RESUMEN
Translation initiation on chloroplast psbA mRNA in plants scales with light intensity, providing its gene product, D1, as needed to replace photodamaged D1 in Photosystem II. The psbA translational activator HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with the psbA 5'-untranslated region (5'-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173's footprint in the psbA 5'-UTR bound the intended site in vivo and partially substituted for HCF173 to activate psbA translation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulate psbA translation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts.
RESUMEN
Chloroplast activities influence nuclear gene expression, a phenomenon referred to as retrograde signaling. Biogenic retrograde signals have been revealed by changes in nuclear gene expression when chloroplast development is disrupted. Research on biogenic signaling has focused on repression of Photosynthesis Associated Nuclear Genes (PhANGs) but this is just one component of a syndrome involving altered expression of thousands of genes involved in diverse processes, many of which are up-regulated. We discuss evidence for a framework that accounts for most of this syndrome. Disruption of chloroplast biogenesis prevents production of signals required to progress through discrete steps in the program of photosynthetic differentiation, causing retention of juvenile states. As a result, expression of PhANGs and other genes that act late during photosynthetic differentiation is not initiated, while expression of genes that act early is retained. The extent of juvenility, and thus the transcriptome, reflects the disrupted process: lack of plastid translation blocks development very early whereas disruption of photosynthesis without compromising plastid translation blocks development at a later stage. We discuss implications of these and other recent observations for the nature of the plastid-derived signals that regulate photosynthetic differentiation, and the role of GUN1, an enigmatic protein involved in biogenic signaling.
RESUMEN
Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5' UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5' UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5' exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5' UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02-25% of the total soluble cellular protein. These findings highlight the potential of employing cis-elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.
Asunto(s)
Regiones no Traducidas 5' , Cloroplastos , Regulación de la Expresión Génica de las Plantas , Nicotiana , Operón , Nicotiana/genética , Nicotiana/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Operón/genética , Regiones no Traducidas 5'/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Genes Reporteros , Plantas Modificadas Genéticamente , Zea mays/genética , Zea mays/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Signals emanating from chloroplasts influence nuclear gene expression, but roles of retrograde signals during chloroplast development are unclear. To address this gap, we analyzed transcriptomes of non-photosynthetic maize mutants and compared them to transcriptomes of stages of normal leaf development. The transcriptomes of two albino mutants lacking plastid ribosomes resembled transcriptomes at very early stages of normal leaf development, whereas the transcriptomes of two chlorotic mutants with thylakoid targeting or plastid transcription defects resembled those at a slightly later stage. We identified â¼2,700 differentially expressed genes, which fall into six major categories based on the polarity and mutant-specificity of the change. Downregulated genes were generally expressed late in normal development and were enriched in photosynthesis genes, whereas upregulated genes act early and were enriched for functions in chloroplast biogenesis and cytosolic translation. We showed further that target-of-rapamycin (TOR) signaling was elevated in mutants lacking plastid ribosomes and declined in concert with plastid ribosome buildup during normal leaf development. Our results implicate three plastid signals as coordinators of photosynthetic differentiation. One signal requires plastid ribosomes and activates photosynthesis genes. A second signal reflects attainment of chloroplast maturity and represses chloroplast biogenesis genes. A third signal, the consumption of nutrients by developing chloroplasts, represses TOR, promoting termination of cell proliferation during leaf development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Zea mays/genética , Zea mays/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulón , Cloroplastos/genética , Cloroplastos/metabolismo , Plastidios/metabolismo , Fotosíntesis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5' end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5' UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPR proteins can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ingeniería de Proteínas/métodos , ARN del Cloroplasto/metabolismo , Motivos de Unión al ARN/genética , Regiones no Traducidas 5' , Arabidopsis/genética , Cloroplastos/genética , Expresión Génica , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair, and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Unión a Clorofila/metabolismo , Cloroplastos/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Luz , Proteínas de la Membrana/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema II/genética , Plantas/genética , Biosíntesis de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/fisiología , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Transcripción Genética , Zea mays/genética , Zea mays/metabolismoRESUMEN
Meiotic double-strand breaks (DSBs) are generated by the evolutionarily conserved SPO11 complex in the context of chromatin loops that are organized along axial elements (AEs) of chromosomes. However, how DSBs are formed with respect to chromosome axes and the SPO11 complex remains unclear in plants. Here, we confirm that DSB and bivalent formation are defective in maize spo11-1 mutants. Super-resolution microscopy demonstrates dynamic localization of SPO11-1 during recombination initiation, with variable numbers of SPO11-1 foci being distributed in nuclei but similar numbers of SPO11-1 foci being found on AEs. Notably, cytological analysis of spo11-1 meiocytes revealed an aberrant AE structure. At leptotene, AEs of wild-type and spo11-1 meiocytes were similarly curly and discontinuous. However, during early zygotene, wild-type AEs become uniform and exhibit shortened axes, whereas the elongated and curly AEs persisted in spo11-1 mutants, suggesting that loss of SPO11-1 compromised AE structural maturation. Our results reveal an interesting relationship between SPO11-1 loading onto AEs and the conformational remodeling of AEs during recombination initiation.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Recombinación Homóloga , Meiosis , Zea mays/citología , Zea mays/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Emparejamiento Cromosómico , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , Genes de Plantas/genética , Meiosis/genética , Mutación , Fenotipo , Zea mays/genéticaRESUMEN
The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.
Asunto(s)
Cloroplastos/genética , Proteínas de Plantas/genética , Empalme del ARN , Setaria (Planta)/genética , Zea mays/genética , Proteínas de Cloroplastos/genética , Intrones , Mutación , Proteínas de Plantas/metabolismo , Biosíntesis de Proteínas , Dominios Proteicos , ARN de TransferenciaRESUMEN
The efficiencies offered by C4 photosynthesis have motivated efforts to understand its biochemical, genetic, and developmental basis. Reactions underlying C4 traits in most C4 plants are partitioned between two cell types, bundle sheath (BS), and mesophyll (M) cells. RNA-seq has been used to catalog differential gene expression in BS and M cells in maize (Zea mays) and several other C4 species. However, the contribution of translational control to maintaining the distinct proteomes of BS and M cells has not been addressed. In this study, we used ribosome profiling and RNA-seq to describe translatomes, translational efficiencies, and microRNA abundance in BS- and M-enriched fractions of maize seedling leaves. A conservative interpretation of our data revealed 182 genes exhibiting cell type-dependent differences in translational efficiency, 31 of which encode proteins with core roles in C4 photosynthesis. Our results suggest that non-AUG start codons are used preferentially in upstream open reading frames of BS cells, revealed mRNA sequence motifs that correlate with cell type-dependent translation, and identified potential translational regulators that are differentially expressed. In addition, our data expand the set of genes known to be differentially expressed in BS and M cells, including genes encoding transcription factors and microRNAs. These data add to the resources for understanding the evolutionary and developmental basis of C4 photosynthesis and for its engineering into C3 crops.