A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

Hibernating brown bears are protected against atherogenic dyslipidemia.

To investigate mechanisms by which hibernators avoid atherogenic hyperlipidemia during hibernation, we assessed lipoprotein and cholesterol metabolisms of free-ranging Scandinavian brown bears (Ursus arctos). In winter- and summer-captured bears, we measured lipoprotein sizes and sub-classes, triglyceride-related plasma-enzyme activities, and muscle lipid composition along with plasma-levels of antioxidant capacities and inflammatory markers. Although hibernating bears increased nearly all lipid levels, a 36%-higher cholesteryl-ester transfer-protein activity allowed to stabilize lipid composition of high-density lipoproteins (HDL). Levels of inflammatory metabolites, i.e., 7-ketocholesterol and 11ß-prostaglandin F2α, declined in winter and correlated inversely with cardioprotective HDL2b-proportions and HDL-sizes that increased during hibernation. Lower muscle-cholesterol concentrations and lecithin-cholesterol acyltransferase activity in winter suggest that hibernating bears tightly controlled peripheral-cholesterol synthesis and/or release. Finally, greater plasma-antioxidant capacities prevented excessive lipid-specific oxidative damages in plasma and muscles of hibernating bears. Hence, the brown bear manages large lipid fluxes during hibernation, without developing adverse atherogenic effects that occur in humans and non-hibernators.

Synthetic biological circuit tested in spaceflight.

Synthetic biology has potential spaceflight applications yet few if any studies have attempted to translate Earth-based synthetic biology tools into spaceflight. An exogenously inducible biological circuit for protein production in Arabidopsis thaliana, pX7-AtPDSi (Guo et al. 2003), was flown to ISS and functionally investigated. Seedlings were grown in a custom built 1.25 U plant greenhouse. Images recorded during the experiment show that leaves of pX7-AtPDSi seedlings photobleached as designed while wild type Col-0 leaves did not, which reveals that the synthetic circuit led to protein production during spaceflight. Polymerase chain reaction analysis post-flight also confirms that the Cre/LoxP (recombination system) portions of the circuit were functional in spaceflight. The subcomponents of the biological circuit, estrogen-responsive transcription factor XVE, Cre/LoxP DNA recombination system, and RNAi post-transcriptional gene silencing system now have flight heritage and can be incorporated in future designs for space applications. To facilitate future plant studies in space, the full payload design and manufacturing files are made available.

Towards plant resistance to viruses using protein-only RNase P.

Plant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5' maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

Determination of protein-only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease.

The essential type of endonuclease that removes 5' leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called 'PRORP', for 'PROtein-only RNase P', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.

Antioxidant capacity is repeatable across years but does not consistently correlate with a marker of peroxidation in a free-living passerine bird.

Oxidative stress occurs when reactive oxygen species (ROS) exceed antioxidant defences, which can have deleterious effects on cell function, health and survival. Therefore, organisms are expected to finely regulate pro-oxidant and antioxidant processes. ROS are mainly produced through aerobic metabolism and vary in response to changes in energetic requirements, whereas antioxidants may be enhanced, depleted or show no changes in response to changes in ROS levels. We investigated the repeatability, within-individual variation and correlation across different conditions of two plasmatic markers of the oxidative balance in 1108 samples from 635 free-living adult collared flycatchers (Ficedula albicollis). We sought to manipulate energy constraints by increasing wing load in 2012 and 2013 and by providing additional food in 2014. We then tested the relative importance of within- and between-individual variation on reactive oxygen metabolites (ROMs), a marker of lipid and protein peroxidation, and on non-enzymatic antioxidant defences (OXY test). We also investigated whether the experimental treatments modified the correlation between markers. Antioxidant defences were repeatable (range of repeatability estimates = 0.128-0.581), whereas ROMs were not (0-0.061). Antioxidants varied neither between incubation and nestling feeding nor between sexes. ROMs increased from incubation to nestling feeding in females and were higher in females than males. Antioxidant defences and ROM concentration were globally positively correlated, but the correlation varied between experimental conditions and between years. Hence, the management of oxidative balance in wild animals appears flexible under variable environmental conditions, an observation which should be confirmed over a wider range of markers.

