. We hypothesize on their features and regulation using bioinformatic approaches.
RESUMO
Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [3H]phosphatidic acid (PA) released from [3H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.
Assuntos
Fosfolipase D/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Streptomyces lividans/enzimologia , Motivos de Aminoácidos , Membrana Celular/enzimologia , Colina/metabolismo , Hidrólise , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipase D/química , Fosfolipase D/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Conformação Proteica , Streptomyces lividans/genéticaRESUMO
A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.
Assuntos
Pyrobaculum/genética , Pyrococcus abyssi/genética , RNA Arqueal/química , RNA Guia de Cinetoplastídeos/química , RNA Nucleolar Pequeno/química , Pareamento de Bases , Euryarchaeota/genética , Regulação da Expressão Gênica em Archaea , Modelos Moleculares , Motivos de Nucleotídeos , Dobramento de RNA , RNA Arqueal/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/metabolismo , Relação Estrutura-AtividadeRESUMO
The genomes of the 21 completely sequenced Thermococcales display a characteristic high level of rearrangements. As a result, the prediction of their origin and termination of replication on the sole basis of chromosomal DNA composition or skew is inoperative. Using a different approach based on biologically relevant sequences, we were able to determine oriC position in all 21 genomes. The position of dif, the site where chromosome dimers are resolved before DNA segregation could be predicted in 19 genomes. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, in sharp contrast with the scrambled nature of their genomes. The active chromosomal reorganization of numerous genes acquired by horizontal transfer, mainly from mobile elements, could explain this phenomenon.
Assuntos
Cromossomos/genética , Genes Arqueais/genética , Thermococcales/genética , Sequência de Bases , Hibridização Genômica Comparativa , Evolução Molecular , Rearranjo Gênico , Genoma , Dados de Sequência MolecularRESUMO
The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure.
Assuntos
Temperatura Alta , Pyrococcus abyssi/genética , RNA Arqueal/química , RNA Arqueal/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Composição de Bases , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Regiões Promotoras Genéticas , Transcrição Gênica , Transcriptoma , Regiões não TraduzidasRESUMO
RNA-seq experiments are now routinely used for the large scale sequencing of transcripts. In bacteria or archaea, such deep sequencing experiments typically produce 10-50 million fragments that cover most of the genome, including intergenic regions. In this context, the precise delineation of the non-coding elements is challenging. Non-coding elements include untranslated regions (UTRs) of mRNAs, independent small RNA genes (sRNAs) and transcripts produced from the antisense strand of genes (asRNA). Here we present a computational pipeline (DETR'PROK: detection of ncRNAs in prokaryotes) based on the Galaxy framework that takes as input a mapping of deep sequencing reads and performs successive steps of clustering, comparison with existing annotation and identification of transcribed non-coding fragments classified into putative 5' UTRs, sRNAs and asRNAs. We provide a step-by-step description of the protocol using real-life example data sets from Vibrio splendidus and Escherichia coli.
Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro/isolamento & purificação , RNA não Traduzido/isolamento & purificação , Archaea/genética , Bactérias/genética , Sequência de Bases , RNA Mensageiro/genética , RNA não Traduzido/genéticaRESUMO
Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio "sRNA core."
Assuntos
Ostreidae/microbiologia , RNA Bacteriano/genética , Vibrio/genética , Vibrio/patogenicidade , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Evolução Molecular , Gammaproteobacteria/genética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Especificidade da Espécie , Vibrio/classificação , Vibrio/metabolismoRESUMO
BACKGROUND: Meiotic exchanges are non-uniformly distributed across the genome of most studied organisms. This uneven distribution suggests that recombination is initiated by specific signals and/or regulations. Some of these signals were recently identified in humans and mice. However, it is unclear whether or not sequence signals are also involved in chromosomal recombination of insects. METHODOLOGY: We analyzed recombination frequencies in the honeybee, in which genome sequencing provided a large amount of SNPs spread over the entire set of chromosomes. As the genome sequences were obtained from a pool of haploid males, which were the progeny of a single queen, an oocyte method (study of recombination on haploid males that develop from unfertilized eggs and hence are the direct reflect of female gametes haplotypes) was developed to detect recombined pairs of SNP sites. Sequences were further compared between recombinant and non-recombinant fragments to detect recombination-specific motifs. CONCLUSIONS: Recombination events between adjacent SNP sites were detected at an average distance of 92 bp and revealed the existence of high rates of recombination events. This study also shows the presence of conversion without crossover (i. e. non-crossover) events, the number of which largely outnumbers that of crossover events. Furthermore the comparison of sequences that have undergone recombination with sequences that have not, led to the discovery of sequence motifs (CGCA, GCCGC, CCGCA), which may correspond to recombination signals.
Assuntos
Abelhas/genética , Troca Genética/genética , Animais , Feminino , Masculino , Meiose/genética , Polimorfismo de Nucleotídeo Único/genética , Recombinação Genética/genéticaRESUMO
BACKGROUND: Due to the continuous improvements of high throughput technologies and experimental procedures, the number of sequenced genomes is increasing exponentially. Ultimately, the task of annotating these data relies on the expertise of biologists. The necessity for annotation to be supervised by human experts is the rate limiting step of the data analysis. To face the deluge of new genomic data, the need for automating, as much as possible, the annotation process becomes critical. RESULTS: We consider annotation of a protein with terms of the functional hierarchy that has been used to annotate Bacillus subtilis and propose a set of rules that predict classes in terms of elements of the functional hierarchy, i.e., a class is a node or a leaf of the hierarchy tree. The rules are obtained through two decision-trees techniques: first-order decision-trees and multilabel attribute-value decision-trees, by using as training data the proteins from two lactic bacteria: Lactobacillus sakei and Lactobacillus bulgaricus. We tested the two methods, first independently, then in a combined approach, and evaluated the obtained results using hierarchical evaluation measures. Results obtained for the two approaches on both genomes are comparable and show a good precision together with a high prediction rate. Using combined approaches increases the recall and the prediction rate. CONCLUSION: The combination of the two approaches is very encouraging and we will further refine these combinations in order to get rules even more useful for the annotators. This first study is a crucial step towards designing a semi-automatic functional annotation tool.
