RESUMEN
Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.
Asunto(s)
Aclimatación , Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Frío , ARN del Cloroplasto , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Aclimatación/genética , ARN del Cloroplasto/genética , ARN del Cloroplasto/metabolismo , Cloroplastos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Regulación de la Expresión Génica de las Plantas , Priones/metabolismo , Priones/genética , Dominios Proteicos , Empalme del ARN/genética , Separación de FasesRESUMEN
Plant organellar RNA metabolism is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. In chloroplasts and mitochondria, these post-transcriptional processes are vital for the production of a small number of essential components of the photosynthetic and respiratory machinery-and consequently for organellar biogenesis and plant survival. Many organellar RBPs have been functionally assigned to individual steps in RNA maturation, often specific to selected transcripts. While the catalog of factors identified is ever-growing, our knowledge of how they achieve their functions mechanistically is far from complete. This review summarizes the current knowledge of plant organellar RNA metabolism taking an RBP-centric approach and focusing on mechanistic aspects of RBP functions and the kinetics of the processes they are involved in.
Asunto(s)
Mitocondrias , ARN , ARN/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Plantas/genética , Plantas/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Núcleo Celular/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismoRESUMEN
The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we used RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatK's preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for splice site selection, and we discovered alternative 5'-splice sites in three MatK target introns. The resulting alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Ribosome profiling analysis of the overexpressor revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.
RESUMEN
Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants. Here, we demonstrate that RNA labeling with the modified, nontoxic uridine analog 5-ethynyl uridine (5-EU) in Arabidopsis (Arabidopsis thaliana) seedlings provides insight into plant transcriptome dynamics. Pulse labeling with 5-EU revealed nascent and unstable RNAs, RNA processing intermediates generated by splicing, and chloroplast RNAs. Pulse-chase experiments with 5-EU allowed us to determine RNA stabilities without the need for chemical transcription inhibitors such as actinomycin and cordycepin. Inhibitor-free, genome-wide analysis of polyadenylated RNA stability via 5-EU pulse-chase experiments revealed RNAs with shorter half-lives than those reported after chemical inhibition of transcription. In summary, our results indicate that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates and suggest that polyadenylated RNAs have low stability in plants. Our technique lays the foundation for easy, affordable, nascent transcriptome analysis and inhibitor-free analysis of RNA stability in plants.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , ARN de Planta/genética , Coloración y Etiquetado , Transcriptoma/genética , Semivida , MicroARNs/genética , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Plantones/genética , Uridina/metabolismoRESUMEN
Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.
Asunto(s)
Hojas de la Planta/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Camellia/genética , Camellia/metabolismo , Camellia/fisiología , Chlamydomonas/genética , Chlamydomonas/metabolismo , Chlamydomonas/fisiología , Hojas de la Planta/genética , Biología de Sistemas/métodos , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/fisiologíaRESUMEN
To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn's disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras , Estudio de Asociación del Genoma Completo , Humanos , Longevidad/genética , Fenotipo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Synthesis of the D1 reaction center protein of Photosystem II is dynamically regulated in response to environmental and developmental cues. In chloroplasts, much of this regulation occurs at the post-transcriptional level, but the proteins responsible are largely unknown. To discover proteins that impact psbA expression, we identified proteins that associate with maize psbA mRNA by: (i) formaldehyde cross-linking of leaf tissue followed by antisense oligonucleotide affinity capture of psbA mRNA; and (ii) co-immunoprecipitation with HCF173, a psbA translational activator that is known to bind psbA mRNA. The S1 domain protein SRRP1 and two RNA Recognition Motif (RRM) domain proteins, CP33C and CP33B, were enriched with both approaches. Orthologous proteins were also among the enriched protein set in a previous study in Arabidopsis that employed a designer RNA-binding protein as a psbA RNA affinity tag. We show here that CP33B is bound to psbA mRNA in vivo, as was shown previously for CP33C and SRRP1. Immunoblot, pulse labeling, and ribosome profiling analyses of mutants lacking CP33B and/or CP33C detected some decreases in D1 protein levels under some conditions, but no change in psbA RNA abundance or translation. However, analogous experiments showed that SRRP1 represses psbA ribosome association in the dark, represses ycf1 ribosome association, and promotes accumulation of ndhC mRNA. As SRRP1 is known to harbor RNA chaperone activity, we postulate that SRRP1 mediates these effects by modulating RNA structures. The uncharacterized proteins that emerged from our analyses provide a resource for the discovery of proteins that impact the expression of psbA and other chloroplast genes.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteoma , Proteínas de Unión al ARN/metabolismo , Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Complejo de Proteína del Fotosistema II/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , ARN Mensajero/genética , ARN de Planta/genética , Proteínas de Unión al ARN/genética , Ribosomas/metabolismoRESUMEN