Small is big in Arabidopsis mitochondrial ribosome.

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.

Differences in the oxidative balance of dispersing and non-dispersing individuals: an experimental approach in a passerine bird.

BACKGROUND: Dispersal is often associated with a suite of phenotypic traits that might reduce dispersal costs, but can be energetically costly themselves outside dispersal. Hence, dispersing and philopatric individuals might differ throughout their life cycle in their management of energy production. Because higher energy expenditure can lead to the production of highly reactive oxidative molecules that are deleterious to the organism if left uncontrolled, dispersing and philopatric individuals might differ in their management of oxidative balance. Here, we experimentally increased flight costs during reproduction via a wing load manipulation in female collared flycatchers (Ficedula albicollis) breeding in a patchy population. We measured the effects of the manipulation on plasmatic markers of oxidative balance and reproductive success in dispersing and philopatric females. RESULTS: The impact of the wing load manipulation on the oxidative balance differed according to dispersal status. The concentration of reactive oxygen metabolites (ROMs), a marker of pro-oxidant status, was higher in philopatric than dispersing females in the manipulated group only. Differences between dispersing and philopatric individuals also depended on habitat quality, as measured by local breeding density. In low quality habitats, ROMs as well as nestling body mass were higher in philopatric females compared to dispersing ones. Independently of the manipulation or of habitat quality, plasma antioxidant capacity differed according to dispersal status: philopatric females showed higher antioxidant capacity than dispersing ones. Nestlings raised by philopatric females also had a higher fledging success. CONCLUSIONS: Our results suggest that dispersing individuals maintain a stable oxidative balance when facing challenging environmental conditions, at the cost of lower reproductive success. Conversely, philopatric individuals increase their effort, and thus oxidative costs, in challenging conditions thereby maintaining their reproductive success. Our study sheds light on energetics and oxidative balance as possible processes underlying phenotypic differences between dispersing and philopatric individuals.

Transfer RNA maturation in Chlamydomonas mitochondria, chloroplast and the nucleus by a single RNase P protein.

The maturation of tRNA precursors involves the 5' cleavage of leader sequences by an essential endonuclease called RNase P. Beyond the ancestral ribonucleoprotein (RNP) RNase P, a second type of RNase P called PRORP (protein-only RNase P) evolved in eukaryotes. The current view on the distribution of RNase P in cells is that multiple RNPs, multiple PRORPs or a combination of both, perform specialised RNase P activities in the different compartments where gene expression occurs. Here, we identify a single gene encoding PRORP in the green alga Chlamydomonas reinhardtii while no RNP is found. We show that its product, CrPRORP, is triple-localised to mitochondria, the chloroplast and the nucleus. Its downregulation results in impaired tRNA biogenesis in both organelles and the nucleus. CrPRORP, as a single-subunit RNase P for an entire organism, makes up the most compact and versatile RNase P machinery described in either prokaryotes or eukaryotes.

Circadian desynchronization triggers premature cellular aging in a diurnal rodent.

Chronic jet lag or shift work is deleterious to human metabolic health, in that such circadian desynchronization is associated with being overweight and the prevalence of altered glucose metabolism. Similar metabolic changes are observed with age, suggesting that chronic jet lag and accelerated cell aging are intimately related, but the association remains to be determined. We addressed whether jet lag induces metabolic and cell aging impairments in young grass rats (2-3 mo old), using control old grass rats (12-18 mo old) as an aging reference. Desynchronized young and control old subjects had impaired glucose tolerance (+60 and +280%) when compared with control young animals. Despite no significant variation in liver DNA damage, shorter telomeres were characterized, not only in old animal liver cells (-18%), but also at an intermediate level in desynchronized young rats (-9%). The same pattern was found for deacetylase sirtuin (SIRT)-1 (-57 and -29%), confirming that jet-lagged young rats have an intermediate aging profile. Our data indicate that an experimental circadian desynchronization in young animals is associated with a precocious aging profile based on 3 well-known markers, as well as a prediabetic phenotype. Such chronic jet lag-induced alterations observed in a diurnal species constitute proof of principle of the need to develop preventive treatments in jet-lagged persons and shift workers.

