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As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.
The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop—a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.
Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.
Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.
In the mouse, most mature olfactory sensory neurons (OSNs) express one allele of one gene from the repertoire of ~1100 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express a given OR coalesce into homogeneous glomeruli, which reside at conserved positions in the olfactory bulb. ORs are intimately involved in ensuring the expression of one OR per OSN and the coalescence of OSN axons into glomeruli. But the mechanisms whereby ORs accomplish these diverse functions remain poorly understood. An experimental approach that has been informative is to substitute an OR genetically with another GPCR that is normally not expressed in OSNs, in order to determine in which aspects this GPCR can serve as surrogate OR in mouse OSNs. Thus far only the β2-adrenergic receptor (β2AR, Ardb2) has been shown to be able to serve as surrogate OR in OSNs; the β2AR could substitute for the M71 OR in all aspects examined. Can other non-olfactory GPCRs function equally well as surrogate ORs in OSNs? Here, we have generated and characterized two novel gene-targeted mouse strains in which the mouse melanocortin 4 receptor (Mc4r) or the mouse dopamine receptor D1 (Drd1a) is coexpressed with tauGFP in OSNs that express the OR locus M71. These alleles and strains are abbreviated as Mc4r→M71-GFP and Drd1a→M71-GFP. We detected strong Mc4r or Drd1a immunoreactivity in axons and dendritic knobs and cilia of OSNs that express Mc4r or Drd1a from the M71 locus. These OSNs responded physiologically to cognate agonists for Mc4r (Ro27-3225) or Drd1a (SKF81297), and not to the M71 ligand acetophenone. Axons of OSNs expressing Mc4r→M71-GFP coalesced into glomeruli. Axons of OSNs expressing Drd1a→M71-GFP converged onto restricted areas of the olfactory bulb but did not coalesce into glomeruli. Thus, OR functions in OSNs can be substituted by Mc4r or Drd1a, but not as well as by β2AR. We attribute the weak performance of Drd1a as surrogate OR to poor OSN maturation.
Hydrogenases are key enzymes of the energy metabolism of many microorganisms. Especially in anoxic habitats where molecular hydrogen (H2) is an important intermediate, these enzymes are used to expel excess reducing power by reducing protons or they are used for the oxidation of H2 as energy and electron source. Despite the fact that hydrogenases catalyze the simplest chemical reaction of reducing two protons with two electrons it turned out that they are often parts of multimeric enzyme complexes catalyzing complex chemical reactions with a multitude of functions in the metabolism. Recent findings revealed multimeric hydrogenases with so far unknown functions particularly in bacteria from the class Clostridia. The discovery of [FeFe] hydrogenases coupled to electron bifurcating subunits solved the enigma of how the otherwise highly endergonic reduction of the electron carrier ferredoxin can be carried out and how H2 production from NADH is possible. Complexes of [FeFe] hydrogenases with formate dehydrogenases revealed a novel enzymatic coupling of the two electron carriers H2 and formate. These novel hydrogenase enzyme complex could also contribute to biotechnological H2 production and H2 storage, both processes essential for an envisaged economy based on H2 as energy carrier.
A major driving force for the adaptation of bacteria to changing environments is the uptake of naked DNA from the environment by natural transformation, which allows the acquisition of new capabilities. Uptake of the high molecular weight DNA is mediated by a complex transport machinery that spans the entire cell periphery. This DNA translocator catalyzes the binding and splitting of double‐stranded DNA and translocation of single‐stranded DNA into the cytoplasm, where it is recombined with the chromosome. The thermophilic bacterium Thermus thermophilus exhibits the highest transformation frequencies reported and is a model system to analyze the structure and function of this macromolecular transport machinery. Transport activity is powered by the traffic ATPase PilF, a soluble protein that forms hexameric complexes. Here, we demonstrate that PilF physically binds to an inner membrane assembly platform of the DNA translocator, comprising PilMNO, via the ATP‐binding protein PilM. Binding to PilMNO or PilMN stimulates the ATPase activity of PilF ~ 2‐fold, whereas there is no stimulation when binding to PilM or PilN alone. A PilMK26A variant defective in ATP binding still binds PilF and, together with PilN, stimulates PilF‐mediated ATPase activity. PilF is unique in having three conserved GSPII (general secretory pathway II) domains (A–C) at its N terminus. Deletion analyses revealed that none of the GSPII domains is essential for binding PilMN, but GSPIIC is essential for PilMN‐mediated stimulation of ATP hydrolysis by PilF. Our data suggest that PilM is a coupling protein that physically and functionally connects the soluble motor ATPase PilF to the DNA translocator via the PilMNO assembly platform.
Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.
Neuropogonoid species in the lichen-forming fungal genus Usnea exhibit great morphological variation that can be misleading for delimitation of species. We specifically focused on the species delimitation of two closely-related, predominantly Antarctic species differing in the reproductive mode and representing a so-called species pair: the asexual U. antarctica and the sexual U. aurantiacoatra. Previous studies have revealed contradicting results. While multi-locus studies based on DNA sequence data provided evidence that these two taxa might be conspecific, microsatellite data suggested they represent distinct lineages. By using RADseq, we generated thousands of homologous markers to build a robust phylogeny of the two species. Furthermore, we successfully implemented these data in fine-scale population genomic analyses such as DAPC and fineRADstructure. Both Usnea species are readily delimited in phylogenetic inferences and, therefore, the hypothesis that both species are conspecific was rejected. Population genomic analyses also strongly confirmed separated genomes and, additionally, showed different levels of co-ancestry and substructure within each species. Lower co-ancestry in the asexual U. antarctica than in the sexual U. aurantiacoatra may be derived from a wider distributional range of the former species. Our results demonstrate the utility of this RADseq method in tracing population dynamics of lichens in future analyses.
Background: Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome.
Methods: The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain.
Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala.
Conclusions: In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.
Lunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle–dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.
Background: Long non-coding RNAs (lncRNAs) represent a novel class of non-coding RNAs having a crucial role in many biological processes. The identification of long non-coding homologs among different species is essential to investigate such roles in model organisms as homologous genes tend to retain similar molecular and biological functions. Alignment–based metrics are able to effectively capture the conservation of transcribed coding sequences and then the homology of protein coding genes. However, unlike protein coding genes the poor sequence conservation of long non-coding genes makes the identification of their homologs a challenging task.
Results: In this study we compare alignment–based and alignment–free string similarity metrics and look at promoter regions as a possible source of conserved information. We show that promoter regions encode relevant information for the conservation of long non-coding genes across species and that such information is better captured by alignment–free metrics. We perform a genome wide test of this hypothesis in human, mouse, and zebrafish.
Conclusions: The obtained results persuaded us to postulate the new hypothesis that, unlike protein coding genes, long non-coding genes tend to preserve their regulatory machinery rather than their transcribed sequence. All datasets, scripts, and the prediction tools adopted in this study are available at https://github.com/bioinformatics-sannio/lncrna-homologs.
Background and Objectives: Valuation of life (VOL) represents a construct capturing individuals’ active attachment to their life. The majority of studies on VOL were conducted in North America and Europe where personal autonomy and independence are highly valued, leaving open the question about the relevance of this construct in interdependence-oriented cultures. Using a framework of cross-cultural and life-span theories, the present study compared levels and predictors of VOL between the young-old and old-old individuals from Germany and Japan.
Research Design and Methods: Two hundred fifty-seven Germans and 248 Japanese, matched by age, gender, education, and IADL, answered a 5-item VOL scale and shared information on sociodemographic, social, and health resources.
Results: Germans’ VOL levels were higher than in Japanese participants. Both culture- and age-moderated predictions of VOL: education was significant only in the young-old Japanese, and close social partners mattered in the old-old, not in the young-old. Health determined VOL irrespective of culture and age.
Discussion and Implications: The findings suggest that cultural values and aging processes should be considered to better understand how individuals value their life and to help older adults to feel that his/her life is meaningful and worth living.
