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• Pentatricopeptide repeat (PPR) proteins are modular RNA-binding proteins involved in different aspects of RNA metabolism in organelles. PPR proteins of the PLS subclass often contain C-terminal domains that are important for their function, but the role of one of these domains, the E domain, is far from resolved. Here, we elucidate the role of the E domain in CRR2 in plastids. • We identified a surprisingly large number of small RNAs that represent in vivo footprints of the Arabidopsis PLS-class PPR protein CRR2. An unexpectedly strong base conservation was found in the nucleotides aligned to the E domain. We used both in vitro and in vivo experiments to reveal the role of the E domain of CRR2. • The E domain of CRR2 can be predictably altered to prefer different nucleotides in its RNA ligand, and position 5 of the E1-motif is biologically important for the PPR–RNA interaction. The ‘code’ of the E domain PPR motifs is different from that of P- and S-motifs. • The findings presented here show that the E domain of CRR2 is involved in sequence-specific interaction with its RNA ligand and have implications for our ability to predict RNA targets for PLS-PPRs and their use as biotechnological tools to manipulate specific RNAs in vivo.

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.

Bacterial group II introns encode maturase proteins required for splicing. In organelles of photosynthetic land plants, most of the group II introns have lost the reading frames for maturases. Here, we show that the plastidial maturase MatK not only interacts with its encoding intron within trnk-UUU, but also with six additional group II introns, all belonging to intron subclass llA. Mapping analyses of RNA binding sites revealed MatK to recognize multiple regions within the trnK intron. Organellar group ll introns are considered to be the ancestors of nuclear spliceosomal introns. That MatK associates with multiple intron ligands makes it an attractive model for an early trans-acting nuclear splicing activity.

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 .

Molecular chaperones are essential throughout a protein’s life and act already during protein synthesis. Bacteria and chloroplasts of plant cells share the ribosome-associated chaperone trigger factor (Tig1 in plastids), facilitating maturation of emerging nascent polypeptides. While typical trigger factor chaperones employ three domains for their task, the here described truncated form, Tig2, contains just the ribosome binding domain. Tig2 is widely present in green plants and appears to have acquired an entirely different task than co-translational nascent polypeptide folding. Tig2 deletion results in remarkable leaf developmental defects of cold-exposed Arabidopsis thaliana plants and specific defects in plastidic ribosomes. Our data indicate that Tig2 functions during ribosome biogenesis by promoting the maturation of the large subunit. We hypothesize that Tig2 binding to the ribosomal tunnel-exit surface aids protecting this sensitive surface during assembly. Tig2 illustrates a fascinating concept of how a chaperone domain evolved individually, serving a completely different molecular task. This study describes the second, shortened chloroplast variant of the ribosome-associated molecular chaperone termed trigger factor. While lacking chaperone activity, it rather contributes to maturation of the 50S ribosome subunit, particularly during plant cold acclimation.

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.

Plastid gene expression is crucial for organelle function, but the factors that control it are still largely unclear. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate organellar gene expression at different levels. Arabidopsis (Arabidopsis thaliana) mTERF6 is localized in chloroplasts and mitochondria, and its knockout perturbs plastid development and results in seedling lethality. In the leakymterf6-1mutant, a defect in photosynthesis is associated with reduced levels of photosystem subunits, although corresponding messenger RNA levels are unaffected, whereas translational capacity and maturation of chloroplast ribosomal RNAs (rRNAs) are perturbed inmterf6-1mutants. Bacterial one-hybrid screening, electrophoretic mobility shift assays, and coimmunoprecipitation experiments reveal a specific interaction between mTERF6 and an RNA sequence in the chloroplast isoleucine transfer RNA gene (trnI.2) located in the rRNA operon. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. At present, it is unclear whether disturbed rRNA maturation is a primary or secondary defect. However, it is clear that mTERF6 is required for the maturation oftrnI.2. This points to an additional function of mTERFs.

The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half- A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5′ untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5′end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain.

The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown to be defective for rps12 trans-splicing. The observations that PPR4 is associated in vivo with rps12-intron 1 and that it is also required for its splicing demonstrate that PPR4 is an rps12 trans-splicing factor. These findings add trans-splicing to the list of RNA-related functions associated with PPR proteins and suggest that plastid group II trans-splicing is performed by different machineries in vascular plants and algae.

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.