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Expresión Génica , Células del Mesófilo/metabolismo , Haz Vascular de Plantas/metabolismo , Ribosomas/metabolismo , Zea mays/genética , Hojas de la Planta/metabolismo , Zea mays/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins bind RNA via a mechanism that facilitates the customization of sequence specificity. However, natural PPR proteins have irregular features that limit the degree to which their specificity can be predicted and customized. We demonstrate here that artificial PPR proteins built from consensus PPR motifs selectively bind the intended RNA in vivo, and we use this property to develop a new tool for ribonucleoprotein characterization. We show by RNA coimmunoprecipitation sequencing (RIP-seq) that artificial PPR proteins designed to bind the Arabidopsis (Arabidopsis thaliana) chloroplast psbA mRNA bind with high specificity to psbA mRNA in vivo. Analysis of coimmunoprecipitating proteins by mass spectrometry showed the psbA translational activator HCF173 and two RNA binding proteins of unknown function (CP33C and SRRP1) to be highly enriched. RIP-seq revealed that these proteins are bound primarily to psbA RNA in vivo, and precise mapping of the HCF173 and CP33C binding sites placed them in different locations on psbA mRNA. These results demonstrate that artificial PPR proteins can be tailored to bind specific endogenous RNAs in vivo, add to the toolkit for characterizing native ribonucleoproteins, and open the door to other applications that rely on the ability to target a protein to a specified RNA sequence.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ARN de Planta/metabolismo , Ribonucleoproteínas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sitios de Unión , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , Ribonucleoproteínas/genéticaRESUMEN
A key characteristic of chloroplast gene expression is the predominance of posttranscriptional control via numerous nucleus-encoded RNA binding factors. Here, we explored the essential roles of the S1-domain-containing protein photosynthetic electron transfer B (petB)/ petD Stabilizing Factor (BSF) in the stabilization and translation of chloroplast mRNAs. BSF binds to the intergenic region of petB-petD, thereby stabilizing 3' processed petB transcripts and stimulating petD translation. BSF also binds to the 5' untranslated region of petA and activates its translation. BSF displayed nucleic-acid-melting activity in vitro, and its absence induces structural changes to target RNAs in vivo, suggesting that BSF functions as an RNA chaperone to remodel RNA structure. BSF physically interacts with the pentatricopeptide repeat protein Chloroplast RNA Processing 1 (AtCRP1) and the ribosomal release factor-like protein Peptide chain Release Factor 3 (PrfB3), whose established RNA ligands overlap with those of BSF. In addition, PrfB3 stimulated the RNA binding ability of BSF in vitro. We propose that BSF and PrfB3 cooperatively reduce the formation of secondary RNA structures within target mRNAs and facilitate AtCRP1 binding. The translation activation function of BSF for petD is conserved in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays), but that for petA operates specifically in Arabidopsis. Our study sheds light on the mechanisms by which RNA binding proteins cooperatively regulate mRNA stability and translation in chloroplasts.
Asunto(s)
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Estabilidad del ARN/fisiología , Zea mays/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidad del ARN/genética , Zea mays/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1007555.].
RESUMEN
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
Chloroplast gene expression involves the participation of hundreds of pentatricopeptide repeat (PPR) RNA binding proteins, and proteins in the PLS subfamily typically specify sites of RNA editing, whereas those in the P-subfamily typically stabilize RNA, activate translation, or promote intron splicing. Several P-type PPR proteins include a small MutS-related (SMR) domain, but the biochemical contribution of the SMR domain remains enigmatic. Here, we describe a rice (Oryza sativa) mutant, osatp4, lacking the ortholog of ATP4, a PPR-SMR protein in maize (Zea mays). osatp4 mutants were chlorotic and had a plastid-ribosome deficiency when grown in the cold. Like maize ATP4, OsATP4 was required for the accumulation of dicistronic rpl16-rpl14 transcripts. Surprisingly, OsATP4 was also required for the editing of a specific nucleotide in the ribosomal protein S8 transcripts, rps8, and this function was conserved in maize. By contrast, rps8 RNA was edited normally in the maize PROTON gradient regulation3 mutant, pgr3, which also lacks rpl16-rpl14 transcripts, indicating that the editing defect in atp4 mutants is not a secondary effect of altered rpl16-rpl14 RNA metabolism. Expression of the edited rps8 isoform in transgenic osatp4 mutants complemented the cold-sensitive phenotype, indicating that a rps8 expression defect accounts for the cold-sensitivity. We suggest that ATP4 stimulates rps8 editing by facilitating access of a previously characterized PLS-type RNA editing factor to its cognate cis-element upstream of the edited nucleotide.
