MINTbase v2.0: a comprehensive database for tRNA-derived fragments that includes nuclear and mitochondrial fragments from all The Cancer Genome Atlas projects Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Venetia Pliatsika, Phillipe Loher, Rogan Magee, Aristeidis G. Telonis, Eric Londin, Megumi Shigematsu, Yohei Kirino, Isidore Rigoutsos
MINTbase is a repository that comprises nuclear and mitochondrial tRNA-derived fragments (‘tRFs’) found in multiple human tissues. The original version of MINTbase comprised tRFs obtained from 768 transcriptomic datasets. We used our deterministic and exhaustive tRF mining pipeline to process all of The Cancer Genome Atlas datasets (TCGA). We identified 23 413 tRFs with abundance of ≥ 1.0 reads-per-million (RPM). To facilitate further studies of tRFs by the community, we just released version 2.0 of MINTbase that contains information about 26 531 distinct human tRFs from 11 719 human datasets as of October 2017. Key new elements include: the ability to filter tRFs on-the-fly by minimum abundance thresholding; the ability to filter tRFs by tissue keywords; easy access to information about a tRF’s maximum abundance and the datasets that contain it; the ability to generate relative abundance plots for tRFs across cancer types and convert them into embeddable figures; MODOMICS information about modifications of the parental tRNA, etc. Version 2.0 of MINTbase contains 15x more datasets and nearly 4x more distinct tRFs than the original version, yet continues to offer fast, interactive access to its contents. Version 2.0 is available freely at http://cm.jefferson.edu/MINTbase/.
The Planteome database: an integrated resource for reference ontologies, plant genomics and phenomics Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Laurel Cooper, Austin Meier, Marie-Angélique Laporte, Justin L. Elser, Chris Mungall, Brandon T. Sinn, Dario Cavaliere, Seth Carbon, Nathan A. Dunn, Barry Smith, Botong Qu, Justin Preece, Eugene Zhang, Sinisa Todorovic, Georgios Gkoutos, John H. Doonan, Dennis W. Stevenson, Elizabeth Arnaud, Pankaj Jaiswal
The Planteome project (http://www.planteome.org) provides a suite of reference and species-specific ontologies for plants and annotations to genes and phenotypes. Ontologies serve as common standards for semantic integration of a large and growing corpus of plant genomics, phenomics and genetics data. The reference ontologies include the Plant Ontology, Plant Trait Ontology and the Plant Experimental Conditions Ontology developed by the Planteome project, along with the Gene Ontology, Chemical Entities of Biological Interest, Phenotype and Attribute Ontology, and others. The project also provides access to species-specific Crop Ontologies developed by various plant breeding and research communities from around the world. We provide integrated data on plant traits, phenotypes, and gene function and expression from 95 plant taxa, annotated with reference ontology terms. The Planteome project is developing a plant gene annotation platform; Planteome Noctua, to facilitate community engagement. All the Planteome ontologies are publicly available and are maintained at the Planteome GitHub site (https://github.com/Planteome) for sharing, tracking revisions and new requests. The annotated data are freely accessible from the ontology browser (http://browser.planteome.org/amigo) and our data repository.
Conformational dynamism for DNA interaction in the Salmonella RcsB response regulator Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Patricia Casino, Laura Miguel-Romero, Juanjo Huesa, Pablo García, Francisco García-del Portillo, Alberto Marina
The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3−. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a β5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic.
Dynamic m6A modification regulates local translation of mRNA in axons Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Jun Yu, Mengxian Chen, Haijiao Huang, Junda Zhu, Huixue Song, Jian Zhu, Jaewon Park, Sheng-Jian Ji
N6-methyladenosine (m6A) is a reversible modification in mRNA and has been shown to regulate processing, translation and decay of mRNA. However, the roles of m6A modification in neuronal development are still not known. Here, we found that the m6A eraser FTO is enriched in axons and can be locally translated. Axon-specific inhibition of FTO by rhein, or compartmentalized siRNA knockdown of Fto in axons led to increases of m6A levels. GAP-43 mRNA is modified by m6A and is a substrate of FTO in axons. Loss-of-function of this non-nuclear pool of FTO resulted in increased m6A modification and decreased local translation of axonal GAP-43 mRNA, which eventually repressed axon elongation. Mutation of a predicted m6A site in GAP-43 mRNA eliminated its m6A modification and exempted regulation of its local translation by axonal FTO. This work showed an example of dynamic internal m6A demethylation of non-nuclear localized mRNA by the demethylase FTO. Regulation of m6A modification of axonal mRNA by axonal FTO might be a general mechanism to control their local translation in neuronal development.
Biochemical and structural characterization of a novel cooperative binding mode by Pit-1 with CATT repeats in the macrophage migration inhibitory factor promoter Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Sorabh Agarwal, Thomas Yoonsang Cho
Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (−794 CATT5–8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent Kd of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders.
Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Ewa Kowalska, Filip Bartnicki, Ryo Fujisawa, Piotr Bonarek, Paweł Hermanowicz, Toshiki Tsurimoto, Klaudia Muszyńska, Wojciech Strzalka
Proliferating cell nuclear antigen (PCNA) is a multifunctional protein present in the nuclei of eukaryotic cells that plays an important role as a component of the DNA replication machinery, as well as DNA repair systems. PCNA was recently proposed as a potential non-oncogenic target for anti-cancer therapy. In this study, using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we developed a short DNA aptamer that binds human PCNA. In the presence of PCNA, the anti-PCNA aptamer inhibited the activity of human DNA polymerase δ and ϵ at nM concentrations. Moreover, PCNA protected the anti-PCNA aptamer against the exonucleolytic activity of these DNA polymerases. Investigation of the mechanism of anti-PCNA aptamer-dependent inhibition of DNA replication revealed that the aptamer did not block formation, but was a component of PCNA/DNA polymerase δ or ϵ complexes. Additionally, the anti-PCNA aptamer competed with the primer-template DNA for binding to the PCNA/DNA polymerase δ or ϵ complex. Based on the observations, a model of anti-PCNA aptamer/PCNA complex-dependent inhibition of DNA replication was proposed.
DNA as UV light–harvesting antenna Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Ivan L. Volkov, Zakhar V. Reveguk, Pavel Yu. Serdobintsev, Ruslan R. Ramazanov, Alexei I. Kononov
The ordered structure of UV chromophores in DNA resembles photosynthetic light-harvesting complexes in which quantum coherence effects play a major role in highly efficient directional energy transfer. The possible role of coherent excitons in energy transport in DNA remains debated. Meanwhile, energy transport properties are greatly important for understanding the mechanisms of photochemical reactions in cellular DNA and for DNA-based artificial nanostructures. Here, we studied energy transfer in DNA complexes formed with silver nanoclusters and with intercalating dye (acridine orange). Steady-state fluorescence measurements with two DNA templates (15-mer DNA duplex and calf thymus DNA) showed that excitation energy can be transferred to the clusters from 21 and 28 nucleobases, respectively. This differed from the DNA–acridine orange complex for which energy transfer took place from four neighboring bases only. Fluorescence up-conversion measurements showed that the energy transfer took place within 100 fs. The efficient energy transport in the Ag–DNA complexes suggests an excitonic mechanism for the transfer, such that the excitation is delocalized over at least four and seven stacked bases, respectively, in one strand of the duplexes stabilizing the clusters. This result demonstrates that the exciton delocalization length in some DNA structures may not be limited to just two bases.
JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Anders Sundqvist, Masato Morikawa, Jiang Ren, Eleftheria Vasilaki, Natsumi Kawasaki, Mai Kobayashi, Daizo Koinuma, Hiroyuki Aburatani, Kohei Miyazono, Carl-Henrik Heldin, Hans van Dam, Peter ten Dijke
It is well established that transforming growth factor-β (TGFβ) switches its function from being a tumor suppressor to a tumor promoter during the course of tumorigenesis, which involves both cell-intrinsic and environment-mediated mechanisms. We are interested in breast cancer cells, in which SMAD mutations are rare and interactions between SMAD and other transcription factors define pro-oncogenic events. Here, we have performed chromatin immunoprecipitation (ChIP)-sequencing analyses which indicate that the genome-wide landscape of SMAD2/3 binding is altered after prolonged TGFβ stimulation. De novo motif analyses of the SMAD2/3 binding regions predict enrichment of binding motifs for activator protein (AP)1 in addition to SMAD motifs. TGFβ-induced expression of the AP1 component JUNB was required for expression of many late invasion-mediating genes, creating a feed-forward regulatory network. Moreover, we found that several components in the WNT pathway were enriched among the late TGFβ-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGFβ-induced breast cancer cell invasion, while inhibition of the WNT pathway reduced this process. Our study thereby helps to explain how accumulation of pro-oncogenic stimuli switches and stabilizes TGFβ-induced cellular phenotypes of epithelial cells.
Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 Ngat T. Tran, Clare E. Stevenson, Nicolle F. Som, Anyarat Thanapipatsiri, Adam S. B. Jalal, Tung B. K. Le
Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA–ParB–parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.
Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-23 A. Marieke Oudelaar, James O.J. Davies, Damien J. Downes, Douglas R. Higgs, Jim R. Hughes
Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000–20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations.
Meta-analysis of DNA double-strand break response kinetics Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-22 Jakub A. Kochan, Emilie C.B. Desclos, Ruben Bosch, Luna Meister, Lianne E.M. Vriend, Haico v. Attikum, Przemek M. Krawczyk
Most proteins involved in the DNA double-strand break response (DSBR) accumulate at the damage sites, where they perform functions related to damage signaling, chromatin remodeling and repair. Over the last two decades, studying the accumulation of many DSBR proteins provided information about their functionality and underlying mechanisms of action. However, comparison and systemic interpretation of these data is challenging due to their scattered nature and differing experimental approaches. Here, we extracted, analyzed and compared the available results describing accumulation of 79 DSBR proteins at sites of DNA damage, which can be further explored using Cumulus (http://www.dna-repair.live/cumulus/)—the accompanying interactive online application. Despite large inter-study variability, our analysis revealed that the accumulation of most proteins starts immediately after damage induction, occurs in parallel and peaks within 15–20 min. Various DSBR pathways are characterized by distinct accumulation kinetics with major non-homologous end joining proteins being generally faster than those involved in homologous recombination, and signaling and chromatin remodeling factors accumulating with varying speeds. Our meta-analysis provides, for the first time, comprehensive overview of the temporal organization of the DSBR in mammalian cells and could serve as a reference for future mechanistic studies of this complex process.
Mechanism of formation of a toroid around DNA by the mismatch sensor protein Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-22 Shivlee Nirwal, Dhananjaya S. Kulkarni, Amit Sharma, Desirazu N. Rao, Deepak T. Nair
The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS–DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS–DNA complex.
Jointly aligning a group of DNA reads improves accuracy of identifying large deletions Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-22 Anish M.S. Shrestha, Martin C. Frith, Kiyoshi Asai, Hugues Richard
Performing sequence alignment to identify structural variants, such as large deletions, from genome sequencing data is a fundamental task, but current methods are far from perfect. The current practice is to independently align each DNA read to a reference genome. We show that the propensity of genomic rearrangements to accumulate in repeat-rich regions imposes severe ambiguities in these alignments, and consequently on the variant calls—with current read lengths, this affects more than one third of known large deletions in the C. Venter genome. We present a method to jointly align reads to a genome, whereby alignment ambiguity of one read can be disambiguated by other reads. We show this leads to a significant improvement in the accuracy of identifying large deletions (≥20 bases), while imposing minimal computational overhead and maintaining an overall running time that is at par with current tools. A software implementation is available as an open-source Python program called JRA at https://bitbucket.org/jointreadalignment/jra-src.
Crystal structure of an anti-CRISPR protein, AcrIIA1 Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-22 Donghyun Ka, So Young An, Jeong-Yong Suh, Euiyoung Bae
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide bacteria with RNA-based adaptive immunity against phage infection. To counteract this defense mechanism, phages evolved anti-CRISPR (Acr) proteins that inactivate the CRISPR-Cas systems. AcrIIA1, encoded by Listeria monocytogenes prophages, is the most prevalent among the Acr proteins targeting type II-A CRISPR-Cas systems and has been used as a marker to identify other Acr proteins. Here, we report the crystal structure of AcrIIA1 and its RNA-binding affinity. AcrIIA1 forms a dimer with a novel two helical-domain architecture. The N-terminal domain of AcrIIA1 exhibits a helix-turn-helix motif similar to transcriptional factors. When overexpressed in Escherichia coli, AcrIIA1 associates with RNAs, suggesting that AcrIIA1 functions via nucleic acid recognition. Taken together, the unique structural and functional features of AcrIIA1 suggest its distinct mode of Acr activity, expanding the diversity of the inhibitory mechanisms employed by Acr proteins.
Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Robert Serfling, Christian Lorenz, Maja Etzel, Gerda Schicht, Thore Böttke, Mario Mörl, Irene Coin
The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.
Modeling RNA secondary structure folding ensembles using SHAPE mapping data Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Aleksandar Spasic, Sarah M. Assmann, Philip C. Bevilacqua, David H. Mathews
RNA secondary structure prediction is widely used for developing hypotheses about the structures of RNA sequences, and structure can provide insight about RNA function. The accuracy of structure prediction is known to be improved using experimental mapping data that provide information about the pairing status of single nucleotides, and these data can now be acquired for whole transcriptomes using high-throughput sequencing. Prior methods for using these experimental data focused on predicting structures for sequences assuming that they populate a single structure. Most RNAs populate multiple structures, however, where the ensemble of strands populates structures with different sets of canonical base pairs. The focus on modeling single structures has been a bottleneck for accurately modeling RNA structure. In this work, we introduce Rsample, an algorithm for using experimental data to predict more than one RNA structure for sequences that populate multiple structures at equilibrium. We demonstrate, using SHAPE mapping data, that we can accurately model RNA sequences that populate multiple structures, including the relative probabilities of those structures. This program is freely available as part of the RNAstructure software package.
MVP: a microbe–phage interaction database Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Na L. Gao, Chengwei Zhang, Zhanbing Zhang, Songnian Hu, Martin J. Lercher, Xing-Ming Zhao, Peer Bork, Zhi Liu, Wei-Hua Chen
Phages invade microbes, accomplish host lysis and are of vital importance in shaping the community structure of environmental microbiota. More importantly, most phages have very specific hosts; they are thus ideal tools to manipulate environmental microbiota at species-resolution. The main purpose of MVP (Microbe Versus Phage) is to provide a comprehensive catalog of phage–microbe interactions and assist users to select phage(s) that can target (and potentially to manipulate) specific microbes of interest. We first collected 50 782 viral sequences from various sources and clustered them into 33 097 unique viral clusters based on sequence similarity. We then identified 26 572 interactions between 18 608 viral clusters and 9245 prokaryotes (i.e. bacteria and archaea); we established these interactions based on 30 321 evidence entries that we collected from published datasets, public databases and re-analysis of genomic and metagenomic sequences. Based on these interactions, we calculated the host range for each of the phage clusters and accordingly grouped them into subgroups such as ‘species-’, ‘genus-’ and ‘family-’ specific phage clusters. MVP is equipped with a modern, responsive and intuitive interface, and is freely available at: http://mvp.medgenius.info.
Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Diego Duchi, Kristofer Gryte, Nicole C. Robb, Zakia Morichaud, Carol Sheppard, Konstantin Brodolin, Sivaramesh Wigneshweraraj, Achillefs N. Kapanidis
Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.
Derivation of nearest-neighbor DNA parameters in magnesium from single molecule experiments Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Josep Maria Huguet, Marco Ribezzi-Crivellari, Cristiano Valim Bizarro, Felix Ritort
DNA hybridization is an essential molecular reaction in biology with many applications. The nearest-neighbor (NN) model for nucleic acids predicts DNA thermodynamics using energy values for the different base pair motifs. These values have been derived from melting experiments in monovalent and divalent salt and applied to predict melting temperatures of oligos within a few degrees. However, an improved determination of the NN energy values and their salt dependencies in magnesium is still needed for current biotechnological applications seeking high selectivity in the hybridization of synthetic DNAs. We developed a methodology based on single molecule unzipping experiments to derive accurate NN energy values and initiation factors for DNA. A new set of values in magnesium is derived, which reproduces unzipping data and improves melting temperature predictions for all available oligo lengths, in a range of temperature and salt conditions where correlation effects between the magnesium bound ions are weak. The NN salt correction parameters are shown to correlate to the GC content of the NN motifs. Our study shows the power of single-molecule force spectroscopy assays to unravel novel features of nucleic acids such as sequence-dependent salt corrections.
Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Evan A. Caton, Erin K. Kelly, Rajashekhar Kamalampeta, Ute Kothe
H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p–Nop10p–Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.
The TFIIE-related Rpc82 subunit of RNA polymerase III interacts with the TFIIB-related transcription factor Brf1 and the polymerase cleft for transcription initiation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-21 Seok-Kooi Khoo, Chih-Chien Wu, Yu-Chun Lin, Hung-Ta Chen
Rpc82 is a TFIIE-related subunit of the eukaryotic RNA polymerase III (pol III) complex. Rpc82 contains four winged-helix (WH) domains and a C-terminal coiled-coil domain. Structural resolution of the pol III complex indicated that Rpc82 anchors on the clamp domain of the pol III cleft to interact with the duplex DNA downstream of the transcription bubble. However, whether Rpc82 interacts with a transcription factor is still not known. Here, we report that a structurally disordered insertion in the third WH domain of Rpc82 is important for cell growth and in vitro transcription activity. Site-specific photo-crosslinking analysis indicated that the WH3 insertion interacts with the TFIIB-related transcription factor Brf1 within the pre-initiation complex (PIC). Moreover, crosslinking and hydroxyl radical probing analyses revealed Rpc82 interactions with the upstream DNA and the protrusion and wall domains of the pol III cleft. Our genetic and biochemical analyses thus provide new molecular insights into the function of Rpc82 in pol III transcription.
Structure modeling of RNA using sparse NMR constraints Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Benfeard Williams, Bo Zhao, Arpit Tandon, Feng Ding, Kevin M. Weeks, Qi Zhang, Nikolay V. Dokholyan
RNAs fold into distinct molecular conformations that are often essential for their functions. Accurate structure modeling of complex RNA motifs, including ubiquitous non-canonical base pairs and pseudoknots, remains a challenge. Here, we present an NMR-guided all-atom discrete molecular dynamics (DMD) platform, iFoldNMR, for rapid and accurate structure modeling of complex RNAs. We show that sparse distance constraints from imino resonances, which can be readily obtained from routine NMR experiments and easier to compile than laborious assignments of non-solvent-exchangeable protons, are sufficient to direct a DMD search for low-energy RNA conformers. Benchmarking on a set of RNAs with complex folds spanning up to 56 nucleotides in length yields structural models that recapitulate experimentally determined structures with all-heavy-atom RMSDs ranging from 2.4 to 6.5 Å. This platform represents an efficient approach for high-throughput RNA structure modeling and will facilitate analysis of diverse, newly discovered functional RNAs.
Gramene 2018: unifying comparative genomics and pathway resources for plant research Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Marcela K. Tello-Ruiz, Sushma Naithani, Joshua C. Stein, Parul Gupta, Michael Campbell, Andrew Olson, Sharon Wei, Justin Preece, Matthew J. Geniza, Yinping Jiao, Young Koung Lee, Bo Wang, Joseph Mulvaney, Kapeel Chougule, Justin Elser, Noor Al-Bader, Sunita Kumari, James Thomason, Vivek Kumar, Daniel M. Bolser, Guy Naamati, Electra Tapanari, Nuno Fonseca, Laura Huerta, Haider Iqbal, Maria Keays, Alfonso Munoz-Pomer Fuentes, Amy Tang, Antonio Fabregat, Peter D’Eustachio, Joel Weiser, Lincoln D. Stein, Robert Petryszak, Irene Papatheodorou, Paul J. Kersey, Patti Lockhart, Crispin Taylor, Pankaj Jaiswal, Doreen Ware
Gramene (http://www.gramene.org) is a knowledgebase for comparative functional analysis in major crops and model plant species. The current release, #54, includes over 1.7 million genes from 44 reference genomes, most of which were organized into 62,367 gene families through orthologous and paralogous gene classification, whole-genome alignments, and synteny. Additional gene annotations include ontology-based protein structure and function; genetic, epigenetic, and phenotypic diversity; and pathway associations. Gramene's Plant Reactome provides a knowledgebase of cellular-level plant pathway networks. Specifically, it uses curated rice reference pathways to derive pathway projections for an additional 66 species based on gene orthology, and facilitates display of gene expression, gene–gene interactions, and user-defined omics data in the context of these pathways. As a community portal, Gramene integrates best-of-class software and infrastructure components including the Ensembl genome browser, Reactome pathway browser, and Expression Atlas widgets, and undergoes periodic data and software upgrades. Via powerful, intuitive search interfaces, users can easily query across various portals and interactively analyze search results by clicking on diverse features such as genomic context, highly augmented gene trees, gene expression anatomograms, associated pathways, and external informatics resources. All data in Gramene are accessible through both visual and programmatic interfaces.
Identification of diverse target RNAs that are functionally regulated by human Pumilio proteins Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Jennifer A. Bohn, Jamie L. Van Etten, Trista L. Schagat, Brittany M. Bowman, Richard C. McEachin, Peter L. Freddolino, Aaron C. Goldstrohm
Human Pumilio proteins, PUM1 and PUM2, are sequence specific RNA-binding proteins that regulate protein expression. We used RNA-seq, rigorous statistical testing and an experimentally derived fold change cut-off to identify nearly 1000 target RNAs—including mRNAs and non-coding RNAs—that are functionally regulated by PUMs. Bioinformatic analysis defined a PUM Response Element (PRE) that was significantly enriched in transcripts that increased in abundance and matches the PUM RNA-binding consensus. We created a computational model that incorporates PRE position and frequency within an RNA relative to the magnitude of regulation. The model reveals significant correlation of PUM regulation with PREs in 3′ untranslated regions (UTRs), coding sequences and non-coding RNAs, but not 5′ UTRs. To define direct, high confidence PUM targets, we cross-referenced PUM-regulated RNAs with all PRE-containing RNAs and experimentally defined PUM-bound RNAs. The results define nearly 300 direct targets that include both PUM-repressed and, surprisingly, PUM-activated target RNAs. Annotation enrichment analysis reveal that PUMs regulate genes from multiple signaling pathways and developmental and neurological processes. Moreover, PUM target mRNAs impinge on human disease genes linked to cancer, neurological disorders and cardiovascular disease. These discoveries pave the way for determining how the PUM-dependent regulatory network impacts biological functions and disease states.
