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Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.
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Bacteriófagos , Poliaminas , Bacteriófagos/genética , Bacterias , Pseudomonas aeruginosa , ADNRESUMEN
Photoautotrophic growth in nature requires the accumulation of energy-containing molecules via photosynthesis during daylight to fuel nighttime catabolism. Many diatoms store photosynthate as the neutral lipid triacylglycerol (TAG). While the pathways of diatom fatty acid and TAG synthesis appear to be well conserved with plants, the pathways of TAG catabolism and downstream fatty acid ß-oxidation have not been characterised in diatoms. We identified a putative mitochondria-targeted, bacterial-type acyl-CoA dehydrogenase (PtMACAD1) that is present in Stramenopile and Hacrobian eukaryotes, but not found in plants, animals or fungi. Gene knockout, protein-YFP tags and physiological assays were used to determine PtMACAD1's role in the diatom Phaeodactylum tricornutum. PtMACAD1 is located in the mitochondria. Absence of PtMACAD1 led to no consumption of TAG at night and slower growth in light : dark cycles compared with wild-type. Accumulation of transcripts encoding peroxisomal-based ß-oxidation did not change in response to day : night cycles or to PtMACAD1 knockout. Mutants also hyperaccumulated TAG after the amelioration of N limitation. We conclude that diatoms utilise mitochondrial ß-oxidation; this is in stark contrast to the peroxisomal-based pathways observed in plants and green algae. We infer that this pattern is caused by retention of catabolic pathways from the host during plastid secondary endosymbiosis.
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Diatomeas , Diatomeas/genética , Ácidos Grasos/metabolismo , Lípidos , Mitocondrias/metabolismo , Oxidación-ReducciónRESUMEN
Gene loss and genome reduction are defining characteristics of endosymbiotic bacteria. The most highly reduced endosymbiont genomes have lost numerous essential genes related to core cellular processes such as replication, transcription, and translation. Computational gene predictions performed for the genomes of the two bacterial symbionts of the cicada Diceroprocta semicincta, "Candidatus Hodgkinia cicadicola" (Alphaproteobacteria) and "Ca Sulcia muelleri" (Bacteroidetes), have found only 26 and 16 tRNA genes and 15 and 10 aminoacyl tRNA synthetase genes, respectively. Furthermore, the original "Ca Hodgkinia cicadicola" genome annotation was missing several essential genes involved in tRNA processing, such as those encoding RNase P and CCA tRNA nucleotidyltransferase as well as several RNA editing enzymes required for tRNA maturation. How these cicada endosymbionts perform basic translation-related processes remains unknown. Here, by sequencing eukaryotic mRNAs and total small RNAs, we show that the limited tRNA set predicted by computational annotation of "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" is likely correct. Furthermore, we show that despite the absence of genes encoding tRNA processing activities in the symbiont genomes, symbiont tRNAs have correctly processed 5' and 3' ends and seem to undergo nucleotide modification. Surprisingly, we found that most "Ca Hodgkinia cicadicola" and "Ca Sulcia muelleri" tRNAs exist as tRNA halves. We hypothesize that "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" tRNAs function in bacterial translation but require host-encoded enzymes to do so.IMPORTANCE The smallest bacterial genomes, in the range of about 0.1 to 0.5 million base pairs, are commonly found in the nutritional endosymbionts of insects. These tiny genomes are missing genes that encode proteins and RNAs required for the translation of mRNAs, one of the most highly conserved and important cellular processes. In this study, we found that the bacterial endosymbionts of cicadas have genomes which encode incomplete tRNA sets and lack genes required for tRNA processing. Nevertheless, we found that endosymbiont tRNAs are correctly processed at their 5' and 3' ends and, surprisingly, that mostly exist as tRNA halves. We hypothesize that the cicada host must supply its symbionts with these missing tRNA processing activities.