Immediate and delayed effects of growth conditions on ageing parameters in nestling zebra finches.

Conditions experienced during development and growth are of crucial importance as they can have a significant influence on the optimisation of life histories. Indeed, the ability of an organism to grow fast and achieve a large body size often confers short- and long-term fitness benefits. However, there is good evidence that organisms do not grow at their maximal rates as growth rates seem to have potential costs on subsequent lifespan. There are several potential proximate causes of such a reduced lifespan. Among them, one emerging hypothesis is that growth impacts adult survival and/or longevity through a shared, end point, ageing mechanism: telomere erosion. In this study, we manipulated brood size in order to investigate whether rapid growth (chicks in reduced broods) is effectively done at the cost of a short- (end of growth) and long-term (at adulthood) increase of oxidative damage and telomere loss. Contrary to what we expected, chicks from the enlarged broods displayed more oxidative damage and had shorter telomeres at the end of the growth period and at adulthood. Our study extends the understanding of the proximate mechanisms involved in the trade-off between growth and ageing. It highlights that adverse environmental conditions during growth can come at a cost via transient increased oxidative stress and pervasive eroded telomeres. Indeed, it suggests that telomeres are not only controlled by intrinsic growth rates per se but also may be under the control of some extrinsic environmental factors, which could complicate our understanding of the growth-ageing interaction.

Leptin levels in free ranging striped mice (Rhabdomys pumilio) increase when food decreases: the ecological leptin hypothesis.

Leptin is a hormone informing the body about its fat stores, reducing appetite and foraging and as such reducing fattening of individuals. In laboratory rodents, leptin secretion is highly correlated to the amount of adipose tissue. We compared this to the alternative ecological leptin hypothesis, which based on the behavioural effects of leptin predicts that leptin levels are disassociated from adipose tissue when fattening is of evolutionary advantage to survive coming periods of low food availability. Studying a species that has to survive a dry season with low food availability, we tested the ecological leptin hypothesis, predicting low leptin levels when food availability and thus adiposity is high promoting foraging and fattening, but high leptin levels in the seasons of low food availability, reducing energetic costs due to foraging. We measured leptin levels in 154 samples of free living African striped mice (Rhabdomys pumilio). Striped mice gain significant body mass during the moist season to survive the following dry season with low food availability. We found a strong seasonal effect, with higher leptin levels in the dry season with low food availability, which was in contrast to the hypothesis deriving from studies on laboratory rodents, but in agreement with ecological leptin hypothesis: leptin levels remained low in the period of high food availability, allowing fattening, but increased during periods of low food availability, possibly suppressing energetically costly foraging in an environment where foraging success would have been very low. Leptin correlated significantly and negatively with testosterone levels, and high testosterone levels in the moist season could explain why leptin levels were low even though food availability was high. However, analysing samples from an experimental laboratory study where testosterone levels were increased via implants found no support for a suppressive role of testosterone on leptin. In sum, our study indicates that in a species with seasonal fattening, leptin levels might be uncoupled from the amount of adipose tissue.

Elevation impacts the balance between growth and oxidative stress in coal tits.