NS3 helicase from dengue virus specifically recognizes viral RNA sequence to ensure optimal replication Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Crystall M. D. Swarbrick, Chandrakala Basavannacharya, Kitti W. K. Chan, Shu-Ann Chan, Daljit Singh, Na Wei, Wint Wint Phoo, Dahai Luo, Julien Lescar, Subhash G. Vasudevan
The protein–RNA interactions within the flavivirus replication complex (RC) are not fully understood. Our structure of dengue virus NS3 adenosine triphosphatase (ATPase)/helicase bound to the conserved 5′ genomic RNA 5′-AGUUGUUAGUCU-3′ reveals that D290 and R538 make specific interactions with G2 and G5 bases respectively. We show that single-stranded 12-mer RNA stimulates ATPase activity of NS3, however the presence of G2 and G5 leads to significantly higher activation. D290 is adjacent to the DEXH motif found in SF2 helicases like NS3 and interacts with R387, forming a molecular switch that activates the ATPase site upon RNA binding. Our structure guided mutagenesis revealed that disruption of D290–R387 interaction increases basal ATPase activity presumably as a result of higher conformational flexibility of the ATPase active site. Mutational studies also showed R538 plays a critical role in RNA interactions affecting translocation of viral RNA through dynamic interactions with bases at positions 4 and 5 of the ssRNA. Restriction of backbone flexibility around R538 through mutation of G540 to proline abolishes virus replication, indicating conformational flexibility around residue R538 is necessary for RNA translocation. The functionally critical sequence-specific contacts in NS3 RNA binding groove in subdomain III reveals potentially novel allosteric anti-viral drug targets.
FunCoup 4: new species, data, and visualization Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Christoph Ogris, Dimitri Guala, Mateusz Kaduk, Erik L.L. Sonnhammer
The regulatory G4 motif of the Kirsten ras (KRAS) gene is sensitive to guanine oxidation: implications on transcription Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Susanna Cogoi, Annalisa Ferino, Giulia Miglietta, Erik B. Pedersen, Luigi E. Xodo
KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.
Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Sridevi Ranganathan, Jonah Cheung, Michael Cassidy, Christopher Ginter, Janice D. Pata, Kathleen A. McDonough
Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr’s DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr’s multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member.
ClinVar: improving access to variant interpretations and supporting evidence Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Melissa J. Landrum, Jennifer M. Lee, Mark Benson, Garth R. Brown, Chen Chao, Shanmuga Chitipiralla, Baoshan Gu, Jennifer Hart, Douglas Hoffman, Wonhee Jang, Karen Karapetyan, Kenneth Katz, Chunlei Liu, Zenith Maddipatla, Adriana Malheiro, Kurt McDaniel, Michael Ovetsky, George Riley, George Zhou, J. Bradley Holmes, Brandi L. Kattman, Donna R. Maglott
ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) is a freely available, public archive of human genetic variants and interpretations of their significance to disease, maintained at the National Institutes of Health. Interpretations of the clinical significance of variants are submitted by clinical testing laboratories, research laboratories, expert panels and other groups. ClinVar aggregates data by variant-disease pairs, and by variant (or set of variants). Data aggregated by variant are accessible on the website, in an improved set of variant call format files and as a new comprehensive XML report. ClinVar recently started accepting submissions that are focused primarily on providing phenotypic information for individuals who have had genetic testing. Submissions may come from clinical providers providing their own interpretation of the variant (‘provider interpretation’) or from groups such as patient registries that primarily provide phenotypic information from patients (‘phenotyping only’). ClinVar continues to make improvements to its search and retrieval functions. Several new fields are now indexed for more precise searching, and filters allow the user to narrow down a large set of search results.
Expression Atlas: gene and protein expression across multiple studies and organisms Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Irene Papatheodorou, Nuno A. Fonseca, Maria Keays, Y. Amy Tang, Elisabet Barrera, Wojciech Bazant, Melissa Burke, Anja Füllgrabe, Alfonso Muñoz-Pomer Fuentes, Nancy George, Laura Huerta, Satu Koskinen, Suhaib Mohammed, Matthew Geniza, Justin Preece, Pankaj Jaiswal, Andrew F. Jarnuczak, Wolfgang Huber, Oliver Stegle, Juan Antonio Vizcaino, Alvis Brazma, Robert Petryszak
Expression Atlas (http://www.ebi.ac.uk/gxa) is an added value database that provides information about gene and protein expression in different species and contexts, such as tissue, developmental stage, disease or cell type. The available public and controlled access data sets from different sources are curated and re-analysed using standardized, open source pipelines and made available for queries, download and visualization. As of August 2017, Expression Atlas holds data from 3,126 studies across 33 different species, including 731 from plants. Data from large-scale RNA sequencing studies including Blueprint, PCAWG, ENCODE, GTEx and HipSci can be visualized next to each other. In Expression Atlas, users can query genes or gene-sets of interest and explore their expression across or within species, tissues, developmental stages in a constitutive or differential context, representing the effects of diseases, conditions or experimental interventions. All processed data matrices are available for direct download in tab-delimited format or as R-data. In addition to the web interface, data sets can now be searched and downloaded through the Expression Atlas R package. Novel features and visualizations include the on-the-fly analysis of gene set overlaps and the option to view gene co-expression in experiments investigating constitutive gene expression across tissues or other conditions.
Structural basis for the GTP specificity of the RNA kinase domain of fungal tRNA ligase Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Barbara S. Remus, Yehuda Goldgur, Stewart Shuman
Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2′,3′-cyclic-PO4 and 5′-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3′-OH,2′-PO4 and 5′-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and are promising targets for antifungal drug discovery because their domain compositions and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. A distinctive feature of Trl1 is its preferential use of GTP as phosphate donor for the RNA kinase reaction. Here we report the 2.2 Å crystal structure of the kinase domain of Trl1 from the fungal pathogen Candida albicans with GDP and Mg2+ in the active site. The P-loop phosphotransferase fold of the kinase is embellished by a unique ‘G-loop’ element that accounts for guanine nucleotide specificity. Mutations of amino acids that contact the guanine nucleobase efface kinase activity in vitro and Trl1 function in vivo. Our findings fortify the case for the Trl1 kinase as an antifungal target.
Nuclear mRNA degradation tunes the gain of the unfolded protein response in Saccharomyces cerevisiae Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Debasish Sarkar, Sunirmal Paira, Biswadip Das
Unfolded protein response (UPR) is triggered by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which is accomplished by a dramatic induction of genes encoding ER chaperones. Activation of these genes involves their rapid transcription by Hac1p, encoded by the HAC1 precursor transcript harboring an intron and a bipartite element (3′-BE) in the 3′-UTR. ER stress facilitates intracellular targeting and recruitment of HAC1 pre-mRNA to Ire1p foci (requiring 3′-BE), leading to its non-spliceosomal splicing mediated by Ire1p/Rlg1p. A critical concentration of the pre-HAC1 harboring a functional 3′-BE element is governed by its 3′→5′ decay by the nuclear exosome/DRN. In the absence of stress, pre-HAC1 mRNA undergoes a rapid and kinetic 3′→5′ decay leading to a precursor pool, the majority of which lack the BE element. Stress, in contrast, causes a diminished decay, thus resulting in the production of a population with an increased abundance of pre-HAC1 mRNA carrying an intact BE, which facilitates its more efficient recruitment to Ire1p foci. This mechanism plays a crucial role in the timely activation of UPR and its prompt attenuation following the accomplishment of homeostasis. Thus, a kinetic mRNA decay provides a novel paradigm for mRNA targeting and regulation of gene expression.