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Bacterias/genética , Genoma Bacteriano , Hemípteros/microbiología , ARN de Transferencia/genética , Simbiosis , Animales , Evolución Molecular , Femenino , Filogenia , Modificación Traduccional de las ProteínasRESUMEN
Soil salinity, a significant problem in agriculture, severely limits the productivity of crop plants. Plants respond to and cope with salt stress by reprogramming gene expression via multiple signaling pathways that converge on transcription factors. To develop strategies to generate salt-tolerant crops, it is necessary to identify transcription factors that modulate salt stress responses in plants. In this study, we investigated the role of VOZ (VASCULAR PLANT ONE-ZINC FINGER PROTEIN) transcription factors (VOZs) in salt stress response. Transcriptome analysis in WT (wild-type), voz1-1, voz2-1 double mutant and a VOZ2 complemented line revealed that many stress-responsive genes are regulated by VOZs. Enrichment analysis for gene ontology terms in misregulated genes in voz double mutant confirmed previously identified roles of VOZs and suggested a new role for them in salt stress. To confirm VOZs role in salt stress, we analyzed seed germination and seedling growth of WT, voz1, voz2-1, voz2-2 single mutants, voz1-1 voz2-1 double mutant and a complemented line under different concentrations of NaCl. Only the double mutant exhibited hypersensitivity to salt stress as compared to WT, single mutants, and a complemented line. Expression analysis showed that hypersensitivity of the double mutant was accompanied by reduced expression of salt-inducible genes. These results suggest that VOZ transcription factors act as positive regulators of several salt-responsive genes and that the two VOZs are functionally redundant in salt stress.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Tolerancia a la Sal , Factores de Transcripción/metabolismo , Biología Computacional/métodos , Perfilación de la Expresión Génica , Ontología de Genes , Mutación con Pérdida de Función , Plantas Modificadas Genéticamente , Elementos de Respuesta , Estrés Fisiológico/genéticaRESUMEN
While boron forms a wide range of metal borides with important industrial applications, there has been relatively little attention devoted to lanthanide boride clusters. Here we report a joint photoelectron spectroscopy and quantum chemical study on two octa-boron di-lanthanide clusters, Ln2B8- (Ln = La, Pr). We found that these clusters form highly stable inverse sandwich structures, [Ln-B8-Ln]-, with strong Ln and B8 bonding via interactions between the Ln 5d orbitals and the delocalized σ and π orbitals on the B8 ring. A (d-p)δ bond, involving the 5dδ and the antibonding π orbital of the B8 ring, is observed to be important in the Ln-B8 interactions. The highly symmetric inverse sandwich structures are overwhelmingly more stable than any other isomers. Upon electron detachment, the (d-p)δ orbitals become half-filled, giving rise to a triplet ground state for neutral La2B8 In addition to the two unpaired electrons in the (d-p)δ orbitals upon electron detachment, the neutral Pr2B8 complex also contains two unpaired 4f electrons on each Pr center. The six unpaired spins in Pr2B8 are ferromagnetically coupled to give rise to a septuplet ground state. The current work suggests that highly magnetic Ln B8 Ln inverse sandwiches or 1D Ln B8 Ln nanowires may be designed with novel electronic and magnetic properties.
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Abiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response.
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Arabidopsis/genética , Tolerancia a la Sal , Transcriptoma , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Silenciador del Gen , Genes de Plantas , Plantones/genética , Plantones/metabolismo , Análisis de Secuencia de ARN , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A thorough theoretical study of the relative energies of various molecular Fe·4O isomers with different oxidation states of both Fe and O atoms is presented, comparing simple Hartree-Fock through many Kohn-Sham approximations up to extended coupled cluster and DMRG multiconfiguration benchmark methods. The ground state of Fe·4O is a singlet, hexavalent iron(VI) complex (1)C2v-[Fe(VI)O2](2+)(O2)(2-), with isomers of oxidation states Fe(II), Fe(III), Fe(IV), Fe(V), and Fe(VIII) all lying slightly higher within the range of 1 eV. The disputed existence of oxidation state Fe(VIII) is discussed for isolated FeO4 molecules. Density functional theory (DFT) at various DF approximation (DFA) levels of local and gradient approaches, Hartree-Fock exchange and meta hybrids, range dependent, DFT-D and DFT+U models do not perform better for the relative stabilities of the geometric and electronic Fe·4O isomers than within 1-5 eV. The Fe·4O isomeric species are an excellent testing and validation ground for the development of density functional and wave function methods for strongly correlated multireference states, which do not seem to always follow chemical intuition.
