Nucleic Acids Research (NUCLEIC ACIDS RES )

Publisher: Oxford University Press


Nucleic Acids Research (NAR) publishes the results of leading edge research into physical, chemical, biochemical and biological aspects of nucleic acids and proteins involved in nucleic acid metabolism and/or interactions. It enables the rapid publication of papers under the following categories: RNA, molecular biology, chemistry, genomics, computational biology and structural biology. A Survey and Summary section provides a format for brief reviews. The first issue of each year is devoted to biological databases.

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    Nucleic acids research
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Oxford University Press

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    • 12 month embargo on science, technology, medicine articles
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    • Articles in some journals can be made Open Access on payment of additional charge
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Publications in this journal

  • Xiaoxia Liu, Wei Wang, Dmitry Samarsky, Li Liu, Qian Xu, Wenqing Zhang, Guangzu Zhu, Ping Wu, Xialin Zuo, Houliang Deng, Jingjing Zhang, Zhuomin Wu, Xiaohui Chen, Lingfeng Zhao, Zhiyong Qiu, Zhongyi Zhang, Qiyi Zeng, Wei Yang, Biliang Zhang, Aimin Ji
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    ABSTRACT: RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvβ3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvβ3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.
    Nucleic Acids Research 09/2014;
  • Anthony J Davis, Linfeng Chi, Sairei So, Kyung-Jong Lee, Eiichiro Mori, Kazi Fattah, Jun Yang, David J Chen
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    ABSTRACT: Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.
    Nucleic Acids Research 09/2014;
  • Xu Peng, Jingyi Wu, Reinhard Brunmeir, Sun-Yee Kim, Qiongyi Zhang, Chunming Ding, Weiping Han, Wei Xie, Feng Xu
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    ABSTRACT: Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (i) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (ii) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (iii) achieved paired-end sequencing of the ChIP-seq libraries; (iv) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq and (v) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies.
    Nucleic Acids Research 09/2014;
  • Pablo Daniel Dans, Ignacio Faustino, Federica Battistini, Krystyna Zakrzewska, Richard Lavery, Modesto Orozco
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    ABSTRACT: We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3'-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change.
    Nucleic Acids Research 09/2014;
  • James E Thornton, Peng Du, Lili Jing, Ljiljana Sjekloca, Shuibin Lin, Elena Grossi, Piotr Sliz, Leonard I Zon, Richard I Gregory
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    ABSTRACT: Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.
    Nucleic Acids Research 09/2014;
  • Maïka Jangal, Jean-Philippe Couture, Stéphanie Bianco, Luca Magnani, Hisham Mohammed, Nicolas Gévry
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    ABSTRACT: Chromatin constitutes a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Nucleosomes prevent the binding of estrogen receptor α (ERα) in absence of ligand and thus represent an important level of transcriptional regulation. Here, we show that in breast cancer MCF-7 cells, TLE3, a co-repressor of the Groucho/Grg/TLE family, interacts with FoxA1 and is detected at regulatory elements of ERα target genes in absence of estrogen. As a result, the chromatin is maintained in a basal state of acetylation, thus preventing ligand-independent activation of transcription. In absence of TLE3, the basal expression of ERα target genes induced by E2 is increased. At the TFF1 gene, the recruitment of TLE3 to the chromatin is FoxA1-dependent and prevents ERα and RNA polymerase II recruitment to TFF1 gene regulatory elements. Moreover, the interaction of TLE3 with HDAC2 results in the maintenance of acetylation at a basal level. We also provide evidence that TLE3 is recruited at several other regulatory elements of ERα target genes and is probably an important co-regulator of the E2 signaling pathway. In sum, our results describe a mechanism by which TLE3 affects ligand dependency in ERα-regulated gene expression via its binding restricting function and its role in gene regulation by histone acetylation.