RESUMEN
Polycomb-group (PcG) proteins are major chromatin complexes that regulate gene expression, mainly described as repressors keeping genes in a transcriptionally silent state during development. Recent studies have nonetheless suggested that PcG proteins might have additional functions, including targeting active genes or acting independently of gene expression regulation. However, the reasons for the implication of PcG proteins and their associated chromatin marks on active genes are still largely unknown. Here, we report that combining mutations for CURLY LEAF (CLF) and LIKE HETEROCHROMATIN PROTEIN1 (LHP1), two Arabidopsis PcG proteins, results in deregulation of expression of active genes that are targeted by PcG proteins or enriched in associated chromatin marks. We show that this deregulation is associated with accumulation of small RNAs corresponding to massive degradation of active gene transcripts. We demonstrate that transcriptionally active genes and especially those targeted by PcG proteins are prone to RNA degradation, even though deregulation of RNA degradation following the loss of function of PcG proteins is not likely to be mediated by a PcG protein-mediated chromatin environment. Therefore, we conclude that PcG protein function is essential to maintain an accurate level of RNA degradation to ensure accurate gene expression.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Regulación de la Expresión Génica de las Plantas , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Estabilidad del ARN/genéticaRESUMEN
In Arabidopsis, transcriptional gene silencing (TGS) can be triggered by 24 nt small-interfering RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. By functional analysis of NERD, a GW repeat- and PHD finger-containing protein, we demonstrate that Arabidopsis harbors a second siRNA-dependent DNA methylation pathway targeting a subset of nonconserved genomic loci. The activity of the NERD-dependent pathway differs from RdDM by the fact that it relies both on silencing-related factors previously implicated only in posttranscriptional gene silencing (PTGS), including RNA-DEPENDENT RNA POLYMERASE1/6 and ARGONAUTE2, and most likely on 21 nt siRNAs. A central role for NERD in integrating RNA silencing and chromatin signals in transcriptional silencing is supported by data showing that it binds both to histone H3 and AGO2 proteins and contributes to siRNA accumulation at a NERD-targeted locus. Our results unravel the existence of a conserved chromatin-based RNA silencing pathway encompassing both PTGS and TGS components in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Interferencia de ARN , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas Argonautas , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Metilación de ADN , Histonas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de la Cápside/metabolismo , Carmovirus/metabolismo , Homeostasis/fisiología , Interacciones Huésped-Patógeno , Ribonucleasa III/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Proteínas de la Cápside/química , Proteínas de Ciclo Celular/genética , Silenciador del Gen , Datos de Secuencia Molecular , Mutación , Enfermedades de las Plantas/virología , Unión Proteica , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/genética , Alineación de SecuenciaRESUMEN
Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Lignina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Procesamiento Proteico-Postraduccional , Pared Celular/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.
Asunto(s)
Arabidopsis/fisiología , Caulimovirus/fisiología , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Interferencia de ARN/fisiología , Proteínas de Unión al ARN/fisiología , Proteínas Virales/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Arabidopsis/virología , Citoplasma/metabolismo , Cuerpos de Inclusión Viral/fisiología , Datos de Secuencia Molecular , Mutación , Señales de Localización Nuclear/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasa III , Ribonucleasas/fisiología , Replicación Viral/fisiologíaRESUMEN
The plastid genome of dicotyledonous plants is transcribed by three different RNA polymerases; an eubacterial-type enzyme, PEP; and two phage-type enzymes, RPOTp and RPOTmp. RPOTp plays an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation. RPOTmp fulfills a specific function in the transcription of the rrn operon in proplasts/amyloplasts during seed imbibition/germination and a more general function in chloroplasts during later developmental stages. In chloroplasts, RPOTmp is tightly associated with thylakoid membranes indicating that functional switching of RPOTmp is connected to thylakoid association. By using the yeast two-hybrid system, we have identified two proteins that interact with RPOTmp. The two proteins are very similar, both characterized by three N-terminal transmembrane domains and a C-terminal RING domain. We show that at least one of these proteins is an intrinsic thylakoid membrane protein that fixes RPOTmp on the stromal side of the thylakoid membrane, probably via the RING domain. A model is presented in which light by triggering the synthesis of the RING protein determines membrane association and functional switching of RPOTmp.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Bacteriófagos/enzimología , ARN Polimerasas Dirigidas por ADN/metabolismo , Tilacoides/enzimología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/química , Proteínas de Unión al ADN , ARN Polimerasas Dirigidas por ADN/química , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Biblioteca de Genes , Membranas Intracelulares/metabolismo , Membranas Intracelulares/efectos de la radiación , Luz , Datos de Secuencia Molecular , Especificidad de Órganos/efectos de la radiación , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de la radiación , Transporte de Proteínas/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 16S/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tilacoides/efectos de la radiación , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5'-cytosine methylation. The RNA-directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence-specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent. The RdDM pathway has been well described, but how DRM2 is recruited onto DNA targets and associates with other RdDM factors remains unknown. To address these questions, we developed biochemical approaches to allow the identification of factors that may escape genetic screens, such as proteins encoded by multigenic families. Through both conventional and affinity purification of DRM2, we identified DEAD box RNA helicases U2AF56 Associated Protein 56 (UAP56a/b), which are widespread among eukaryotes, as new DRM2 partners. We have shown that, similar to DRM2 and other RdDM actors, UAP56 has chromatin-associated protein properties. We confirmed this association both in vitro and in vivo in reproductive tissues. In addition, our experiments also suggest that UAP56 may exhibit differential distribution in cells depending on plant organ. While originally identified for its role in splicing, our study suggests that UAP56 may also have other roles, and our findings allow us to initiate discussion about its potential role in the RdDM pathway.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , ARN Helicasas DEAD-box/genética , Metiltransferasas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , ARN Helicasas DEAD-box/metabolismo , Metilación de ADN , Metiltransferasas/metabolismoRESUMEN
Global, segmental, and gene duplication-related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)-assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3'-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.