RESUMEN
Plant SR45 and its metazoan ortholog RNPS1 are serine/arginine-rich (SR)-like RNA binding proteins that function in splicing/postsplicing events and regulate diverse processes in eukaryotes. Interactions of SR45 with both RNAs and proteins are crucial for regulating RNA processing. However, in vivo RNA targets of SR45 are currently unclear. Using RNA immunoprecipitation followed by high-throughput sequencing, we identified over 4000 Arabidopsis thaliana RNAs that directly or indirectly associate with SR45, designated as SR45-associated RNAs (SARs). Comprehensive analyses of these SARs revealed several roles for SR45. First, SR45 associates with and regulates the expression of 30% of abscisic acid (ABA) signaling genes at the postsplicing level. Second, although most SARs are derived from intron-containing genes, surprisingly, 340 SARs are derived from intronless genes. Expression analysis of the SARs suggests that SR45 differentially regulates intronless and intron-containing SARs. Finally, we identified four overrepresented RNA motifs in SARs that likely mediate SR45's recognition of its targets. Therefore, SR45 plays an unexpected role in mRNA processing of intronless genes, and numerous ABA signaling genes are targeted for regulation at the posttranscriptional level. The diverse molecular functions of SR45 uncovered in this study are likely applicable to other species in view of its conservation across eukaryotes.
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Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Transcriptoma , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arginina/metabolismo , Intrones/genética , Motivos de Nucleótidos , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ARN , Serina/metabolismoRESUMEN
Genome-wide studies revealed the prevalence of multiple transcripts resulting from alternative polyadenylation (APA) of a single given gene in higher eukaryotes. Several studies in the past few years attempted to address how those APA events are regulated and what the biological consequences of those regulations are. Common to these efforts is the comparison of unbiased transcriptome data, either derived from whole-genome tiling array or next generation sequencing, to identify the specific APA events in a given condition. RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression) is an R program, developed to serve such a purpose using data from the whole-genome tilling array. RADPRE took a set of tilling array data as input, performed a series of calculation including a correction of the probe affinity variation, a hierarchy of statistical tests and an estimation of the false discovery rate (FDR) of the differentially processed genes (DPG). The result was an output of a few tabular files including DPG and their corresponding FDR. This chapter is written for scientists with limited programming experiences.
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Arabidopsis/genética , Genoma de Planta , Poliadenilación , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Programas Informáticos , Arabidopsis/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN de Planta/genética , TranscriptomaRESUMEN
BACKGROUND: The yeast and human Pcf11 functions in both constitutive and regulated transcription and pre-mRNA processing. The constitutive roles of PCF11 are largely mediated by its direct interaction with RNA Polymerase II C-terminal domain and a polyadenylation factor, Clp1. However, little is known about the mechanism of the regulatory roles of Pcf11. Though similar to Pcf11 in multiple aspects, Arabidopsis Pcf11-similar-4 protein (PCFS4) plays only a regulatory role in Arabidopsis gene expression. Towards understanding how PCFS4 regulates the expression of its direct target genes in a genome level, ChIP-Seq approach was employed in this study to identify PCFS4 enrichment sites (ES) and the ES-linked genes within the Arabidopsis genome. RESULTS: A total of 892 PCFS4 ES sites linked to 839 genes were identified. Distribution analysis of the ES sites along the gene bodies suggested that PCFS4 is preferentially located on the coding sequences of the genes, consistent with its regulatory role in transcription and pre-mRNA processing. Gene ontology (GO) analysis revealed that the ES-linked genes were specifically enriched in a few GO terms, including those categories of known PCFS4 functions in Arabidopsis development. More interestingly, GO analysis suggested novel roles of PCFS4. An example is its role in circadian rhythm, which was experimentally verified herein. ES site sequences analysis identified some over-represented sequence motifs shared by subsets of ES sites. The motifs may explain the specificity of PCFS4 on its target genes and the PCFS4's functions in multiple aspects of Arabidopsis development and behavior. CONCLUSIONS: Arabidopsis PCFS4 has been shown to specifically target on, and physically interact with, the subsets of genes. Its targeting specificity is likely mediated by cis-elements shared by the genes of each subset. The potential regulation on both transcription and mRNA processing levels of each subset of the genes may explain the functions of PCFS4 in multiple aspects of Arabidopsis development and behavior.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Inmunoprecipitación de Cromatina , Ritmo Circadiano/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Motivos de Nucleótidos , Plantas Modificadas Genéticamente/genética , Elementos Reguladores de la TranscripciónRESUMEN