The processing of chloroplast RNA requires a large number of nuclear-encoded RNA-binding proteins (RBPs) that are imported post-translationally into the organelle. The chloroplast ribonucleoprotein 31A (CP31A) supports RNA editing at 13 sites and also supports the accumulation of multiple chloroplast mRNAs. In cp31a mutants it is the ndhF mRNA (coding for a subunit of the NDH complex) that is most strongly affected. CP31A becomes particularly important at low temperatures, where it is essential for chloroplast development in young tissue. Next to two RNA-recognition motifs (RRMs), CP31A has an N-terminal acidic domain that is phosphorylated at several sites. We investigated the function of the acidic domain in the role of CP31A in RNA metabolism and cold resistance. Using point mutagenesis, we demonstrate that the known phosphorylation sites within the acidic domain are irrelevant for any of the known functions of CP31A, both at normal and at low temperatures. Even when the entire acidic domain is removed, no effects on RNA editing were observed. By contrast, loss of the acidic domain reduced the ability of CP31A to stabilize the ndhF mRNA, which was associated with reduced NDH complex activity. Most importantly, acidic domain-less CP31A lines displayed bleached young tissue in the cold. Together, these data show that the different functions of CP31A can be assigned to different regions of the protein: the RRMs are sufficient to maintain RNA editing and to allow the accumulation of basal amounts of ndhF mRNA, while chloroplast development under cold conditions critically depends on the acidic domain.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , ARN del Cloroplasto , Proteínas de Unión al ARN/genéticaRESUMEN
Recently, nine Caenorhabditis elegans genes, grouped into two pathways/clusters, were found to be implicated in healthspan in C. elegans and their homologues in humans, based on literature curation, WormBase data mining and bioinformatics analyses. Here, we further validated these genes experimentally in C. elegans. We downregulated the nine genes via RNA interference (RNAi), and their effects on physical function (locomotion in a swim assay) and on physiological function (survival after heat stress) were analysed in aged nematodes. Swim performance was negatively affected by the downregulation of acox-1.1, pept-1, pak-2, gsk-3 and C25G6.3 in worms with advanced age (twelfth day of adulthood) and heat stress resistance was decreased by RNAi targeting of acox-1.1, daf-22, cat-4, pig-1, pak-2, gsk-3 and C25G6.3 in moderately (seventh day of adulthood) or advanced aged nematodes. Only one gene, sad-1, could not be linked to a health-related function in C. elegans with the bioassays we selected. Thus, most of the healthspan genes could be re-confirmed by health measurements in old worms.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Estrés Fisiológico , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Humanos , Longevidad/genéticaRESUMEN
Plants are constantly exposed to temperature fluctuations, which have direct effects on all cellular reactions because temperature influences reaction likelihood and speed. Chloroplasts are crucial to temperature acclimation responses of plants, due to their photosynthetic reactions whose products play a central role in plant metabolism. Consequently, chloroplasts serve as sensors of temperature changes and are simultaneously major targets of temperature acclimation. The core subunits of the complexes involved in the light reactions of photosynthesis are encoded in the chloroplast. As a result, it is assumed that temperature acclimation in plants requires regulatory responses in chloroplast gene expression and protein turnover. We conducted western blot experiments to assess changes in the accumulation of two photosynthetic complexes (PSII, and Cytb6f complex) and the ATP synthase in tobacco plants over two days of acclimation to low temperature. Surprisingly, the concentration of proteins within the chloroplast varied negligibly compared to controls. To explain this observation, we used a simplified Ordinary Differential Equation (ODE) model of transcription, translation, mRNA degradation and protein degradation to explain how the protein concentration can be kept constant. This model takes into account temperature effects on these processes. Through simulations of the ODE model, we show that mRNA and protein degradation are possible targets for control during temperature acclimation. Our model provides a basis for future directions in research and the analysis of future results.
