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The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
Spatial genome organization is tightly controlled by several regulatory mechanisms and is essential for gene expression control. Nuclear receptors are ligand-activated transcription factors that modulate physiological and pathophysiological processes and are primary pharmacological targets. DNA binding of the important loop-forming insulator protein CCCTC-binding factor (CTCF) was modulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We performed CTCF HiChIP assays to produce the first genome-wide dataset of CTCF long-range interactions in 1,25(OH)2D3-treated cells, and to determine whether dynamic changes of spatial chromatin interactions are essential for fine-tuning of nuclear receptor signaling. We detected changes in 3D chromatin organization upon vitamin D receptor (VDR) activation at 3.1% of all observed CTCF interactions. VDR binding was enriched at both differential loop anchors and within differential loops. Differential loops were observed in several putative functional roles including TAD border formation, promoter-enhancer looping, and establishment of VDR-responsive insulated neighborhoods. Vitamin D target genes were enriched in differential loops and at their anchors. Secondary vitamin D effects related to dynamic chromatin domain changes were linked to location of downstream transcription factors in differential loops. CRISPR interference and loop anchor deletion experiments confirmed the functional relevance of nuclear receptor ligand-induced adjustments of the chromatin 3D structure for gene expression regulation.
Nonmycorrhizal root-colonizing fungi are key determinants of plant growth, driving processes ranging from pathogenesis to stress alleviation. Evidence suggests that they might also facilitate host access to soil nutrients in a mycorrhiza-like manner, but the extent of their direct contribution to plant nutrition is unknown. To study how widespread such capacity is across root-colonizing fungi, we surveyed soils in nutrient-limiting habitats using plant baits to look for fungal community changes in response to nutrient conditions. We established a fungal culture collection and used Arabidopsis thaliana inoculation bioassays to assess the ability of fungi to facilitate host’s growth in the presence of organic nutrients unavailable to plants. Plant baits captured a representation of fungal communities extant in natural habitats and showed that nutrient limitation has little influence on community assembly. Arabidopsis thaliana inoculated with 31 phylogenetically diverse fungi exhibited a consistent fungus-driven growth promotion when supplied with organic nutrients compared to untreated plants. However, direct phosphorus measurement and RNA-seq data did not support enhanced nutrient uptake but rather that growth effects may result from changes in the plant’s immune response to colonization. The widespread and consistent host responses to fungal colonization suggest that distinct, locally adapted nonmycorrhizal fungi affect plant performance across habitats.
IMPORTANCE: Recent studies have shown that root-associated fungi that do not engage in classical mycorrhizal associations can facilitate the hosts’ access to nutrients in a mycorrhiza-like manner. However, the generality of this capacity remains to be tested. Root-associated fungi are frequently deemed major determinants of plant diversity and performance, but in the vast majority of cases their ecological roles in nature remain unknown. Assessing how these plant symbionts affect plant productivity, diversity, and fitness is important to understanding how plant communities function. Recent years have seen important advances in the understanding of the main drivers of the diversity and structure of plant microbiomes, but a major challenge is still linking community properties with function. This study contributes to the understanding of the cryptic function of root-associated fungi by testing their ability to participate in a specific process: nutrient acquisition by plants.
Kálmán Vánky (15th of June 1930–18th of October 2021) was arguably the most prolific researcher of smut fungi so far. He published more than 1000 taxonomic novelties, and crowned his outstanding oeuvre with the most comprehensive monograph of the smut fungi (Smut Fungi of the World) written to date.
Acetogenic bacteria such as Acetobacterium woodii use the Wood–Ljungdahl pathway (WLP) for fixation of CO2 and energy conservation. This pathway enables conversion of diverse substrates to the main product of acetogenesis, acetate. Methyl group containing substrates such as methanol or methylated compounds, derived from pectin, are abundant in the environment and a source for CO2. Methyl groups enter the WLP at the level of methyltetrahydrofolic acid (methyl-THF). For methyl transfer from methanol to THF a substrate-specific methyltransferase system is required. In this study, we used genetic methods to identify mtaBC2A (Awo_c22760-Awo_c22740) as the methanol-specific methyltransferase system of A. woodii. After methyl transfer, methyl-THF serves as carbon and/or electron source and the respiratory Rnf complex is required for redox homeostasis if methanol + CO2 is the substrate. Resting cells fed with methanol + CO2, indeed converted methanol to acetate in a 4:3 stoichiometry. When methanol was fed in combination with other electron sources such as H2 + CO2 or CO, methanol was converted Rnf-independently and the methyl group was condensed with CO to build acetate. When fed in combination with alternative electron sinks such as caffeate methanol was oxidized only and resulting electrons were used for non-acetogenic growth. These different pathways for the conversion of methyl-group containing substrates enable acetogens to adapt to various ecological niches and to syntrophic communities.