BACKGROUND: Polyadenylation, an essential step in eukaryotic gene expression, requires both cis-elements and a plethora of trans-acting polyadenylation factors. The polyadenylation factors are largely conserved across mammals and fungi. The conservation seems also extended to plants based on the analyses of Arabidopsis polyadenylation factors. To extend this observation, we systemically identified the orthologs of yeast and human polyadenylation factors from 10 plant species chosen based on both the availability of their genome sequences and their positions in the evolutionary tree, which render them representatives of different plant lineages. RESULTS: The evolutionary trajectories revealed several interesting features of plant polyadenylation factors. First, the number of genes encoding plant polyadenylation factors was clearly increased from "lower" to "higher" plants. Second, the gene expansion in higher plants was biased to some polyadenylation factors, particularly those involved in RNA binding. Finally, while there are clear commonalities, the differences in the polyadenylation apparatus were obvious across different species, suggesting an ongoing process of evolutionary change. These features lead to a model in which the plant polyadenylation complex consists of a conserved core, which is rather rigid in terms of evolutionary conservation, and a panoply of peripheral subunits, which are less conserved and associated with the core in various combinations, forming a collection of somewhat distinct complex assemblies. CONCLUSIONS: The multiple forms of plant polyadenylation complex, together with the diversified polyA signals may explain the intensive alternative polyadenylation (APA) and its regulatory role in biological functions of higher plants.
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Secuencia Conservada , Variación Genética , Plantas/genética , Poliadenilación , ARN Mensajero/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Secuencia de Bases , Evolución Biológica , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Filogenia , Plantas/clasificación , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Functioning as an essential step of pre-mRNA processing, polyadenylation has been realized in recent years to play an important regulatory role during eukaryotic gene expression. Such regulation occurs mostly through the use of alternative polyadenylation (APA) sites and generates different transcripts with altered coding capacity for proteins and/or RNA. However, the molecular mechanisms that underlie APAs are poorly understood. Besides APA cases demonstrated in animal embryo development, cancers, and other diseases, there are a number of APA examples reported in plants. The best-known ones are related to flowering time control pathways and stress responses. Genome-wide studies have revealed that plants use APA extensively to generate diversity in their transcriptomes. Although each transcript produced by RNA polymerase II has a poly(A) tail, over 50% of plant genes studied possess multiple APA sites in their transcripts. The signals defining poly(A) sites in plants were mostly studied through classical genetic means. Our understanding of these poly(A) signals is enhanced by the tallies of whole plant transcriptomes. The profiles of these signals have been used to build computer models that can predict poly(A) sites in newly sequenced genomes, potential APA sites in genes of interest, and/or to identify, and then mutate, unwanted poly(A) sites in target transgenes to facilitate crop improvements. In this review, we provide readers an update on recent research advances that shed light on the understanding of polyadenylation, APA, and its role in gene expression regulation in plants.
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Regulación de la Expresión Génica de las Plantas , Plantas/genética , Plantas/metabolismo , Poliadenilación , Empalme Alternativo , Arabidopsis/genética , Arabidopsis/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Flores/genética , Flores/metabolismo , Genoma de Planta , Modelos Biológicos , Estrés Oxidativo , Señales de Poliadenilación de ARN 3' , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismoRESUMEN
BACKGROUND: Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development. METHODOLOGY/PRINCIPAL FINDINGS: To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data--variations caused by differential probe affinity--was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation. CONCLUSION/SIGNIFICANCE: We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.
