An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1.

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.

Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

Hepcidin deficiency impairs hippocampal neurogenesis and mediates brain atrophy and memory decline in mice.

BACKGROUND: Hepcidin is the master regulator of iron homeostasis. Hepcidin downregulation has been demonstrated in the brains of Alzheimer's disease (AD) patients. However, the mechanism underlying the role of hepcidin downregulation in cognitive impairment has not been elucidated. METHODS: In the present study, we generated GFAP-Cre-mediated hepcidin conditional knockout mice (HampGFAP cKO) to explore the effect of hepcidin deficiency on hippocampal structure and neurocognition. RESULTS: We found that the HampGFAP cKO mice developed AD-like brain atrophy and memory deficits. In particular, the weight of the hippocampus and the number of granule neurons in the dentate gyrus were significantly reduced. Further investigation demonstrated that the morphological change in the hippocampus of HampGFAP cKO mice was attributed to impaired neurogenesis caused by decreased proliferation of neural stem cells. Regarding the molecular mechanism, increased iron content after depletion of hepcidin followed by an elevated level of the inflammatory factor tumor necrosis factor-α accounted for the impairment of hippocampal neurogenesis in HampGFAP cKO mice. These observations were further verified in GFAP promoter-driven hepcidin knockdown mice and in Nestin-Cre-mediated hepcidin conditional knockout mice. CONCLUSIONS: The present findings demonstrated a critical role for hepcidin in hippocampal neurogenesis and validated the importance of iron and associated inflammatory cytokines as key modulators of neurodevelopment, providing insights into the potential pathogenesis of cognitive dysfunction and related treatments.

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Morphology-Controlled Synthesis of V1.11S2 for Electrocatalytic Hydrogen Evolution Reaction in Acid Media.

High-performance low-cost catalysts are in high demand for the hydrogen evolution reaction (HER). In the present study, we reported that V1.11S2 materials with flower-like, flake-like, and porous morphologies were successfully synthesized by hydrothermal synthesis and subsequent calcination. The effects of morphology on hydrogen evolution performance were studied. Results show that flower-like V1.11S2 exhibits the best electrocatalytic activity for HER, achieving both high activity and preferable stability in 0.5 M H2SO4 solution. The main reason can be ascribed to the abundance of catalytically active sites and low charge transfer resistance.

RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation.

Emerging evidence suggests that the ribosome has a regulatory function in directing how the genome is translated in time and space. However, how this regulation is encoded in the messenger RNA sequence remains largely unknown. Here we uncover unique RNA regulons embedded in homeobox (Hox) 5' untranslated regions (UTRs) that confer ribosome-mediated control of gene expression. These structured RNA elements, resembling viral internal ribosome entry sites (IRESs), are found in subsets of Hox mRNAs. They facilitate ribosome recruitment and require the ribosomal protein RPL38 for their activity. Despite numerous layers of Hox gene regulation, these IRES elements are essential for converting Hox transcripts into proteins to pattern the mammalian body plan. This specialized mode of IRES-dependent translation is enabled by an additional regulatory element that we term the translation inhibitory element (TIE), which blocks cap-dependent translation of transcripts. Together, these data uncover a new paradigm for ribosome-mediated control of gene expression and organismal development.

Updates to the RNA mapping database (RMDB), version 2.

Chemical mapping is a broadly utilized technique for probing the structure and function of RNAs. The volume of chemical mapping data continues to grow as more researchers routinely employ this information and as experimental methods increase in throughput and information content. To create a central location for these data, we established an RNA mapping database (RMDB) 5 years ago. The RMDB, which is available at http://rmdb.stanford.edu, now contains chemical mapping data for over 800 entries, involving 134 000 natural and engineered RNAs, in vitro and in cellulo. The entries include large data sets from multidimensional techniques that focus on RNA tertiary structure and co-transcriptional folding, resulting in over 15 million residues probed. The database interface has been redesigned and now offers interactive graphical browsing of structural, thermodynamic and kinetic data at single-nucleotide resolution. The front-end interface now uses the force-directed RNA applet for secondary structure visualization and other JavaScript-based views of bar graphs and annotations. A new interface also streamlines the process for depositing new chemical mapping data to the RMDB.