Asunto(s)
Cloroplastos/genética , Productos Agrícolas/genética , Oryza/genética , Edición de ARN , ARN Mensajero/genética , ARN de Planta/genética , Zea mays/genética , Regulación de la Expresión Génica de las Plantas , Variación Genética , Genotipo , Mutación , Plantas Modificadas GenéticamenteRESUMEN
Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The psbA mRNA exhibits a dramatic gain of ribosomes within minutes after shifting plants to the light and reverts to low ribosome occupancy within one hour in the dark, correlating with the need to replace damaged PsbA in Photosystem II. (ii) Ribosome occupancy on all other chloroplast mRNAs remains similar to that at midday even after 12 hours in the dark. (iii) Analysis of ribosome dynamics in the presence of lincomycin revealed a global decrease in the translation elongation rate shortly after shifting plants to the dark. The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Luz , Complejo de Proteína del Fotosistema II/metabolismo , Ribosomas/efectos de la radiación , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Cloroplastos/genética , Lincomicina/farmacología , Sistemas de Lectura Abierta , Fotosíntesis , Complejo de Proteína del Fotosistema II/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Biosíntesis de Proteínas , ARN de Planta/genética , Ribosomas/metabolismo , Análisis de Secuencia de ARN , Zea mays/genéticaRESUMEN
The expression of chloroplast genes relies on a host of nucleus-encoded proteins. Identification of such proteins and elucidation of their functions are ongoing challenges. We used ribosome profiling to revisit the function of the pentatricopeptide repeat protein LPE1, reported to stimulate translation of the chloroplast psbA mRNA in Arabidopsis. Mutation of the maize LPE1 ortholog causes a photosystem II (PSII) deficiency and a defect in translation of the chloroplast psbJ open reading frame (ORF) but has no effect on psbA expression. To reflect this function, we named the maize LPE1 ortholog Translation of psbJ 1 (TPJ1). Arabidopsis lpe1 mutants likewise exhibit a loss of psbJ translation, and have, in addition, a decrease in psbN translation. We detected a small decrease in ribosome occupancy on the psbA mRNA in Arabidopsis lpe1 mutants, but ribosome profiling analyses of other PSII mutants (hcf107 and hcf173) in conjunction with in vitro RNA binding data strongly suggest that this is a secondary effect of their PSII deficiency. We conclude that maize TPJ1 promotes PSII synthesis by activating translation of the psbJ ORF, that this function is conserved in Arabidopsis LPE1, and that an additional role for LPE1 in psbN translation contributes to the PSII deficiency in lpe1 mutants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ARN del Cloroplasto/metabolismo , Zea mays/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Sistemas de Lectura Abierta/genética , Complejo de Proteína del Fotosistema II/metabolismo , ARN de Planta/metabolismo , Ribosomas/metabolismoRESUMEN
Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that offer a promising scaffold for the engineering of proteins to bind specified RNAs. PPR tracts bind RNA in a modular 1-repeat, 1-nucleotide fashion. An amino acid code specifying the bound nucleotide has been elucidated. However, this code does not fully explain the sequence specificity of native PPR proteins. Furthermore, it does not address nuances such as the contribution toward binding affinity of various repeat-nucleotide pairs or the impact of mismatches between a repeat and aligning nucleotide. We used an in vitro bind-n-seq approach to describe the population of sequences bound by four artificial PPR proteins built from consensus scaffolds. The specificity of these proteins can be accounted for by canonical code-based nucleotide recognition. The results show, however, that interactions near the 3'-end of binding sites make less contribution to binding affinity than do those near the 5'-end, that proteins with 11 and 14 repeats exhibit similar affinity for their intended targets but 14-repeats are more permissive for mismatches, and that purine-binding repeats are less tolerant of transversion mismatches than are pyrimidine-binding motifs. These findings have implications for mechanisms that establish PPR-RNA interactions and for optimizing PPR design to minimize off-target interactions.
Asunto(s)
Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/química , ARN/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Unión Proteica , Ingeniería de Proteínas , Proteínas de Unión al ARN/genética , Secuencias Repetitivas de AminoácidoRESUMEN
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
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 comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat-one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR-RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of â¼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10-RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR-RNA interactions that pose challenges for binding site prediction and design.