Asunto(s)
Cloroplastos , Fotosíntesis , Aclimatación , Cloroplastos/metabolismo , Frío , Luz , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins are a large family of helical repeat proteins that bind RNA in mitochondria and chloroplasts. Sites of PPR action have been inferred primarily from genetic data, which have led to the view that most PPR proteins act at a very small number of sites in vivo. Here, we report new functions for three chloroplast PPR proteins that had already been studied in depth. Maize PPR5, previously shown to promote trnG splicing, is also required for rpl16 splicing. Maize PPR10, previously shown to bind the atpI-atpH and psaJ-rpl33 intercistronic regions, also stabilizes a 3'-end downstream from psaI. Arabidopsis PGR3, shown previously to bind upstream of petL, also binds the rpl14-rps8 intercistronic region where it stabilizes a 3'-end and stimulates rps8 translation. These functions of PGR3 are conserved in maize. The discovery of new functions for three proteins that were already among the best characterized members of the PPR family implies that functional repertoires of PPR proteins are more complex than have been appreciated. The diversity of sequences bound by PPR10 and PGR3 in vivo highlights challenges of predicting binding sites of native PPR proteins based on the amino acid code for nucleotide recognition by PPR motifs.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Cloroplastos/genética , Proteínas de Plantas/genética , Proteínas de Unión al ARN/genética , Zea mays/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Proteínas de Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Proteínas de Plantas/metabolismo , Unión Proteica , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Ácido Nucleico , Zea mays/metabolismoRESUMEN
Gene expression in mitochondria of Plasmodium falciparum is essential for parasite survival. The molecular mechanisms of Plasmodium organellar gene expression remain poorly understood. This includes the enigmatic assembly of the mitochondrial ribosome from highly fragmented rRNAs. Here, we present the identification of clustered organellar short RNA fragments (cosRNAs) that are possible footprints of RNA-binding proteins (RBPs) in Plasmodium organelles. In plants, RBPs of the pentatricopeptide repeat (PPR) class produce footprints as a consequence of their function in processing organellar RNAs. Intriguingly, many of the Plasmodium cosRNAs overlap with 5'-ends of rRNA fragments. We hypothesize that these are footprints of RBPs involved in assembling the rRNA fragments into a functioning ribosome. A bioinformatics search of the Plasmodium nuclear genome identified a hitherto unrecognized organellar helical-hairpin-repeat protein family that we term heptatricopeptide repeat (HPR) proteins. We demonstrate that selected HPR proteins are targeted to mitochondria in P. berghei and that one of them, PbHPR1, associates with RNA, but not DNA in vitro. A phylogenetic search identified HPR proteins in a wide variety of eukaryotes. We hypothesize that HPR proteins are required for processing and stabilizing RNAs in Apicomplexa and other taxa.