Protective Effects of Anthocyanins from Coreopsis tinctoria against Oxidative Stress Induced by Hydrogen Peroxide in MIN6 Cells.

Anthocyanins (AC) from Coreopsis tinctoria possesses strong antioxidant properties, while the effects of AC on cells damage induced by reactive oxygen species (ROS) in diabetes mellitus diseases progression have not been reported. The present study was carried out to evaluate the protective property of AC against cellular oxidative stress with an experimental model, H2 O2 -exposed MIN6 cells. AC could reverse the decrease of cell viability induced by H2 O2 and efficiently suppressed cellular ROS production and cell apoptosis. In addition, Real-time PCR and Western blot analyses indicated that AC could protect MIN6 cells against oxidative injury through increasing the translocation of Nrf2 into nuclear, decreasing the phosphorylation level of p38 and up-regulating the protein expression of antioxidant enzyme (SOD1, SOD2 and CAT). Thus, this study provides evidence to support the beneficial effect of AC in inhibiting MIN6 cells from H2 O2 -induced oxidative injury.

The Identification of Anthocyanins from Padus racemosa and Its Protective Effects on H2 O2 -Induced INS-1 Cells Damage and STZ-Induced Diabetes Mice.

Oxidative damage in cells induced by reactive oxygen species (ROS) is a main factor in diabetes mellitus diseases progression. The composition of anthocyanins from Padus racemosa (APR) and the protective effects of APR on H2 O2 -induced rat insulinoma (INS-1) cells damage and streptozocin (STZ)-induced diabetes mice were investigated in this study. The main components of APR were cyanidin-cyanidin glucosyl-rutinoside, cyanidin-cyanidin xylosyl-rutinoside, cyanidin-xylosyl-glucoside and cyanidin-rutinoside, which were determined by liquid chromatography-mass spectrometry (LC/MS). APR could scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical and superoxide radical in vitro. ROS level was decreased and the cell viability was increased in INS-1 cells after treated with APR. Cell apoptosis induced by H2 O2 in INS-1 cells was decreased after incubation with APR. APR could decrease the phosphorylation of p38 and the nuclear translocation of p65, which indicated that APR could inhibit the activation of p38 Mitogen-activated protein kinase (MAPK) and Nuclear factor kappa B (NF-κB) cell signaling pathways. Meanwhile, APR could effectively reduce the blood glucose and blood lipid in STZ-induced diabetic mice. These results suggested that APR might be a potential agent for diabetes mellitus diseases treatment.

Resveratrol protects human bronchial epithelial cells against nickel-induced toxicity via suppressing p38 MAPK, NF-κB signaling, and NLRP3 inflammasome activation.

Nickel is a common environmental pollutant that can impair the lung, but the underlying mechanisms have not yet been fully elucidated. Furthermore, natural products are generally used to inhibit cell damage induced by heavy metal. Resveratrol possesses wide biological activities, including anti-inflammation and antioxidative stress. This study was conducted to explore the toxicity of nickel on human bronchial epithelial (BEAS-2B) cells and evaluate the protective effect of resveratrol. The results showed that nickel could induce cell apoptosis, increase oxidative stress, and promote the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-8, C-reaction protein. Western blot analysis showed that nickel activated p38 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B, and nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 pathways, while resveratrol could reverse these effects. Our results suggested that resveratrol could protect BEAS-2B cells from nickel-induced cytotoxicity. Therefore, resveratrol is a potential chemopreventive agent against nickel-induced lung disease.

Oxysterol-binding protein homologs mediate sterol transport from the endoplasmic reticulum to mitochondria in yeast.