The short favorable period of time available for the growth in seasonal environments could constrain the resources allocation between growth and other life-history traits, and the short-term fitness benefits of increased growth rate may prevail over other functions. Accelerated growth rates have been associated with long-term deleterious consequences (e.g., decreased lifespan), and recently oxidative stress (the imbalance between pro-oxidants generation and antioxidant defenses) has been suggested as a mediator of these effects. Here, we examined the impact of elevation on growth rate and self-maintenance parameters (resting metabolism, oxidative damage, and antioxidant defenses) of coal tit chicks (Periparus ater). We predicted that the shorter favorable season at the higher-elevation site could lead to a reallocation of resources towards growth at the expense of self-maintenance processes. We found that chicks at high elevation grew significantly faster in terms of body mass and body size. Chicks from the high-elevation site presented higher resting metabolism, higher oxidative damage level, but similar antioxidant defenses, compared to low-elevation chicks. Interestingly, the chicks exhibiting the better antioxidant defenses at 7 days were also those with the highest resting metabolic rate, and the chicks that grew at the faster rate within the high-elevation site were those with the highest levels of oxidative damage on DNA. Our study supports the idea that increasing elevation leads to a higher growth rate in coal tit chicks, possibly in response to a shorter favorable season. In accordance with life-history theory, a bigger investment in growth was done at the expense of body maintenance, at least in terms of oxidative stress.

Experimental increase in telomere length leads to faster feather regeneration.

Telomeres - the protective ends of linear chromosomes - reveal themselves not only as a good proxy in terms of longevity, but more recently also as a marker of healthy ageing in laboratory rodents. Telomere erosion is prevented by the activation of telomerase, an enzyme suspected to be also vital for tissue regeneration and which experimental activation improves health state in mice. One emerging hypothesis is that telomerase activity accounts for the frequently reported positive links between telomere lengths and individual quality in a wide range of organisms. Still, we lack an experimental approach testing the exact impact of inter-individual differences in telomere length on individual trait variability. In a first step study, we tested the impact of the TA-65, a plant-derived product stimulating the expression and the activity of telomerase, on telomere lengths and flight feather renewal capacity of captive zebra finches (Taenopygia guttata). Telomere length was longer in TA-65 treated finches while their feather grew faster than in controls. Our data support the idea that long telomeres could reflect high telomerase activity, and in so doing be a good predictor of greater telomerase-dependent tissue regeneration, which may ultimately explain variation in organism quality and longevity.

Genetic compensation in a human genomic disorder.

Cytogenetic studies of the parents of a girl with the DiGeorge (or velocardiofacial) syndrome, who carried a deletion at 22q11.2, revealed an unexpected rearrangement of both 22q11.2 regions in the unaffected father. He carried a 22q11.2 deletion on one copy of chromosome 22 and a reciprocal 22q11.2 duplication on the other copy of chromosome 22. Genetic compensation, which is consistent with the normal phenotype of the father, was shown through quantitative-expression analyses of genes located within the genetic region associated with the DiGeorge syndrome. This finding has implications for genetic counseling and represents a case of genetic compensation in a human genomic disorder.

Temporal analysis of French Bordetella pertussis isolates by comparative whole-genome hybridization.

Bordetella pertussis, a gram-negative beta-proteobacterium, is the agent of whooping cough in humans. Whooping cough remains a public health problem worldwide, despite well-implemented infant/child vaccination programs. It continues to be endemic and is observed cyclically in vaccinated populations. Classical molecular subtyping methods indicate that genome diversity among B. pertussis isolates is limited. Although the whole bacterial genome has been studied by pulsed-field gel electrophoresis, the genes implicated in the diversity have not been identified. We developed a B. pertussis whole-genome DNA microarray representing over 91% of the predicted coding sequences of the sequenced strain Tohama I. Genomic DNA from clinical isolates with various pulsed-field gel electrophoresis profile patterns was competitively hybridized with the DNA microarray and coding sequences were classified as present, absent or duplicated. Our data strongly suggest that the B. pertussis population is dynamic. In France, with highly vaccinated population, the genetic diversity is low and decreasing with time, and clonal expansion correlates with cycles of the disease. This decrease in diversity is essentially due to loss of genes and pseudogenes. The genes deleted are most of the time flanked by insertion sequences.