GENOMES UNCOUPLED PROTEIN1 binds to plastid RNAs and promotes their maturation.

Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development. GENOMES UNCOUPLED1 (GUN1) plays a central role in retrograde signaling during early plant development. The putative function of GUN1 has been extensively studied, but its molecular function remains controversial. Here, we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling-relevant conditions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling. Our study of the plastid (post)transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome. By combining this result with a pentatricopeptide repeat code-based prediction and experimental validation by RNA immunoprecipitation experiments, we identified several putative targets of GUN1, including tRNAs and RNAs derived from ycf1.2, rpoC1, and rpoC2 and the ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD gene cluster. The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants account for the cotyledon phenotype. Our study provides evidence for RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies. We anticipate that our findings will serve as a basis for subsequent studies on mechanisms of plastid gene expression and will help to elucidate the function of GUN1 in retrograde signaling.

MatK impacts differential chloroplast translation by limiting spliced tRNA-K(UUU) abundance.

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.

A prion-like domain is required for phase separation and chloroplast RNA processing during cold acclimation in Arabidopsis.

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.

Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes.

The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression.

A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts.

Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. The objective is to demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.

Plant organellar RNA maturation.

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.

Identification of a Hydroxygallic Acid Derivative, Zingibroside R1 and a Sterol Lipid as Potential Active Ingredients of Cuscuta chinensis Extract That Has Neuroprotective and Antioxidant Effects in Aged Caenorhabditis elegans.

We examined the effects of the extracts from two traditional Chinese medicine plants, Cuscuta chinensis and Eucommia ulmoides, on the healthspan of the model organism Caenorhabditis elegans. C. chinensis increased the short-term memory and the mechanosensory response of aged C. elegans. Furthermore, both extracts improved the resistance towards oxidative stress, and decreased the intracellular level of reactive oxygen species. Chemical analyses of the extracts revealed the presence of several bioactive compounds such as chlorogenic acid, cinnamic acid, and quercetin. A fraction from the C. chinensis extract enriched in zingibroside R1 improved the lifespan, the survival after heat stress, and the locomotion in a manner similar to the full C. chinensis extract. Thus, zingibroside R1 could be (partly) responsible for the observed health benefits of C. chinensis. Furthermore, a hydroxygallic acid derivative and the sterol lipid 4-alpha-formyl-stigmasta-7,24(241)-dien-3-beta-ol are abundantly present in the C. chinensis extract and its most bioactive fraction, but hardly in E. ulmoides, making them good candidates to explain the overall healthspan benefits of C. chinensis compared to the specific positive effects on stress resistance by E. ulmoides. Our findings highlight the overall anti-aging effects of C. chinensis in C. elegans and provide first hints about the components responsible for these effects.

Identification of healthspan-promoting genes in Caenorhabditis elegans based on a human GWAS study.

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.

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development.

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

Genes implicated in Caenorhabditis elegans and human health regulate stress resistance and physical abilities in aged Caenorhabditis elegans.

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.

The acidic domain of the chloroplast RNA-binding protein CP31A supports cold tolerance in Arabidopsis thaliana.

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.

Modeling indicates degradation of mRNA and protein as a potential regulation mechanisms during cold acclimation.

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.

Enhanced Healthspan in Caenorhabditis elegans Treated With Extracts From the Traditional Chinese Medicine Plants Cuscuta chinensis Lam. and Eucommia ulmoides Oliv.

To uncover potential anti-aging capacities of Traditional Chinese Medicine (TCM), the nematode Caenorhabditis elegans was used to investigate the effects of Eucommia ulmoides and Cuscuta chinensis extracts, selected by screening seven TCM extracts, on different healthspan parameters. Nematodes exposed to E. ulmoides and C. chinensis extracts, starting at the young adult stage, exhibited prolonged lifespan and increased survival after heat stress as well as upon exposure to the pathogenic bacterium Photorhabdus luminescens, whereby the survival benefits were monitored after stress initiation at different adult stages. However, only C. chinensis had the ability to enhance physical fitness: the swimming behavior and the pharyngeal pumping rate of C. elegans were improved at day 7 and especially at day 12 of adulthood. Finally, monitoring the red fluorescence of aged worms revealed that only C. chinensis extracts caused suppression of intestinal autofluorescence, a known marker of aging. The results underline the different modes of action of the tested plants extracts. E. ulmoides improved specifically the physiological fitness by increasing the survival probability of C. elegans after stress, while C. chinensis seems to be an overall healthspan enhancer, reflected in the suppressed autofluorescence, with beneficial effects on physical as well as physiological fitness. The C. chinensis effects may be hormetic: this is supported by increased gene expression of hsp-16.1 and by trend, also of hsp-12.6.

Acclimation in plants - the Green Hub consortium.

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.

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

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.

Healthspan Enhancement by Olive Polyphenols in C. elegans Wild Type and Parkinson's Models.

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.

Healthspan Maintenance and Prevention of Parkinson's-like Phenotypes with Hydroxytyrosol and Oleuropein Aglycone in C. elegans.

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.

The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA.

Chloroplast RNAs are stabilized and processed by a multitude of nuclear-encoded RNA-binding proteins, often in response to external stimuli like light and temperature. A particularly interesting RNA-based regulation occurs with the psbA mRNA, which shows light-dependent translation. Recently, the chloroplast ribonucleoprotein CP33B was identified as a ligand of the psbA mRNA. We here characterized the interaction of CP33B with chloroplast RNAs in greater detail using a combination of RIP-chip, quantitative dot-blot, and RNA-Bind-n-Seq experiments. We demonstrate that CP33B prefers psbA over all other chloroplast RNAs and associates with the vast majority of the psbA transcript pool. The RNA sequence target motif, determined in vitro, does not fully explain CP33B's preference for psbA, suggesting that there are other determinants of specificity in vivo.

Metabolic Labeling of RNAs Uncovers Hidden Features and Dynamics of the Arabidopsis Transcriptome.

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.

Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts.

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.