Sterols are present in eukaryotic membranes and significantly affect membrane fluidity, permeability, and microdomain formation. They are synthesized in the endoplasmic reticulum (ER) and transported to other organelles and the plasma membrane. Sterols play important roles in the biogenesis and maintenance of mitochondrial membranes. However, the mechanisms underlying ER-to-mitochondrion sterol transport remain to be identified. Here, using purified yeast membrane fractions enriched in ER and mitochondria, we show that the oxysterol-binding protein homologs encoded by the OSH genes in the yeast Saccharomyces cerevisiae mediate sterol transport from the ER to mitochondria. Combined depletion of all seven Osh proteins impaired sterol transport from the ER to mitochondria in vitro; however, sterol transport was recovered at different levels upon adding one of the Osh proteins. Of note, the sterol content in the mitochondrial fraction was significantly decreased in vivo after Osh4 inactivation in a genetic background in which all the other OSH genes were deleted. We also found that Osh5-Osh7 bind cholesterol in vitro We propose a model in which Osh proteins share a common function to transport sterols between membranes, with varying contributions by these proteins, depending on the target membranes. In summary, we have developed an in vitro system to examine intracellular sterol transport and provide evidence for involvement of Osh proteins in sterol transport from the ER to mitochondria in yeast.

Skipping of an exon with a nonsense mutation in the DMD gene is induced by the conversion of a splicing enhancer to a splicing silencer.

Modulation of dystrophin pre-mRNA splicing is an attractive strategy to ameliorate the severe phenotype of Duchenne muscular dystrophy (DMD), although this requires a better understanding of the mechanism of splicing regulation. Aberrant splicing caused by gene mutations provides a good model to study splicing regulatory cis-elements and binding proteins. In this study, we identified skipping of in-frame exon 25 induced by a nonsense mutation (NM_004006.2:c.3340A > T;p.Lys1114*) in the DMD gene. Site-directed mutagenesis study in minigenes suggested that c.3340A > T converts an exonic splicing enhancer sequence (ESE) to a silencer element (ESS). Indeed, RNA pull-down and functional study provided evidence that c.3340A > T abolishes the binding of the splicing enhancer protein Tra2ß and promotes interactions with the repressor proteins hnRNP A1, hnRNP A2, and hnRNP H. By carefully analyzing the sequence motif encompassing the mutation site, we concluded that the skipping of exon 25 was due to disruption of a Tra2ß-dependent ESE and the creation of a new ESS associated with hnRNP A1 and hnRNP A2, which in turn increased the recruitment of hnRNP H to a nearby binding site. Finally, we demonstrated that c.3340A > T impairs the splicing of upstream intron 24 in a splicing minigene assay. In addition, we showed that the correct splicing of exon 25 is finely regulated by multiple splicing regulators that function in opposite directions by binding to closely located ESE and ESS. Our results clarify the detailed molecular mechanism of exon skipping induced by the nonsense mutation c.3340A > T and also provide information on exon 25 splicing.

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

RNA structure through multidimensional chemical mapping.

The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed •OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to 200 nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-cross-link-map (MXM) expansion to overcome this and other current limitations to resolving the in vivo 'RNA structurome'.

Primerize-2D: automated primer design for RNA multidimensional chemical mapping.

Summary: Rapid RNA synthesis of comprehensive single mutant libraries and targeted multiple mutant libraries is enabling new multidimensional chemical approaches to solve RNA structures. PCR assembly of DNA templates and in vitro transcription allow synthesis and purification of hundreds of RNA mutants in a cost-effective manner, with sharing of primers across constructs allowing significant reductions in expense. However, these protocols require organization of primer locations across numerous 96 well plates and guidance for pipetting, non-trivial tasks for which informatics and visualization tools can prevent costly errors. We report here an online tool to accelerate synthesis of large libraries of desired mutants through design and efficient organization of primers. The underlying program and graphical interface have been experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides and libraries encompassing up to 960 variants. In addition to the freely available Primerize-2D server, the primer design code is available as a stand-alone Python package for broader applications. Availability and Implementation: http://primerize2d.stanford.edu. Contact: rhiju@stanford.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Triticum urartu MTP1: its ability to maintain Zn2+ and Co2+ homeostasis and metal selectivity determinants.