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Arabidopsis , Perfilación de la Expresión Génica/métodos , Análisis por Micromatrices/métodos , Procesamiento Postranscripcional del ARN/fisiología , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Algoritmos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Perfilación de la Expresión Génica/normas , Regulación de la Expresión Génica de las Plantas , Análisis por Micromatrices/normas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Plantas Modificadas Genéticamente , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Estándares de Referencia , Estudios de Validación como AsuntoRESUMEN
Based on comparative genome analyses, the increases in protein-coding gene number could not account for the increases of morphological and behavioral complexity of higher eukaryotes. Transcriptional regulations, alternative splicing and the involvement of non-coding RNA in gene expression regulations have been credited for the drastic increase of transcriptome complexity. However, an emerging theme of another mechanism that contributes to the formation of alternative mRNA 3'-ends is alternative polyadenylation (APA). First, recent studies indicated that APA is a wide spread phenomenon across the transcriptomes of higher eukaryotes and being regulated by developmental and environmental cues. Secondly, our characterization of the Arabidopsis polyadenylation factors suggested that plant polyadenylation has also evolved to regulate the expression of specific genes by means of APA and therefore the specific biological functions. Finally, Phylogenetic analyses of eukaryotic polyadenylation factors from several organisms revealed that the number of polyadenylation factors tends to increase in higher eukaryotes, which provides the potential for their functional differentiation in regulating gene expression through APA. Based on above evidence, we, thus, hypothesize that APA, serving as an additional mechanism, contributes to the complexity of higher eukaryotes.
RESUMEN
Cleavage and polyadenylation of precursor mRNA is an essential process for mRNA maturation. Among the 15 to 20 protein factors required for this process, a subgroup of proteins is needed for both cleavage and polyadenylation in plants and animals. This subgroup of proteins is known as the cleavage and polyadenylation specificity factor (CPSF). To explore the in vivo structural features of plant CPSF, we used tandem affinity purification methods to isolate the interacting protein complexes for each component of the CPSF subunits using Arabidopsis (Arabidopsis thaliana ecotype Landsberg erecta) suspension culture cells. The proteins in these complexes were identified by mass spectrometry and western immunoblots. By compiling the in vivo interaction data from tandem affinity purification tagging as well as other available yeast two-hybrid data, we propose an in vivo plant CPSF model in which the Arabidopsis CPSF possesses AtCPSF30, AtCPSF73-I, AtCPSF73-II, AtCPSF100, AtCPSF160, AtFY, and AtFIPS5. Among them, AtCPSF100 serves as a core with which all other factors, except AtFIPS5, are associated. These results show that plant CPSF possesses distinct features, such as AtCPSF73-II and AtFY, while sharing other ortholog components with its yeast and mammalian counterparts. Interestingly, these two unique plant CPSF components have been associated with embryo development and flowering time controls, both of which involve plant-specific biological processes.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Células Cultivadas , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/aislamiento & purificación , Espectrometría de Masas , Proteómica , Precursores del ARN/metabolismo , ARN de Planta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Ribonucleases (RNases) play a variety of cellular and biological roles in all three domains of life. In an attempt to perform RNA immuno-precipitation assays of Arabidopsis proteins, we found an EDTA-dependent RNase activity from Arabidopsis suspension tissue cultures. Further investigations proved that the EDTA-dependent RNase activity was plant specific. Characterization of the RNase activity indicated that it was insensitive to low pH and high concentration of NaCl. In the process of isolating the activity with cation exchange chromatography, we found that the EDTA dependency of the activity was lost. This led us to speculate that some metal ions, which inhibited the RNase activity, may be removed during cation exchange chromatography so that the nuclease activity was released. The EDTA dependency of the activity could be due to the ability of the EDTA chelating those metal ions, mimicking the effect of the cation exchange chromatography. Indeed, Zn(2+) strongly inhibited the activity, and the inhibition could be released by EDTA based on both in-solution and in-gel assays. In-gel assays identified two RNase activity bands. Mass spectrometry assays of those activity bands revealed more than 20 proteins. However, none of them has an apparent known nuclease domain, suggesting that one or more of those proteins might possess a currently uncharacterized nuclease domain. Our results may shed light on RNA metabolism in plants by introducing a novel plant-specific RNase activity.