A Thermus phage protein inhibits host RNA polymerase by preventing template DNA strand loading during open promoter complex formation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Wei-Yang Ooi, Yuko Murayama, Vladimir Mekler, Leonid Minakhin, Konstantin Severinov, Shigeyuki Yokoyama, Shun-ichi Sekine
RNA polymerase (RNAP) is a major target of gene regulation. Thermus thermophilus bacteriophage P23–45 encodes two RNAP binding proteins, gp39 and gp76, which shut off host gene transcription while allowing orderly transcription of phage genes. We previously reported the structure of the T. thermophilus RNAP•σA holoenzyme complexed with gp39. Here, we solved the structure of the RNAP•σA holoenzyme bound with both gp39 and gp76, which revealed an unprecedented inhibition mechanism by gp76. The acidic protein gp76 binds within the RNAP cleft and occupies the path of the template DNA strand at positions –11 to –4, relative to the transcription start site at +1. Thus, gp76 obstructs the formation of an open promoter complex and prevents transcription by T. thermophilus RNAP from most host promoters. gp76 is less inhibitory for phage transcription, as tighter RNAP interaction with the phage promoters allows the template DNA to compete with gp76 for the common binding site. gp76 also inhibits Escherichia coli RNAP highlighting the template–DNA binding site as a new target site for developing antibacterial agents.
Structure of mycobacterial 3′-to-5′ RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Anam Ejaz, Heather Ordonez, Agata Jacewicz, Ryan Ferrao, Stewart Shuman
Mycobacterial Lhr is a DNA damage-inducible superfamily 2 helicase that uses adenosine triphosphate (ATP) hydrolysis to drive unidirectional 3′-to-5′ translocation along single-stranded DNA (ssDNA) and to unwind RNA:DNA duplexes en route. ATPase, translocase and helicase activities are encompassed within the N-terminal 856-amino acid segment. The crystal structure of Lhr-(1–856) in complex with AMPPNP•Mg2+ and ssDNA defines a new helicase family. The enzyme comprises two N-terminal RecA-like modules, a winged helix (WH) domain and a unique C-terminal domain. The 3′ ssDNA end binds in a crescent-shaped groove at the interface between the first RecA domain and the WH domain and tracks 5′ into a groove between the second RecA and C domains. A kissing interaction between the second RecA and C domains forms an aperture that demarcates a putative junction between the loading strand tail and the duplex, with the first duplex nucleoside bookended by stacking on Trp597. Intercalation of Ile528 between nucleosides of the loading strand creates another bookend. Coupling of ATP hydrolysis to RNA:DNA unwinding is dependent on Trp597 and Ile528, and on Thr145 and Arg279 that contact phosphates of the loading strand. The structural and functional data suggest a ratchet mechanism of translocation and unwinding coupled to ATP-driven domain movements.
The mechano-chemistry of a monomeric reverse transcriptase Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Omri Malik, Hadeel Khamis, Sergei Rudnizky, Ariel Kaplan
Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.
Protein complex scaffolding predicted as a prevalent function of long non-coding RNAs Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Diogo M. Ribeiro, Andreas Zanzoni, Andrea Cipriano, Riccardo Delli Ponti, Lionel Spinelli, Monica Ballarino, Irene Bozzoni, Gian Gaetano Tartaglia, Christine Brun
The human transcriptome contains thousands of long non-coding RNAs (lncRNAs). Characterizing their function is a current challenge. An emerging concept is that lncRNAs serve as protein scaffolds, forming ribonucleoproteins and bringing proteins in proximity. However, only few scaffolding lncRNAs have been characterized and the prevalence of this function is unknown. Here, we propose the first computational approach aimed at predicting scaffolding lncRNAs at large scale. We predicted the largest human lncRNA–protein interaction network to date using the catRAPID omics algorithm. In combination with tissue expression and statistical approaches, we identified 847 lncRNAs (∼5% of the long non-coding transcriptome) predicted to scaffold half of the known protein complexes and network modules. Lastly, we show that the association of certain lncRNAs to disease may involve their scaffolding ability. Overall, our results suggest for the first time that RNA-mediated scaffolding of protein complexes and modules may be a common mechanism in human cells.
Crystal structure of NucB, a biofilm-degrading endonuclease Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Arnaud Baslé, Lorraine Hewitt, Alan Koh, Heather K. Lamb, Paul Thompson, J. Grant Burgess, Michael J. Hall, Alastair R. Hawkins, Heath Murray, Richard J. Lewis
Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ββα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.
Separase prevents genomic instability by controlling replication fork speed Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Francesco Cucco, Elisa Palumbo, Serena Camerini, Barbara D’Alessio, Valentina Quarantotti, Maria Luisa Casella, Ilaria Maria Rizzo, Dubravka Cukrov, Domenico Delia, Antonella Russo, Marco Crescenzi, Antonio Musio
Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2–7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis.
Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Fang Yang, Yingying Lei, Meiling Zhou, Qili Yao, Yichao Han, Xiang Wu, Wanshun Zhong, Chenghang Zhu, Weize Xu, Ran Tao, Xi Chen, Da Lin, Khaista Rahman, Rohit Tyagi, Zeshan Habib, Shaobo Xiao, Dang Wang, Yang Yu, Huanchun Chen, Zhenfang Fu, Gang Cao
Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based ‘library vs library’ Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome–lysosome fusion. This concept can also be applied to other systems to screen protein–RNA and protein–DNA interactions and delineate signaling landscape in cells.
Translation can affect the antisense activity of RNase H1-dependent oligonucleotides targeting mRNAs Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Xue-Hai Liang, Joshua G. Nichols, Hong Sun, Stanley T. Crooke
RNase H1-dependent antisense oligonucleotides (ASOs) can degrade complementary RNAs in both the nucleus and the cytoplasm. Since cytoplasmic mRNAs are actively engaged in translation, ASO activity may thus be affected by translating ribosomes that scan the mRNAs. Here we show that mRNAs associated with ribosomes can be cleaved using ASOs and that translation can alter ASO activity. Translation inhibition tends to increase ASO activity when targeting the coding regions of efficiently translated mRNAs, but not nuclear non-coding RNAs or less efficiently translated mRNAs. Increasing the level of RNase H1 protein eliminated the enhancing effects of translation inhibition on ASO activity, suggesting that RNase H1 recruitment to ASO/mRNA heteroduplexes is a rate limiting step and that translating ribosomes can inhibit RNase H1 recruitment. Consistently, ASO activity was not increased by translation inhibition when targeting the 3′ UTRs, independent of the translation efficiency of the mRNAs. Contrarily, the activity of 3′ UTR-targeting ASOs tended to be reduced upon translation inhibition, likely due to decreased accessibility. These results indicate that ASO activity can be affected by the translation process, and the findings also provide important information toward helping better ASO drug design.