KEY MESSAGE: TuMTP1 maintains Zn2+ and Co2+ homeostasis by sequestering excess Zn2+ and Co2+ into vacuoles. The mutations NSEDD/VTVTT in the His-rich loop and I119F in TMD3 of TuMTP1 restrict metal selectivity. Mineral nutrients, such as zinc (Zn) and cobalt (Co), are essential or beneficial for plants but can be toxic at elevated levels. Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. However, the determinants of substrate selectivity have not been clarified due to the diversity of MTP1 substrates in various plants. In this study, Triticum urartu MTP1 was characterized. When expressed in yeast, TuMTP1 conferred tolerance to Zn2+ and Co2+ but not Fe2+, Cu2+, Ni2+ or Cd2+ in solid and liquid culture and localized on the vacuolar membrane. Furthermore, TuMTP1-expressing yeast accumulated more Zn2+ and Co2+ when treated. TuMTP1 expression in T. urartu roots was significantly increased under Zn2+ and Co2+ stresses. Determinants of substrate selectivity were then examined through site-directed mutagenesis. The exchange of NSEDD with VTVTT in the His-rich loop of TuMTP1 restricted its metal selectivity to Zn2+, whereas the I119F mutation confined specificity to Co2+. The mutations H74, D78, H268 and D272 (in the Zn2+-binding site) and Leu322 (in the C-terminal Leu-zipper) partially or completely abolished the transport function of TuMTP1. These results show that TuMTP1 might sequester excess cytosolic Zn2+ and Co2+ into yeast vacuoles to maintain Zn2+ and Co2+ homeostasis. The NSEDD/VTVTT and I119F mutations are crucially important for restricting the substrate specificity of TuMTP1, and the Zn2+-binding site and Leu322 are essential for its ion selectivity and transport function. These results can be employed to change metal selectivity for biofortification or phytoremediation applications.

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

Primerize: automated primer assembly for transcribing non-coding RNA domains.

Customized RNA synthesis is in demand for biological and biotechnological research. While chemical synthesis and gel or chromatographic purification of RNA is costly and difficult for sequences longer than tens of nucleotides, a pipeline of primer assembly of DNA templates, in vitro transcription by T7 RNA polymerase and kit-based purification provides a cost-effective and fast alternative for preparing RNA molecules. Nevertheless, designing template primers that optimize cost and avoid mispriming during polymerase chain reaction currently requires expert inspection, downloading specialized software or both. Online servers are currently not available or maintained for the task. We report here a server named Primerize that makes available an efficient algorithm for primer design developed and experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides. Free access: http://primerize.stanford.edu.

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states.

The three-dimensional conformations of noncoding RNAs underpin their biochemical functions but have largely eluded experimental characterization. Here, we report that integrating a classic mutation/rescue strategy with high-throughput chemical mapping enables rapid RNA structure inference with unusually strong validation. We revisit a 16S rRNA domain for which SHAPE (selective 2'-hydroxyl acylation with primer extension) and limited mutational analysis suggested a conformational change between apo- and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M(2)) experiments highlight issues of prior methodology and instead give a near-crystallographic secondary structure. Systematic interrogation of single base pairs via a high-throughput mutation/rescue approach then permits incisive validation and refinement of the M(2)-based secondary structure. The data further uncover the functional conformation as an excited state (20 ± 10% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change, expose critical limitations of conventional structure mapping methods, and illustrate practical steps for more incisively dissecting RNA dynamic structure landscapes.