RESUMO
BACKGROUND: The Wuschel related homeobox (WOX) family proteins are key regulators implicated in the determination of cell fate in plants by preventing cell differentiation. A recent WOX phylogeny, based on WOX homeodomains, showed that all of the Physcomitrella patens and Selaginella moellendorffii WOX proteins clustered into a single orthologous group. We hypothesized that members of this group might preferentially share a significant part of their function in phylogenetically distant organisms. Hence, we first validated the limits of the WOX13 orthologous group (WOX13 OG) using the occurrence of other clade specific signatures and conserved intron insertion sites. Secondly, a functional analysis using expression data and mutants was undertaken. RESULTS: The WOX13 OG contained the most conserved plant WOX proteins including the only WOX detected in the highly proliferating basal unicellular and photosynthetic organism Ostreococcus tauri. A large expansion of the WOX family was observed after the separation of mosses from other land plants and before monocots and dicots have arisen. In Arabidopsis thaliana, AtWOX13 was dynamically expressed during primary and lateral root initiation and development, in gynoecium and during embryo development. AtWOX13 appeared to affect the floral transition. An intriguing clade, represented by the functional AtWOX14 gene inside the WOX13 OG, was only found in the Brassicaceae. Compared to AtWOX13, the gene expression profile of AtWOX14 was restricted to the early stages of lateral root formation and specific to developing anthers. A mutational insertion upstream of the AtWOX14 homeodomain sequence led to abnormal root development, a delay in the floral transition and premature anther differentiation. CONCLUSION: Our data provide evidence in favor of the WOX13 OG as the clade containing the most conserved WOX genes and established a functional link to organ initiation and development in Arabidopsis, most likely by preventing premature differentiation. The future use of Ostreococcus tauri and Physcomitrella patens as biological models should allow us to obtain a better insight into the functional importance of WOX13 OG genes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Sequência Conservada , Flores/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Raízes de Plantas/crescimento & desenvolvimento , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Eucariotos/genética , Eucariotos/fisiologia , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Ordem dos Genes , Genoma de Planta/genética , Proteínas de Homeodomínio/química , Dados de Sequência Molecular , Mutação , Filogenia , Raízes de Plantas/metabolismo , Plantas/classificação , Plantas/genética , Regiões Promotoras Genéticas/genéticaRESUMO
In Arabidopsis (Arabidopsis thaliana) the 466 pentatricopeptide repeat (PPR) proteins are putative RNA-binding proteins with essential roles in organelles. Roughly half of the PPR proteins form the plant combinatorial and modular protein (PCMP) subfamily, which is land-plant specific. PCMPs exhibit a large and variable tandem repeat of a standard pattern of three PPR variant motifs. The association or not of this repeat with three non-PPR motifs at their C terminus defines four distinct classes of PCMPs. The highly structured arrangement of these motifs and the similar repartition of these arrangements in the four classes suggest precise relationships between motif organization and substrate specificity. This study is an attempt to reconstruct an evolutionary scenario of the PCMP family. We developed an innovative approach based on comparisons of the proteins at two levels: namely the succession of motifs along the protein and the amino acid sequence of the motifs. It enabled us to infer evolutionary relationships between proteins as well as between the inter- and intraprotein repeats. First, we observed a polarized elongation of the repeat from the C terminus toward the N-terminal region, suggesting local recombinations of motifs. Second, the most N-terminal PPR triple motif proved to evolve under different constraints than the remaining repeat. Altogether, the evidence indicates different evolution for the PPR region and the C-terminal one in PCMPs, which points to distinct functions for these regions. Moreover, local sequence homogeneity observed across PCMP classes may be due to interclass shuffling of motifs, or to deletions/insertions of non-PPR motifs at the C terminus.
Assuntos
Arabidopsis/genética , Evolução Molecular , Família Multigênica , Proteínas de Ligação a RNA/genética , Genes de Plantas , Homologia de Sequência de AminoácidosRESUMO
The model genome of Arabidopsis thaliana contains a DEAD-box RNA helicase family (RH) of 58 members, i.e. almost twice as many as in the animal or yeast genomes. Transcript profiling using real-time quantitative polymerase chain reaction (PCR) has been obtained for 20 AtRHs from nine different organs. Two AtRHs exhibited plant-specific profiles associated with photosynthetic and sink organs. The other 18 AtRHs had the same transcript profile, and the levels of transcription of these 'housekeeping'AtRHs were under strict quantitative control over a large range of values. Transcript levels may be very different between the most recently duplicated genes. The master regulatory element in the definition of the transcript level is the simultaneous presence of a TATA-box and an intron in the 5' untranslated region (UTR). There is a positive and highly significant correlation between the size of the 5' UTR intron and the transcription level, as long as a characteristic TATA-box is present. Our work on the housekeeping AtRHs suggests a scenario for the evolution of duplicated genes, leading to both highly and poorly transcribed genes in the same terminal branch of the phylogenetic tree. The general evolutionary drive of the AtRH family, after duplication of a highly transcribed ancestral AtRH, was towards an alteration of the transcriptional activity of the divergent duplicates through successive events of suppression of the TATA-box and/or the 5' UTR intron.