Climate forecasts show that in many regions the temporal distribution of precipitation events will become less predictable. Root traits may play key roles in dealing with changes in precipitation predictability, but their functional plastic responses, including transgenerational processes, are scarcely known. We investigated root trait plasticity of Papaver rhoeas with respect to higher versus lower intra-seasonal and inter-seasonal precipitation predictability (i.e., the degree of temporal autocorrelation among precipitation events) during a four-year outdoor multi-generation experiment. We first tested how the simulated predictability regimes affected intra-generational plasticity of root traits and allocation strategies of the ancestors, and investigated the selective forces acting on them. Second, we exposed three descendant generations to the same predictability regime experienced by their mothers or to a different one. We then investigated whether high inter-generational predictability causes root trait differentiation, whether transgenerational root plasticity existed and whether it was affected by the different predictability treatments. We found that the number of secondary roots, root biomass and root allocation strategies of ancestors were affected by changes in precipitation predictability, in line with intra-generational plasticity. Lower predictability induced a root response, possibly reflecting a fast-acquisitive strategy that increases water absorbance from shallow soil layers. Ancestors’ root traits were generally under selection, and the predictability treatments did neither affect the strength nor the direction of selection. Transgenerational effects were detected in root biomass and root weight ratio (RWR). In presence of lower predictability, descendants significantly reduced RWR compared to ancestors, leading to an increase in performance. This points to a change in root allocation in order to maintain or increase the descendants’ fitness. Moreover, transgenerational plasticity existed in maximum rooting depth and root biomass, and the less predictable treatment promoted the lowest coefficient of variation among descendants’ treatments in five out of six root traits. This shows that the level of maternal predictability determines the variation in the descendants’ responses, and suggests that lower phenotypic plasticity evolves in less predictable environments. Overall, our findings show that roots are functional plastic traits that rapidly respond to differences in precipitation predictability, and that the plasticity and adaptation of root traits may crucially determine how climate change will affect plants.
Alternative splicing (AS) is a major mechanism for gene expression in eukaryotes, increasing proteome diversity but also regulating transcriptome abundance. High temperatures have a strong impact on the splicing profile of many genes and therefore AS is considered as an integral part of heat stress response. While many studies have established a detailed description of the diversity of the RNAome under heat stress in different plant species and stress regimes, little is known on the underlying mechanisms that control this temperature-sensitive process. AS is mainly regulated by the activity of splicing regulators. Changes in the abundance of these proteins through transcription and AS, post-translational modifications and interactions with exonic and intronic cis-elements and core elements of the spliceosomes modulate the outcome of pre-mRNA splicing. As a major part of pre-mRNAs are spliced co-transcriptionally, the chromatin environment along with the RNA polymerase II elongation play a major role in the regulation of pre-mRNA splicing under heat stress conditions. Despite its importance, our understanding on the regulation of heat stress sensitive AS in plants is scarce. In this review, we summarize the current status of knowledge on the regulation of AS in plants under heat stress conditions. We discuss possible implications of different pathways based on results from non-plant systems to provide a perspective for researchers who aim to elucidate the molecular basis of AS under high temperatures.
Understanding how species relate mechanistically to their environment via traits is a central goal in ecology. Many macroecological rules were found for macroorganisms, however, whether they can explain microorganismal macroecological patterns still requires investigation. Further, whether macroecological rules are also applicable in microclimates is largely unexplored. Here we use fruit body-forming fungi to understand both aspects better. A recent study showed first evidence for the thermal-melanism hypothesis (Bogert’s rule) in fruit body-forming fungi and relied on a continental spatial scale with large grid size. At large spatial extent and grid sizes, other factors like dispersal limitation or local microclimatic variability might influence observed patterns besides the rule of interest. Therefore, we test fungal assemblage fruit body color lightness along a local elevational gradient (mean annual temperature gradient of 7°C) while considering the vegetation cover as a proxy for local variability in microclimate. Using multivariate linear modeling, we found that fungal fruiting assemblages are significantly darker at lower mean annual temperatures supporting the thermal-melanism hypothesis. Further, we found a non-significant trend of assemblage color lightness with vegetation cover. Our results support Bogert’s rule for microorganisms with macroclimate, which was also found for macroorganisms.
Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.
The ability of wild animals to navigate and survive in complex and dynamic environments depends on their ability to store relevant information and place it in a spatial context. Despite the centrality of spatial memory, and given our increasing ability to observe animal movements in the wild, it is perhaps surprising how difficult it is to demonstrate spatial memory empirically. We present a cognitive analysis of movements of several wolves (Canis lupus) in Finland during a summer period of intensive hunting and den-centered pup-rearing. We tracked several wolves in the field by visiting nearly all GPS locations outside the den, allowing us to identify the species, location and timing of nearly all prey killed. We then developed a model that assigns a spatially explicit value based on memory of predation success and territorial marking. The framework allows for estimation of multiple cognitive parameters, including temporal and spatial scales of memory. For most wolves, fitted memory-based models outperformed null models by 20 to 50% at predicting locations where wolves chose to forage. However, there was a high amount of individual variability among wolves in strength and even direction of responses to experiences. Some wolves tended to return to locations with recent predation success—following a strategy of foraging site fidelity—while others appeared to prefer a site switching strategy. These differences are possibly explained by variability in pack sizes, numbers of pups, and features of the territories. Our analysis points toward concrete strategies for incorporating spatial memory in the study of animal movements while providing nuanced insights into the behavioral strategies of individual predators.
Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.
The opportunistic human pathogen Acinetobacter baumannii can grow with carnitine but its metabolism, regulation and role in virulence remained elusive. Recently, we identified a carnitine transporter encoded by a gene closely associated with potential carnitine degradation genes. Among those is a gene coding for a putative d-malate dehydrogenase (Mdh). Deletion of the mdh gene led to a loss of growth with carnitine but not l-malate; growth with d-malate was strongly reduced. Therefore, it is hypothesized that d-malate is formed during carnitine oxidation and further oxidized to CO2 and pyruvate and, that not, as previously suggested, l-malate is the product and funnelled directly into the TCA cycle. Mutant analyses revealed that the hydrolase in this cluster funnels acetylcarnitine into the degradation pathway by deacetylation. A transcriptional regulator CarR bound in a concentration-dependent manner to the intergenic region between the mdh gene, the first gene of the carnitine catabolic operon and the carR gene in the presence and absence of carnitine. Both carnitine and d-malate induced CarR-dependent expression of the carnitine operon. Infection studies with Galleria mellonella larvae demonstrated a strong increase in virulence by addition of carnitine indicating that carnitine degradation plays a pivotal role in virulence of A. baumannii.
Establishing management programs to preserve the benthic communities along the NW Pacific and the Arctic Ocean (AO) requires a deep understanding of the composition of communities and their responses to environmental stressors. In this study, we thus examine patterns of benthic community composition and patterns of species richness along the NW Pacific and Arctic Seas and investigate the most important environmental drivers of those patterns. Overall we found a trend of decreasing species richness toward higher latitudes and deeper waters, peaking in coastal waters of the eastern Philippines. The most dominant taxa along the entire study area were Arthropoda, Mollusca, Cnidaria, Echinodermata, and Annelida. We found that depth, not temperature, was the main driver of community composition along the NW Pacific and neighboring Arctic Seas. Depth has been previously suggested as a factor driving species distribution in benthic fauna. Following depth, the most influential environmental drivers of community composition along the NW Pacific and the Arctic Ocean were silicate, light, and currents. For example, silicate in Hexactinellida, Holothuroidea, and Ophiuroidea; and light in Cephalopoda and Gymnolaemata had the highest correlations with community composition. In this study, based on a combination of new samples and open-access data, we show that different benthic communities might respond differently to future climatic changes based on their taxon-specific biological, physiological, and ecological characteristics. International conservation efforts and habitat preservation should take an adaptive approach and apply measures that take the differences among benthic communities in responding to future climate change into account. This facilitates implementing appropriate conservation management strategies and sustainable utilization of the NW Pacific and Arctic marine ecosystems.
Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.
To fight the global problems of humanity, the United Nations has adopted 17 Sustainable Development Goals (SDGs). To achieve these goals, it is necessary that future decision-makers and stakeholders in society consider these goals to be important. Therefore, in this study, we examined how important students in 41 countries directly related to the environmental sector rated each of the 17 SDGs. Based on the analysis of these ratings, it was possible to categorize the SDGs into three higher-level factors that reflect the three pillars of sustainability (social, economic, environmental). These three pillars are considered to be of varying importance in different countries. We also correlated the ratings of these higher-level factors with country-specific indicators, such as the Human Development Index. The correlations between the indicators and the higher-level factors revealed that in countries with higher indices, the SDGs are rated as less important compared to in countries with lower indices. These results provide stakeholders with important guidance on how the SDGs should be promoted in their country.
Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
Acinetobacter baumannii can thrive on a broad range of substrates such as sugars, alcohols, lipids, amino acids and aromatic compounds. The latter three are abundant in the human host and are potential candidates as carbon sources for the metabolic adaptation of A. baumannii to the human host. In this study we determined the biodegradative activities of A. baumannii AYE with monocyclic aromatic compounds. Deletion of genes encoding the key enzymes of the ß-ketoadipate pathway, the protocatechuate-3,4-dioxygenase (ΔpcaHG) and the catechol-1,2-dioxygenase (ΔcatA), led to a complete loss of growth on benzoate and p-hydroxybenzoate, suggesting that these substrates are metabolized via the two distinct branches (pca and cat) of this pathway. Furthermore, we investigated the potential role of these gene products in host adaptation by analyzing the capability of the mutants to resist complement-mediated killing. These studies revealed that the mutants exhibit a decreased complement resistance, but a dramatic increase in survival in normal human serum in the presence of p-hydroxybenzoate or protocatechuate. These results indicate that the ß-ketoadipate pathway plays a role in adaptation of A. baumannii to the human host. Moreover, the single and double mutants exhibited increased antibiotic resistances indicating a link between the two dioxygenases and antibiotic resistance.