Asunto(s)
Adenosina/análogos & derivados , Regulación de la Expresión Génica de las Plantas , Plantas/genética , Plantas/metabolismo , Transducción de Señal , Transcriptoma , Adenosina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Perfilación de la Expresión Génica , Sitios Genéticos , Mutación , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Plant plastids contain a circular genome of approximately 150 kb organized into approximately 35 transcription units. The plastid genome is organized into nucleoids and attached to plastid membranes. This relatively small genome is transcribed by at least two different RNA polymerases, one being of the prokaryotic type and plastid-encoded (PEP), the other one being of the phage-type and nucleus-encoded (NEP). The presumed localization of a second phage-type RNA polymerase in plastids is still questionable. There is strong evidence for a sequential action of NEP and PEP enzymes during plant development attributing a prevailing role of NEP during early plant and plastid development, although NEP is present in mature chloroplasts. In the present paper, we have analysed two different NEP enzymes from spinach with respect to subcellular and intra-plastidial localization in mature chloroplasts with the help of specific antibodies. Results show the presence of the two different NEP enzymes in mature chloroplasts. Both enzymes are entirely membrane bound but, unlike previously thought, this membrane binding is not mediated via DNA. This finding indicates that NEP enzymes are not found as elongating transcription complexes on the template DNA in mature chloroplasts and raises the question of their function in mature chloroplasts.
Asunto(s)
Cloroplastos/enzimología , ARN Polimerasas Dirigidas por ADN/análisis , Proteínas de Plantas/análisis , Spinacia oleracea/enzimología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Biología Computacional , ADN de Plantas/fisiología , ARN Polimerasas Dirigidas por ADN/inmunología , Membranas Intracelulares/enzimología , Datos de Secuencia Molecular , Proteínas de Plantas/inmunologíaRESUMEN
Argonautes are central and common components of crucial effectors of RNA silencing pathways. Although earlier steps in these pathways, such as small RNA biogenesis and their loading into AGO, have been quite well described, our knowledge on regulation of the action of AGO and their partners is still poor. Recent breakthroughs have highlighted the existence in many eukaryotes of an evolutionarily conserved motif, the Ago-hook, in factors implicated in AGO action. Furthermore, it has been shown that certain plant pathogen proteins have co-opted the Ago-hook as a means of evasion of plant defense systems. Here we discuss the roles and properties of Ago-hook proteins in divergent RNAi-related pathways.
Asunto(s)
Proteínas de Plantas/genética , Plantas/genética , Interferencia de ARN , Proteínas de Unión al ARN/genética , Complejo Silenciador Inducido por ARN/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Transducción de SeñalRESUMEN
The plastid genome is transcribed by three different RNA polymerases, one is called plastid-encoded RNA polymerase (PEP) and two are called nucleus-encoded RNA polymerases (NEPs). PEP transcribes preferentially photosynthesis-related genes in mature chloroplasts while NEP transcribes preferentially housekeeping genes during early phases of plant development, and it was generally thought that during plastid differentiation the building up of the NEP transcription system precedes the building up of the PEP transcription system. We have now analyzed in detail the establishment of the two different transcription systems, NEP and PEP, during germination and early seedling development on the mRNA and protein level. Experiments have been performed with two different plant species, Arabidopsis (Arabidopsis thaliana) and spinach (Spinacia oleracea). Results show that the building up of the two different transcription systems is different in the two species. However, in both species NEP as well as PEP are already present in seeds, and results using Tagetin as a specific inhibitor of PEP activity demonstrate that PEP is important for efficient germination, i.e. PEP is already active in not yet photosynthetically active seed plastids.