Human cancer tissues exhibit reduced A-to-I editing of miRNAs coupled with elevated editing of their targets Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Yishay Pinto, Ilana Buchumenski, Erez Y. Levanon, Eli Eisenberg
A-to-I RNA editing is an important post-transcriptional modification, known to be altered in tumors. It targets dozens of sites within miRNAs, some of which impact miRNA biogenesis and function, as well as many miRNA recognition sites. However, the full extent of the effect of editing on regulation by miRNAs and its behavior in human cancers is still unknown. Here we systematically characterized miRNA editing in 10 593 human samples across 32 cancer types and normal controls. We find that the majority of previously reported sites show little to no evidence for editing in this dataset, compile a list of 58 reliable miRNA editing sites, and study them across normal and cancer samples. Edited miRNA versions tend to suppress expression of known oncogenes, and, consistently, we observe a clear global tendency for hypo-editing in tumors, in strike contrast to the behavior for mRNA editing, allowing an accurate classification of normal/tumor samples based on their miRNA editing profile. In many cancers this profile correlates with patients' survival. Finally, thousands of miRNA binding sites are differentially edited in cancer. Our study thus establishes the important effect of RNA editing on miRNA-regulation in the tumor cell, with prospects for diagnostic and prognostic applications.
Expanding the repertoire of DNA shape features for genome-scale studies of transcription factor binding Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-20 Jinsen Li, Jared M. Sagendorf, Tsu-Pei Chiu, Marco Pasi, Alberto Perez, Remo Rohs
Uncovering the mechanisms that affect the binding specificity of transcription factors (TFs) is critical for understanding the principles of gene regulation. Although sequence-based models have been used successfully to predict TF binding specificities, we found that including DNA shape information in these models improved their accuracy and interpretability. Previously, we developed a method for modeling DNA binding specificities based on DNA shape features extracted from Monte Carlo (MC) simulations. Prediction accuracies of our models, however, have not yet been compared to accuracies of models incorporating DNA shape information extracted from X-ray crystallography (XRC) data or Molecular Dynamics (MD) simulations. Here, we integrated DNA shape information extracted from MC or MD simulations and XRC data into predictive models of TF binding and compared their performance. Models that incorporated structural information consistently showed improved performance over sequence-based models regardless of data source. Furthermore, we derived and validated nine additional DNA shape features beyond our original set of four features. The expanded repertoire of 13 distinct DNA shape features, including six intra-base pair and six inter-base pair parameters and minor groove width, is available in our R/Bioconductor package DNAshapeR and enables a comprehensive structural description of the double helix on a genome-wide scale.
Europe PMC in 2017 Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Maria Levchenko, Yuci Gou, Florian Graef, Audrey Hamelers, Zhan Huang, Michele Ide-Smith, Anusha Iyer, Oliver Kilian, Jyothi Katuri, Jee-Hyub Kim, Nikos Marinos, Rakesh Nambiar, Michael Parkin, Xingjun Pi, Frances Rogers, Francesco Talo, Vid Vartak, Aravind Venkatesan, Johanna McEntyre
Europe PMC (https://europepmc.org) is a comprehensive resource of biomedical research publications that offers advanced tools for search, retrieval, and interaction with the scientific literature. This article outlines new developments since 2014. In addition to delivering the core database and services, Europe PMC focuses on three areas of development: individual user services, data integration, and infrastructure to support text and data mining. Europe PMC now provides user accounts to save search queries and claim publications to ORCIDs, as well as open access profiles for authors based on public ORCID records. We continue to foster connections between scientific data and literature in a number of ways. All the data behind the paper - whether in structured archives, generic archives or as supplemental files - are now available via links to the BioStudies database. Text-mined biological concepts, including database accession numbers and data DOIs, are highlighted in the text and linked to the appropriate data resources. The SciLite community annotation platform accepts text-mining results from various contributors and overlays them on research articles as licence allows. In addition, text miners and developers can access all open content via APIs or via the FTP site.
Partial bisulfite conversion for unique template sequencing Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Vijay Kumar, Julie Rosenbaum, Zihua Wang, Talitha Forcier, Michael Ronemus, Michael Wigler, Dan Levy
We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone.
MethBank 3.0: a database of DNA methylomes across a variety of species Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Rujiao Li, Fang Liang, Mengwei Li, Dong Zou, Shixiang Sun, Yongbing Zhao, Wenming Zhao, Yiming Bao, Jingfa Xiao, Zhang Zhang
MethBank (http://bigd.big.ac.cn/methbank) is a database that integrates high-quality DNA methylomes across a variety of species and provides an interactive browser for visualization of methylation data. Here, we present an updated implementation of MethBank (version 3.0) by incorporating more DNA methylomes from multiple species and equipping with more enhanced functionalities for data annotation and more friendly web interfaces for data presentation, search and visualization. MethBank 3.0 features large-scale integration of high-quality methylomes, involving 34 consensus reference methylomes derived from a large number of human samples, 336 single-base resolution methylomes from different developmental stages and/or tissues of five plants, and 18 single-base resolution methylomes from gametes and early embryos at multiple stages of two animals. Additionally, it is enhanced by improving the functionalities for data annotation, which accordingly enables systematic identification of methylation sites closely associated with age, sites with constant methylation levels across different ages, differentially methylated promoters, age-specific differentially methylated cytosines/regions, and methylated CpG islands. Moreover, MethBank provides tools to estimate human methylation age online and to identify differentially methylated promoters, respectively. Taken together, MethBank is upgraded with significant improvements and advances over the previous version, which is of great help for deciphering DNA methylation regulatory mechanisms for epigenetic studies.
DNA polymerase beta participates in DNA End-joining Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Sreerupa Ray, Gregory Breuer, Michelle DeVeaux, Daniel Zelterman, Ranjit Bindra, Joann B. Sweasy
DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol μ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol β), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol β, fewer small deletions are observed. In addition, depletion of Pol β results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol β in functions in aNHEJ and provide mechanistic insight into its role in this process.
NRF2 regulates endothelial glycolysis and proliferation with miR-93 and mediates the effects of oxidized phospholipids on endothelial activation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Suvi M. Kuosmanen, Emilia Kansanen, Minna U. Kaikkonen, Virve Sihvola, Kati Pulkkinen, Henna-Kaisa Jyrkkänen, Pauli Tuoresmäki, Juha Hartikainen, Mikko Hippeläinen, Hannu Kokki, Pasi Tavi, Sami Heikkinen, Anna-Liisa Levonen
Phospholipids, such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), are the major components of cell membranes. Their exposure to reactive oxygen species creates oxidized phospholipids, which predispose to the development of chronic inflammatory diseases and metabolic disorders through endothelial activation and dysfunction. Although the effects of oxidized PAPC (oxPAPC) on endothelial cells have been previously studied, the underlying molecular mechanisms evoking biological responses remain largely unknown. Here, we investigated the molecular mechanisms of oxPAPC function with a special emphasis on NRF2-regulated microRNAs (miRNAs) in human umbilical vein endothelial cells (HUVECs) utilizing miRNA profiling, global run-on sequencing (GRO-seq), genome-wide NRF2 binding model, and RNA sequencing (RNA-seq) with miRNA overexpression and silencing. We report that the central regulators of endothelial activity, KLF2 for quiescence, PFKFB3 for glycolysis, and VEGFA, FOXO1 and MYC for growth and proliferation, are regulated by transcription factor NRF2 and the NRF2-regulated miR-106b∼25 cluster member, miR-93, in HUVECs. Mechanistically, oxPAPC was found to induce glycolysis and proliferation NRF2-dependently, and oxPAPC-dependent induction of the miR-106b∼25 cluster was mediated by NRF2. Additionally, several regulatory loops were established between NRF2, miR-93 and the essential regulators of healthy endothelium, collectively implying that NRF2 controls the switch between the quiescent and the proliferative endothelial states together with miR-93.