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Arabidopsis/enzimología , Ribonucleasas/metabolismo , Zinc/farmacología , Ácido Edético/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/aislamiento & purificación , Cloruro de Sodio/farmacología , Especificidad de la EspecieRESUMEN
Polyadenylation factor CLP1 is essential for mRNA 3'-end processing in yeast and mammals. The Arabidopsis (Arabidopsis thaliana) CLP1-SIMILAR PROTEIN3 (CLPS3) is an ortholog of human hCLP1. CLPS3 was previously found to be a subunit in the affinity-purified PCFS4-TAP (tandem affinity purification) complex involved in the alternative polyadenylation of FCA and flowering time control in Arabidopsis. In this article, we further explored the components in the affinity-purified CLPS3-TAP complex, from which Arabidopsis cleavage and polyadenylation specificity factor (CPSF) subunits AtCPSF100 and AtCPSF160 were found. This result implies that CLPS3 may bridge CPSF to the PCFS4 complex. Characterization of the CLPS3 mutant revealed that CLPS3 was essential for embryo development and important for female gametophyte transmission. Overexpression of CLPS3-TAP fusion caused a range of postembryonic development abnormalities, including early flowering time, altered phyllotaxy, and abnormal numbers and shapes of flower organs. These phenotypes are associated with the altered gene expression levels of FCA, WUS, and CUC1. The decreased ratio of FCA-beta to FCA-gamma in the overexpression plants suggests that CLPS3 favored the usage of FCA regular poly(A) site over the alternative site. These observations indicate that Arabidopsis CLPS3 might be involved in the processing of pre-mRNAs encoded by a distinct subset of genes that are important in plant development.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Semillas/crecimiento & desarrollo , Factores de Escisión y Poliadenilación de ARNm/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular , Desarrollo Embrionario/genética , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Meristema/citología , Meristema/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfotransferasas/química , Fosfotransferasas/genética , Filogenia , Poliadenilación/genética , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Semillas/genética , Semillas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Perfilación de la Expresión Génica , Poliadenilación , ARN Mensajero/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Arabidopsis/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
The timely transition from vegetative to reproductive growth is vital for reproductive success in plants. It has been suggested that messenger RNA 3'-end processing plays a role in this transition. Specifically, two autonomous factors in the Arabidopsis thaliana flowering time control pathway, FY and FCA, are required for the alternative polyadenylation of FCA pre-mRNA. In this paper we provide evidence that Pcf11p-similar protein 4 (PCFS4), an Arabidopsis homologue of yeast polyadenylation factor Protein 1 of Cleavage Factor 1 (Pcf11p), regulates FCA alternative polyadenylation and promotes flowering as a novel factor in the autonomous pathway. First, the mutants of PCFS4 show delayed flowering under both long-day and short-day conditions and still respond to vernalization treatment. Next, gene expression analyses indicate that the delayed flowering in pcfs4 mutants is mediated by Flowering Locus C (FLC). Moreover, the expression profile of the known FCA transcripts, which result from alternative polyadenylation, was altered in the pcfs4 mutants, suggesting the role of PCFS4 in FCA alternative polyadenylation and control of flowering time. In agreement with these observations, using yeast two-hybrid assays and TAP-tagged protein pull-down analyses, we also revealed that PCFS4 forms a complex in vivo with FY and other polyadenylation factors. The PCFS4 promoter activity assay indicated that the transcription of PCFS4 is temporally and spatially regulated, suggesting its non-essential nature in plant growth and development.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Flores , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Escisión y Poliadenilación de ARNm/fisiología , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fracciones Subcelulares/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Plant WRKY transcription factors can function as either positive or negative regulators of plant basal disease resistance. Arabidopsis WRKY48 is induced by mechanical and/or osmotic stress due to infiltration and pathogen infection and, therefore, may play a role in plant defense responses. WRKY48 is localized to the nucleus, recognizes the TTGACC W-box sequence with a high affinity in vitro and functions in plant cells as a strong transcriptional activator. To determine the biological functions directly, we have isolated loss-of-function T-DNA insertion mutants and generated gain-of-function transgenic overexpression plants for WRKY48 in Arabidopsis. Growth of a virulent strain of the bacterial pathogen Pseudomonas syringae was decreased in the wrky48 T-DNA insertion mutants. The enhanced resistance of the loss-of-function mutants was associated with increased induction of salicylic acid-regulated PR1 by the bacterial pathogen. By contrast, transgenic WRKY48-overexpressing plants support enhanced growth of P. syringae and the enhanced susceptibility was associated with reduced expression of defense-related PR genes. These results suggest that WRKY48 is a negative regulator of PR gene expression and basal resistance to the bacterial pathogen P. syringae.