Asunto(s)
Malaria Falciparum/genética , Orgánulos/genética , Plasmodium falciparum/genética , Proteínas de Unión al ARN/genética , Cloroplastos/genética , Genoma/genética , Malaria Falciparum/parasitología , Mitocondrias/química , Mitocondrias/genética , Péptidos/química , Péptidos/genética , Filogenia , Plasmodium falciparum/patogenicidad , ARN Ribosómico/química , ARN Ribosómico/genética , Proteínas de Unión al ARN/química , Ribosomas/química , Ribosomas/genéticaRESUMEN
Numerous studies highlighted the beneficial effects of the Mediterranean diet (MD) in maintaining health, especially during ageing. Even neurodegeneration, which is part of the natural ageing process, as well as the foundation of ageing-related neurodegenerative disorders like Alzheimer's and Parkinson's disease (PD), was successfully targeted by MD. In this regard, olive oil and its polyphenolic constituents have received increasing attention in the last years. Thus, this study focuses on two main olive oil polyphenols, hydroxytyrosol (HT) and oleuropein aglycone (OLE), and their effects on ageing symptoms with special attention to PD. In order to avoid long-lasting, expensive, and ethically controversial experiments, the established invertebrate model organism Caenorhabditis elegans was used to test HT and OLE treatments. Interestingly, both polyphenols were able to increase the survival after heat stress, but only HT could prolong the lifespan in unstressed conditions. Furthermore, in aged worms, HT and OLE caused improvements of locomotive behavior and the attenuation of autofluorescence as a marker for ageing. In addition, by using three different C. elegans PD models, HT and OLE were shown i) to enhance locomotion in worms suffering from α-synuclein-expression in muscles or rotenone exposure, ii) to reduce α-synuclein accumulation in muscles cells, and iii) to prevent neurodegeneration in α-synuclein-containing dopaminergic neurons. Hormesis, antioxidative capacities and an activity-boost of the proteasome & phase II detoxifying enzymes are discussed as potential underlying causes for these beneficial effects. Further biological and medical trials are indicated to assess the full potential of HT and OLE and to uncover their mode of action.
Asunto(s)
Acetatos/uso terapéutico , Monoterpenos Ciclopentánicos/uso terapéutico , Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/prevención & control , Alcohol Feniletílico/análogos & derivados , Piranos/uso terapéutico , alfa-Sinucleína , Acetatos/farmacología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/efectos de los fármacos , Monoterpenos Ciclopentánicos/farmacología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/fisiología , Alcohol Feniletílico/farmacología , Alcohol Feniletílico/uso terapéutico , Polifenoles/farmacología , Piranos/farmacología , Resultado del TratamientoRESUMEN
Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Cloroplastos/metabolismo , NADPH Deshidrogenasa/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Mutación/genética , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Parkinson's disease (PD) is the second most prevalent late-age onset neurodegenerative disorder, affecting 1% of the population after the age of about 60 years old and 4% of those over 80 years old, causing motor impairments and cognitive dysfunction. Increasing evidence indicates that Mediterranean diet (MD) exerts beneficial effects in maintaining health, especially during ageing and by the prevention of neurodegenerative disorders. In this regard, olive oil and its biophenolic constituents like hydroxytyrosol (HT) have received growing attention in the past years. Thus, in the current study we test the health-promoting effects of two hydroxytyrosol preparations, pure HT and Hidrox® (HD), which is hydroxytyrosol in its "natural" environment, in the established invertebrate model organism Caenorhabditis elegans. HD exposure led to much stronger beneficial locomotion effects in wild type worms compared to HT in the same concentration. Consistent to this finding, in OW13 worms, a PD-model characterized by α-synuclein expression in muscles, HD exhibited a significant higher effect on α-synuclein accumulation and swim performance than HT, an effect partly confirmed also in swim assays with the UA44 strain, which features α-synuclein expression in DA-neurons. Interestingly, beneficial effects of HD and HT treatment with similar strength were detected in the lifespan and autofluorescence of wild-type nematodes, in the neuronal health of UA44 worms as well as in the locomotion of rotenone-induced PD-model. Thus, the hypothesis that HD features higher healthspan-promoting abilities than HT was at least partly confirmed. Our study demonstrates that HD polyphenolic extract treatment has the potential to partly prevent or even treat ageing-related neurodegenerative diseases and ageing itself. Future investigations including mammalian models and human clinical trials are needed to uncover the full potential of these olive compounds.