JASPAR 2018: update of the open-access database of transcription factor binding profiles and its web framework Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Aziz Khan, Oriol Fornes, Arnaud Stigliani, Marius Gheorghe, Jaime A. Castro-Mondragon, Robin van der Lee, Adrien Bessy, Jeanne Chèneby, Shubhada R. Kulkarni, Ge Tan, Damir Baranasic, David J. Arenillas, Albin Sandelin, Klaas Vandepoele, Boris Lenhard, Benoît Ballester, Wyeth W. Wasserman, François Parcy, Anthony Mathelier
Nucleic Acids Research (2017), https://doi.org/10.1093/nar/gkx1126
Linnorm: improved statistical analysis for single cell RNA-seq expression data Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-17 Shun H. Yip, Panwen Wang, Jean-Pierre A. Kocher, Pak Chung Sham, Junwen Wang
Nucleic Acids Research (2017), https://doi.org/10.1093/nar/gkx828
Ensembl 2018 Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Daniel R. Zerbino, Premanand Achuthan, Wasiu Akanni, M. Ridwan Amode, Daniel Barrell, Jyothish Bhai, Konstantinos Billis, Carla Cummins, Astrid Gall, Carlos García Girón, Laurent Gil, Leo Gordon, Leanne Haggerty, Erin Haskell, Thibaut Hourlier, Osagie G. Izuogu, Sophie H. Janacek, Thomas Juettemann, Jimmy Kiang To, Matthew R. Laird, Ilias Lavidas, Zhicheng Liu, Jane E. Loveland, Thomas Maurel, William McLaren, Benjamin Moore, Jonathan Mudge, Daniel N. Murphy, Victoria Newman, Michael Nuhn, Denye Ogeh, Chuang Kee Ong, Anne Parker, Mateus Patricio, Harpreet Singh Riat, Helen Schuilenburg, Dan Sheppard, Helen Sparrow, Kieron Taylor, Anja Thormann, Alessandro Vullo, Brandon Walts, Amonida Zadissa, Adam Frankish, Sarah E. Hunt, Myrto Kostadima, Nicholas Langridge, Fergal J. Martin, Matthieu Muffato, Emily Perry, Magali Ruffier, Dan M. Staines, Stephen J. Trevanion, Bronwen L. Aken, Fiona Cunningham, Andrew Yates, Paul Flicek
The Ensembl project has been aggregating, processing, integrating and redistributing genomic datasets since the initial releases of the draft human genome, with the aim of accelerating genomics research through rapid open distribution of public data. Large amounts of raw data are thus transformed into knowledge, which is made available via a multitude of channels, in particular our browser (http://www.ensembl.org). Over time, we have expanded in multiple directions. First, our resources describe multiple fields of genomics, in particular gene annotation, comparative genomics, genetics and epigenomics. Second, we cover a growing number of genome assemblies; Ensembl Release 90 contains exactly 100. Third, our databases feed simultaneously into an array of services designed around different use cases, ranging from quick browsing to genome-wide bioinformatic analysis. We present here the latest developments of the Ensembl project, with a focus on managing an increasing number of assemblies, supporting efforts in genome interpretation and improving our browser.
The human papillomavirus DNA helicase E1 binds, stimulates, and confers processivity to cellular DNA polymerase epsilon Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Michaelle Chojnacki, Thomas Melendy
The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly specific and occurs even in the absence of the known pol ϵ cofactors Replication Factor C (RFC), Proliferating Cell Nuclear Antigen (PCNA) and RPA. This stimulation is due to an increase in the processivity of pol ϵ and occurs independently of pol ϵ’s replication cofactors. This increase in processivity is dependent on the ability of the E1 helicase to hydrolyze ATP, suggesting it is dependent on E1’s helicase action. In addition, RPA, thought to be vital for processive DNA synthesis by both pol ϵ and pol δ, was found to be dispensable for processive synthesis by pol ϵ in the presence of E1. Overall, E1 appears to be conferring processivity to pol ϵ by directly tethering pol ϵ to the DNA parental strand and towing ϵ behind the E1 helicase as the replication fork progresses; and thereby apparently obviating the need for RPA for leading strand synthesis. Thus far only pol α and pol δ have been implicated in the DNA replication of mammalian viruses; this is the first reported example of a virus recruiting pol ϵ. Furthermore, this demonstrates a unique capacity of a viral helicase having evolved to stimulate a cellular replicative DNA polymerase.
GPCRdb in 2018: adding GPCR structure models and ligands Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Gáspár Pándy-Szekeres, Christian Munk, Tsonko M. Tsonkov, Stefan Mordalski, Kasper Harpsøe, Alexander S. Hauser, Andrzej J. Bojarski, David E. Gloriam
G protein-coupled receptors are the most abundant mediators of both human signalling processes and therapeutic effects. Herein, we report GPCRome-wide homology models of unprecedented quality, and roughly 150 000 GPCR ligands with data on biological activities and commercial availability. Based on the strategy of ’Less model – more Xtal’, each model exploits both a main template and alternative local templates. This achieved higher similarity to new structures than any of the existing resources, and refined crystal structures with missing or distorted regions. Models are provided for inactive, intermediate and active states—except for classes C and F that so far only have inactive templates. The ligand database has separate browsers for: (i) target selection by receptor, family or class, (ii) ligand filtering based on cross-experiment activities (min, max and mean) or chemical properties, (iii) ligand source data and (iv) commercial availability. SMILES structures and activity spreadsheets can be downloaded for further processing. Furthermore, three recent landmark publications on GPCR drugs, G protein selectivity and genetic variants have been accompanied with resources that now let readers view and analyse the findings themselves in GPCRdb. Altogether, this update will enable scientific investigation for the wider GPCR community. GPCRdb is available at http://www.gpcrdb.org.
The discovery potential of RNA processing profiles Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Amadís Pagès, Ivan Dotu, Joan Pallarès-Albanell, Eulàlia Martí, Roderic Guigó, Eduardo Eyras
Small non-coding RNAs (sncRNAs) are highly abundant molecules that regulate essential cellular processes and are classified according to sequence and structure. Here we argue that read profiles from size-selected RNA sequencing capture the post-transcriptional processing specific to each RNA family, thereby providing functional information independently of sequence and structure. We developed SeRPeNT, a new computational method that exploits reproducibility across replicates and uses dynamic time-warping and density-based clustering algorithms to identify, characterize and compare sncRNAs by harnessing the power of read profiles. We applied SeRPeNT to: (i) generate an extended human annotation with 671 new sncRNAs from known classes and 131 from new potential classes, (ii) show pervasive differential processing of sncRNAs between cell compartments and (iii) predict new molecules with miRNA-like behaviour from snoRNA, tRNA and long non-coding RNA precursors, potentially dependent on the miRNA biogenesis pathway. Furthermore, we validated experimentally four predicted novel non-coding RNAs: a miRNA, a snoRNA-derived miRNA, a processed tRNA and a new uncharacterized sncRNA. SeRPeNT facilitates fast and accurate discovery and characterization of sncRNAs at an unprecedented scale. SeRPeNT code is available under the MIT license at https://github.com/comprna/SeRPeNT.
Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Yanhui Hu, Arunachalam Vinayagam, Ankita Nand, Aram Comjean, Verena Chung, Tong Hao, Stephanie E. Mohr, Norbert Perrimon
Model organism and human databases are rich with information about genetic and physical interactions. These data can be used to interpret and guide the analysis of results from new studies and develop new hypotheses. Here, we report the development of the Molecular Interaction Search Tool (MIST; http://fgrtools.hms.harvard.edu/MIST/). The MIST database integrates biological interaction data from yeast, nematode, fly, zebrafish, frog, rat and mouse model systems, as well as human. For individual or short gene lists, the MIST user interface can be used to identify interacting partners based on protein–protein and genetic interaction (GI) data from the species of interest as well as inferred interactions, known as interologs, and to view a corresponding network. The data, interologs and search tools at MIST are also useful for analyzing ‘omics datasets. In addition to describing the integrated database, we also demonstrate how MIST can be used to identify an appropriate cut-off value that balances false positive and negative discovery, and present use-cases for additional types of analysis. Altogether, the MIST database and search tools support visualization and navigation of existing protein and GI data, as well as comparison of new and existing data.
PRGdb 3.0: a comprehensive platform for prediction and analysis of plant disease resistance genes Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Cristina M. O. Cruz, Andreu Paytuvi-Gallart, Antimo Di Donato, Vicky Sundesha, Giuseppe Andolfo, Riccardo Aiese Cigliano, Walter Sanseverino, Maria R. Ercolano
The Plant Resistance Genes database (PRGdb; http://prgdb.org) has been redesigned with a new user interface, new sections, new tools and new data for genetic improvement, allowing easy access not only to the plant science research community but also to breeders who want to improve plant disease resistance. The home page offers an overview of easy-to-read search boxes that streamline data queries and directly show plant species for which data from candidate or cloned genes have been collected. Bulk data files and curated resistance gene annotations are made available for each plant species hosted. The new Gene Model view offers detailed information on each cloned resistance gene structure to highlight shared attributes with other genes. PRGdb 3.0 offers 153 reference resistance genes and 177 072 annotated candidate Pathogen Receptor Genes (PRGs). Compared to the previous release, the number of putative genes has been increased from 106 to 177 K from 76 sequenced Viridiplantae and algae genomes. The DRAGO 2 tool, which automatically annotates and predicts (PRGs) from DNA and amino acid with high accuracy and sensitivity, has been added. BLAST search has been implemented to offer users the opportunity to annotate and compare their own sequences. The improved section on plant diseases displays useful information linked to genes and genomes to connect complementary data and better address specific needs. Through, a revised and enlarged collection of data, the development of new tools and a renewed portal, PRGdb 3.0 engages the plant science community in developing a consensus plan to improve knowledge and strategies to fight diseases that afflict main crops and other plants.
Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Carika Weldon, Justine G. Dacanay, Vijay Gokhale, Peda Venkat L. Boddupally, Isabelle Behm-Ansmant, Glenn A. Burley, Christiane Branlant, Laurence H. Hurley, Cyril Dominguez, Ian C. Eperon
Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5′ splice site (5′SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5′SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5′SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5′SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.
Structural rearrangements in mRNA upon its binding to human 80S ribosomes revealed by EPR spectroscopy Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Alexey A. Malygin, Dmitri M. Graifer, Maria I. Meschaninova, Alya G. Venyaminova, Ivan O. Timofeev, Andrey A. Kuzhelev, Olesya A. Krumkacheva, Matvey V. Fedin, Galina G. Karpova, Elena G. Bagryanskaya
The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5′- and 3′-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.
DIANA-TarBase v8: a decade-long collection of experimentally supported miRNA–gene interactions Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Dimitra Karagkouni, Maria D. Paraskevopoulou, Serafeim Chatzopoulos, Ioannis S. Vlachos, Spyros Tastsoglou, Ilias Kanellos, Dimitris Papadimitriou, Ioannis Kavakiotis, Sofia Maniou, Giorgos Skoufos, Thanasis Vergoulis, Theodore Dalamagas, Artemis G. Hatzigeorgiou
DIANA-TarBase v8 (http://www.microrna.gr/tarbase) is a reference database devoted to the indexing of experimentally supported microRNA (miRNA) targets. Its eighth version is the first database indexing >1 million entries, corresponding to ∼670 000 unique miRNA-target pairs. The interactions are supported by >33 experimental methodologies, applied to ∼600 cell types/tissues under ∼451 experimental conditions. It integrates information on cell-type specific miRNA–gene regulation, while hundreds of thousands of miRNA-binding locations are reported. TarBase is coming of age, with more than a decade of continuous support in the non-coding RNA field. A new module has been implemented that enables the browsing of interactions through different filtering combinations. It permits easy retrieval of positive and negative miRNA targets per species, methodology, cell type and tissue. An incorporated ranking system is utilized for the display of interactions based on the robustness of their supporting methodologies. Statistics, pie-charts and interactive bar-plots depicting the database content are available through a dedicated result page. An intuitive interface is introduced, providing a user-friendly application with flexible options to different queries.
DGIdb 3.0: a redesign and expansion of the drug–gene interaction database Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Kelsy C. Cotto, Alex H. Wagner, Yang-Yang Feng, Susanna Kiwala, Adam C. Coffman, Gregory Spies, Alex Wollam, Nicholas C. Spies, Obi L. Griffith, Malachi Griffith
The drug–gene interaction database (DGIdb, www.dgidb.org) consolidates, organizes and presents drug–gene interactions and gene druggability information from papers, databases and web resources. DGIdb normalizes content from 30 disparate sources and allows for user-friendly advanced browsing, searching and filtering for ease of access through an intuitive web user interface, application programming interface (API) and public cloud-based server image. DGIdb v3.0 represents a major update of the database. Nine of the previously included 24 sources were updated. Six new resources were added, bringing the total number of sources to 30. These updates and additions of sources have cumulatively resulted in 56 309 interaction claims. This has also substantially expanded the comprehensive catalogue of druggable genes and anti-neoplastic drug–gene interactions included in the DGIdb. Along with these content updates, v3.0 has received a major overhaul of its codebase, including an updated user interface, preset interaction search filters, consolidation of interaction information into interaction groups, greatly improved search response times and upgrading the underlying web application framework. In addition, the expanded API features new endpoints which allow users to extract more detailed information about queried drugs, genes and drug–gene interactions, including listings of PubMed IDs, interaction type and other interaction metadata.
aBiofilm: a resource of anti-biofilm agents and their potential implications in targeting antibiotic drug resistance Nucleic Acids Res. (IF 10.162) Pub Date : 2017-11-16 Akanksha Rajput, Anamika Thakur, Shivangi Sharma, Manoj Kumar
Biofilms play an important role in the antibiotic drug resistance, which is threatening public health globally. Almost, all microbes mimic multicellular lifestyle to form biofilm by undergoing phenotypic changes to adapt adverse environmental conditions. Many anti-biofilm agents have been experimentally validated to disrupt the biofilms during last three decades. To organize this data, we developed the ‘aBiofilm’ resource (http://bioinfo.imtech.res.in/manojk/abiofilm/) that harbors a database, a predictor, and the data visualization modules. The database contains biological, chemical, and structural details of 5027 anti-biofilm agents (1720 unique) reported from 1988–2017. These agents target over 140 organisms including Gram-negative, Gram-positive bacteria, and fungus. They are mainly chemicals, peptides, phages, secondary metabolites, antibodies, nanoparticles and extracts. They show the diverse mode of actions by attacking mainly signaling molecules, biofilm matrix, genes, extracellular polymeric substances, and many more. The QSAR based predictor identifies the anti-biofilm potential of an unknown chemical with an accuracy of ∼80.00%. The data visualization section summarized the biofilm stages targeted (Circos plot); interaction maps (Cytoscape) and chemicals diversification (CheS-Mapper) of the agents. This comprehensive platform would help the researchers to understand the multilevel communication in the microbial consortium. It may aid in developing anti-biofilm therapeutics to deal with antibiotic drug resistance menace.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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