Experimental evidence supports that cortical oscillations represent multiscale temporal modulations existent in natural stimuli, yet little is known about the processing of these multiple timescales at a neuronal level. Here, using extracellular recordings from the auditory cortex (AC) of awake bats (Carollia perspicillata), we show the existence of three neuronal types which represent different levels of the temporal structure of conspecific vocalizations, and therefore constitute direct evidence of multiscale temporal processing of naturalistic stimuli by neurons in the AC. These neuronal subpopulations synchronize differently to local-field potentials, particularly in theta- and high frequency bands, and are informative to a different degree in terms of their spike rate. Interestingly, we also observed that both low and high frequency cortical oscillations can be highly informative about the listened calls. Our results suggest that multiscale neuronal processing allows for the precise and non-redundant representation of natural vocalizations in the AC.
Neurons collect their inputs from other neurons by sending out arborized dendritic structures. However, the relationship between the shape of dendrites and the precise organization of synaptic inputs in the neural tissue remains unclear. Inputs could be distributed in tight clusters, entirely randomly or else in a regular grid-like manner. Here, we analyze dendritic branching structures using a regularity index R, based on average nearest neighbor distances between branch and termination points, characterizing their spatial distribution. We find that the distributions of these points depend strongly on cell types, indicating possible fundamental differences in synaptic input organization. Moreover, R is independent of cell size and we find that it is only weakly correlated with other branching statistics, suggesting that it might reflect features of dendritic morphology that are not captured by commonly studied branching statistics. We then use morphological models based on optimal wiring principles to study the relation between input distributions and dendritic branching structures. Using our models, we find that branch point distributions correlate more closely with the input distributions while termination points in dendrites are generally spread out more randomly with a close to uniform distribution. We validate these model predictions with connectome data. Finally, we find that in spatial input distributions with increasing regularity, characteristic scaling relationships between branching features are altered significantly. In summary, we conclude that local statistics of input distributions and dendrite morphology depend on each other leading to potentially cell type specific branching features.
Acinetobacter baumannii is a nosocomial pathogen which can persist in the hospital environment not only due to the acquirement of multiple antibiotic resistances, but also because of its exceptional resistance against disinfectants and desiccation. A suitable desiccation assay was established in which A. baumannii ATCC 19606T survived for ca. 1 month. The growth medium slightly influenced survival after subsequent desiccation. A significant effect could be attributed to the growth phase in which bacteria were dried: In exponential phase, cells were much more desiccation sensitive. The main focus of the present study was the elucidation of the role of compatible solutes, which are known to protect many bacteria under low water activity conditions, in desiccation survival of A. baumannii. Exogenous trehalose was shown to efficiently protect A. baumannii on dry surfaces, in contrast to other compatible solutes tested such as mannitol or glycine betaine. To analyze the importance of intracellularly accumulated solutes, a double mutant lacking biosynthesis pathways for mannitol and trehalose was generated. This mutant accumulated glutamate as sole solute in the presence of high NaCl concentrations and showed severe growth defects under osmotic stress conditions. However, no effect on desiccation tolerance could be seen, neither when cells were dried in water nor in the presence of NaCl.
Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID.
Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions.
Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin–positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane.
Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.
Low-frequency spike-field coherence is a fingerprint of periodicity coding in the auditory cortex
(2018)
The extraction of temporal information from sensory input streams is of paramount importance in the auditory system. In this study, amplitude-modulated sounds were used as stimuli to drive auditory cortex (AC) neurons of the bat species Carollia perspicillata, to assess the interactions between cortical spikes and local-field potentials (LFPs) for the processing of temporal acoustic cues. We observed that neurons in the AC capable of eliciting synchronized spiking to periodic acoustic envelopes were significantly more coherent to theta- and alpha-band LFPs than their non-synchronized counterparts. These differences occurred independently of the modulation rate tested and could not be explained by power or phase modulations of the field potentials. We argue that the coupling between neuronal spiking and the phase of low-frequency LFPs might be important for orchestrating the coding of temporal acoustic structures in the AC.