Asunto(s)
Caenorhabditis elegans/fisiología , Olea/química , Enfermedad de Parkinson/dietoterapia , Enfermedad de Parkinson/fisiopatología , Polifenoles/farmacología , Envejecimiento , Animales , Animales Modificados Genéticamente , Biomarcadores/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Dieta Mediterránea , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Humanos , Longevidad , Microscopía Fluorescente , Aceite de Oliva/química , Rotenona/toxicidad , alfa-Sinucleína/metabolismoRESUMEN
The chloroplast is a prime target for genetic engineering in plants, offering various advantages over nuclear transformation. For example, chloroplasts allow the expression of polycistronic transcripts and thus to engineer complex metabolic pathways. Each cistron within such a longer transcript needs its own expression elements. Within the 5'-UTR, such expression elements are needed for stabilizing mRNAs and for translation of the downstream reading frame. One of the few effective expression elements used so far in transplastomic approaches is the intercistronic expression element (IEE). The IEE is derived from the psbT-psbH intergenic region and includes a target sequence of the RNA binding protein HCF107. We here show that excessive expression of the IEE can lead to specific defects of endogenous chloroplast mRNA stabilization, likely via depletion of HCF107. Key players in chloroplast transcript stabilization and translation are pentatricopeptide repeat (PPR) proteins, which are structurally related to HCF107. PPR proteins that stabilize mRNAs leave behind short RNA footprints that are indicators of their activity. We identified such sRNAs in tobacco, and demonstrate that they are sufficient to stabilize and stimulate translation of mRNAs from synthetic dicistronic transgenes in chloroplasts. Thus, minimal sequence elements are generally adequate to support key steps in chloroplast gene expression, i.e. RNA stability and translation. Furthermore, our analysis expands the repertoire of available expression elements to facilitate the assembly and expression of multi-gene ensembles in the chloroplast.
Asunto(s)
Cloroplastos/metabolismo , Operón/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Transgenes/genética , Sitios de Unión , Cloroplastos/genética , ADN Intergénico/genética , Plantas Modificadas Genéticamente , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids. We identified a surprisingly large number of small RNAs that represent in vivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both in vitro and in vivo experiments to reveal the role of the E domain of CRR2. The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR-RNA interaction. The 'code' of the E domain PPR motifs is different from that of P- and S-motifs. The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs in vivo.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/metabolismo , Plastidios/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Glicina/metabolismo , Mutación/genética , Dominios ProteicosRESUMEN
The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half-A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5' untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5'end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain.
Asunto(s)
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Complejo de Proteína del Fotosistema I/genética , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Fotosíntesis/genética , Complejo de Proteína del Fotosistema I/metabolismo , Unión ProteicaRESUMEN
Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA-binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP CP33A localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or to strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , ARN del Cloroplasto/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Immunoblotting , Mutación , Plantas Modificadas Genéticamente , Plastidios/genética , Plastidios/metabolismo , Unión Proteica , Empalme del ARN , ARN del Cloroplasto/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismoRESUMEN
Land plant organellar genomes encode a small number of genes, many of which are essential for respiration and photosynthesis. Organellar gene expression is characterized by a multitude of RNA processing events that lead to stable, translatable transcripts. RNA binding proteins (RBPs), have been shown to generate and protect transcript termini and eventually induce the accumulation of short RNA footprints. We applied knowledge of such RBP-derived footprints to develop software (sRNA miner) that enables identification of RBP footprints, or other clusters of small RNAs, in organelles. We used this tool to determine mitochondrial and chloroplast cosRNAs (clustered organellar sRNAs) in Arabidopsis. We found that in mitochondria, cosRNAs coincide with transcript 3'-ends, but are largely absent from 5'-ends. In chloroplasts this bias is absent, suggesting a different mode of 5' processing, possibly owing to different sets of RNases. Furthermore, we identified a large number of cosRNAs that represent silenced insertions of mitochondrial DNA in the nuclear genome of Arabidopsis. Steady-state RNA analyses demonstrate that cosRNAs display differential accumulation during development. Finally, we demonstrate that the chloroplast RBP PPR10 associates in vivo with its cognate cosRNA. A hypothetical role of cosRNAs as competitors of mRNAs for PPR proteins is discussed.