ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Zea mays/genetics , Zea mays/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant/genetics , Regulon , Chloroplasts/genetics , Chloroplasts/metabolism , Plastids/metabolism , Photosynthesis/genetics , Arabidopsis Proteins/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation, Plant , Gene Expression , Mesophyll Cells/metabolism , Plant Vascular Bundle/metabolism , Ribosomes/metabolism , Zea mays/genetics , Plant Leaves/metabolism , Zea mays/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chloroplasts/metabolism , Protein Engineering/methods , RNA, Chloroplast/metabolism , RNA-Binding Motifs/genetics , 5' Untranslated Regions , Arabidopsis/genetics , Chloroplasts/genetics , Gene Expression , Plants, Genetically Modified , Protein Binding , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins , Ribulose-Bisphosphate Carboxylase/geneticsABSTRACT
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.
Subject(s)
Chloroplasts/genetics , Plant Proteins/genetics , RNA Splicing , Setaria Plant/genetics , Zea mays/genetics , Chloroplast Proteins/genetics , Introns , Mutation , Plant Proteins/metabolism , Protein Biosynthesis , Protein Domains , RNA, TransferABSTRACT
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.
Subject(s)
Chloroplasts/genetics , Crops, Agricultural/genetics , Oryza/genetics , RNA Editing , RNA, Messenger/genetics , RNA, Plant/genetics , Zea mays/genetics , Gene Expression Regulation, Plant , Genetic Variation , Genotype , Mutation , Plants, Genetically ModifiedABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Photosystem II Protein Complex/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Chlorophyll Binding Proteins/metabolism , Chloroplasts/metabolism , Eukaryotic Initiation Factors/metabolism , Light , Membrane Proteins/metabolism , Photosynthesis , Photosystem II Protein Complex/genetics , Plants/genetics , Protein Biosynthesis/physiology , Protein Processing, Post-Translational/physiology , RNA, Messenger/metabolism , Ribosomes/metabolism , Transcription, Genetic , Zea mays/genetics , Zea mays/metabolismABSTRACT
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.
Subject(s)
Endodeoxyribonucleases/metabolism , Homologous Recombination , Meiosis , Zea mays/cytology , Zea mays/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosome Pairing , DNA Breaks, Double-Stranded , Endodeoxyribonucleases/genetics , Genes, Plant/genetics , Meiosis/genetics , Mutation , Phenotype , Zea mays/geneticsABSTRACT
Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and in response to stress. The cyanobacterial ribosome hibernation factor LrtA has been suggested to inactivate ribosomes in the dark and to be important for post-stress survival. In this study, we addressed the hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), the chloroplast-localized LrtA homolog in plants, contributes to the global repression of chloroplast translation that occurs when plants are shifted from light to dark. We found that the abundance of PSRP1 and its association with ribosomes were similar in the light and the dark. Maize mutants lacking PSRP1 were phenotypically normal under standard laboratory growth conditions. Furthermore, the absence of PSRP1 did not alter the distribution of chloroplast ribosomes among monosomes and polysomes in the light or in the dark, and did not affect the light-regulated synthesis of the chloroplast psbA gene product. These results suggest that PSRP1 does not play a significant role in the regulation of chloroplast translation by light. As such, the physiological driving force for the retention of PSRP1 during chloroplast evolution remains unclear.
ABSTRACT
Photosystem II (PSII) in chloroplasts and cyanobacteria contains approximately fifteen core proteins, which organize numerous pigments and prosthetic groups that mediate the light-driven water-splitting activity that drives oxygenic photosynthesis. The PSII reaction center protein D1 is subject to photodamage, whose repair requires degradation of damaged D1 and its replacement with nascent D1. Mechanisms that couple D1 synthesis with PSII assembly and repair are poorly understood. We address this question by using ribosome profiling to analyze the translation of chloroplast mRNAs in maize and Arabidopsis mutants with defects in PSII assembly. We found that OHP1, OHP2, and HCF244, which comprise a recently elucidated complex involved in PSII assembly and repair, are each required for the recruitment of ribosomes to psbA mRNA, which encodes D1. By contrast, HCF136, which acts upstream of the OHP1/OHP2/HCF244 complex during PSII assembly, does not have this effect. The fact that the OHP1/OHP2/HCF244 complex brings D1 into proximity with three proteins with dual roles in PSII assembly and psbA ribosome recruitment suggests that this complex is the hub of a translational autoregulatory mechanism that coordinates D1 synthesis with need for nascent D1 during PSII biogenesis and repair.
ABSTRACT
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.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Photosystem II Protein Complex/metabolism , Proteome , RNA-Binding Proteins/metabolism , Arabidopsis/genetics , Chloroplasts/genetics , Chloroplasts/metabolism , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Photosystem II Protein Complex/genetics , Plant Leaves/genetics , Plant Leaves/metabolism , RNA, Messenger/genetics , RNA, Plant/genetics , RNA-Binding Proteins/genetics , Ribosomes/metabolismABSTRACT
The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.
Subject(s)
Arabidopsis Proteins/genetics , Chloroplasts/genetics , Gene Expression Regulation, Plant/genetics , Genes, Switch/genetics , Protein Engineering , Transgenes/genetics , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Protein Engineering/methods , RNA-Binding Proteins/geneticsABSTRACT
Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.
Subject(s)
Plant Proteins/genetics , Plastids/genetics , Protein Engineering/methods , RNA-Binding Proteins/genetics , Transgenes/genetics , Gene Expression Regulation, Plant/genetics , Green Fluorescent Proteins/genetics , Plant Leaves/metabolism , Plant Proteins/metabolism , Solanum tuberosum/genetics , Zea mays/genetics , Zea mays/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , RNA, Plant/metabolism , Ribonucleoproteins/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Binding Sites , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , Ribonucleoproteins/geneticsABSTRACT
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.
Subject(s)
Arabidopsis/metabolism , Chloroplasts/metabolism , RNA Stability/physiology , Zea mays/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , RNA Stability/genetics , Zea mays/geneticsABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , RNA, Chloroplast/metabolism , Zea mays/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Open Reading Frames/genetics , Photosystem II Protein Complex/metabolism , RNA, Plant/metabolism , Ribosomes/metabolismABSTRACT
[This corrects the article DOI: 10.1371/journal.pgen.1007555.].
ABSTRACT
Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA sequences via modular 1-repeat:1-nucleotide interactions. Binding specificity is dictated, in part, by hydrogen bonds between the amino acids at two positions in each PPR motif and the Watson-Crick face of the aligned nucleobase. There is evidence that PPR-RNA interactions can compete with RNA-RNA interactions in vivo, and that this competition underlies some effects of PPR proteins on gene expression. Conversely, RNA secondary structure can inhibit the binding of a PPR protein to its specific binding site. The parameters that influence whether PPR-RNA or RNA-RNA interactions prevail are unknown. Understanding these parameters will be important for understanding the functions of natural PPR proteins and for the design of engineered PPR proteins for synthetic biology purposes. We addressed this question by analyzing the effects of RNA structures of varying stability and position on the binding of the model protein PPR10 to its atpH RNA ligand. Our results show that even very weak RNA structures (ΔG° ~ 0 kcal/mol) involving only one nucleotide at either end of the minimal binding site impede PPR10 binding. Analysis of binding kinetics using Surface Plasmon Resonance showed that RNA structures reduce PPR10's on-rate and increase its off-rate. Complexes between the PPR proteins PPR10 and HCF152 and their respective RNA ligands have long half-lives (one hour or more), correlating with their functions as barriers to exonucleolytic RNA decay in vivo. The effects of salt concentration on PPR10-RNA binding kinetics showed that electrostatic interactions play an important role in establishing PPR10-RNA interactions but play a relatively small role in maintaining specific interactions once established.