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Recent studies have shown that basic steric and connectivity constraints encoded at the secondary structure level are key determinants of 3D structure and dynamics in simple two-way RNA junctions. However, the role of these topological constraints in higher order RNA junctions remains poorly understood. Here, we use a specialized coarse-grained molecular dynamics model to directly probe the thermodynamic contributions of topological constraints in defining the 3D architecture and dynamics of transfer RNA (tRNA). Topological constraints alone restrict tRNA's allowed conformational space by over an order of magnitude and strongly discriminate against formation of non-native tertiary contacts, providing a sequence independent source of folding specificity. Topological constraints also give rise to long-range correlations between the relative orientation of tRNA's helices, which in turn provides a mechanism for encoding thermodynamic cooperativity between distinct tertiary interactions. These aspects of topological constraints make it such that only several tertiary interactions are needed to confine tRNA to its native global structure and specify functionally important 3D dynamics. We further show that topological constraints are conserved across tRNA's different naturally occurring secondary structures. Taken together, our results emphasize the central role of secondary-structure-encoded topological constraints in defining RNA 3D structure, dynamics and folding.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells, revealing two prominent genetic networks. Genes largely associated with muscle development were down-regulated by loss of MEF2A while up-regulated genes reveal a previously unrecognized function of MEF2A in suppressing growth/proliferative genes. Several up-regulated (Tprg, Mctp2, Kitl, Prrx1, Dusp6) and down-regulated (Atp1a2, Hspb7, Tmem182, Sorbs2, Lmod3) MEF2A target genes were chosen for further investigation. Interestingly, siRNA targeting of the MEF2A/D heterodimer revealed a somewhat divergent role in the regulation of Dusp6, a MAPK phosphatase, in cardiac and skeletal myogenic lineages. Furthermore, MEF2D functions as a p38MAPK-dependent repressor of Dusp6 in myoblasts. These data illustrate that MEF2 orchestrates both common and non-overlapping programs of signal-dependent gene expression in skeletal and cardiac muscle lineages.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Post-transcriptional gene regulation mechanisms decide on cellular mRNA activities. Essential gatekeepers of post-transcriptional mRNA regulation are broadly conserved mRNA-modifying enzymes, such as cytoplasmic poly(A) polymerases (cytoPAPs). Although these non-canonical nucleotidyltransferases efficiently elongate mRNA poly(A) tails in artificial tethering assays, we still know little about their global impact on poly(A) metabolism and their individual molecular roles in promoting protein production in organisms. Here, we use the animal model Caenorhabditis elegans to investigate the global mechanisms of two germline-enriched cytoPAPs, GLD-2 and GLD-4, by combining polysome profiling with RNA sequencing. Our analyses suggest that GLD-2 activity mediates mRNA stability of many translationally repressed mRNAs. This correlates with a general shortening of long poly(A) tails in gld-2-compromised animals, suggesting that most if not all targets are stabilized via robust GLD-2-mediated polyadenylation. By contrast, only mild polyadenylation defects are found in gld-4-compromised animals and few mRNAs change in abundance. Interestingly, we detect a reduced number of polysomes in gld-4 mutants and GLD-4 protein co-sediments with polysomes, which together suggest that GLD-4 might stimulate or maintain translation directly. Our combined data show that distinct cytoPAPs employ different RNA-regulatory mechanisms to promote gene expression, offering new insights into translational activation of mRNAs.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: The nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2'-deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the associated adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to determine the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helices were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic separation between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixture of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity and mutagenic outcomes, and support recent experiments suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochemical experiments on this potentially genotoxic lesion.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor expressed in metazoan cells that is responsible for eliciting the production of type I interferons and pro-inflammatory cytokines upon detection of intracellular, non-self RNA. Structural studies of RIG-I have identified a novel Pincer domain composed of two alpha helices that physically tethers the C-terminal domain to the SF2 helicase core. We find that the Pincer plays an important role in mediating the enzymatic and signaling activities of RIG-I. We identify a series of mutations that additively decouple the Pincer motif from the ATPase core and show that this decoupling results in impaired signaling. Through enzymological and biophysical analysis, we further show that the Pincer domain controls coupled enzymatic activity of the protein through allosteric control of the ATPase core. Further, we show that select regions of the HEL1 domain have evolved to potentiate interactions with the Pincer domain, resulting in an adapted ATPase cleft that is now responsive to adjacent domains that selectively bind viral RNA.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (σ(A)) or any other σ factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of σ(A). The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with σ(A). Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Repeat-associated disorders caused by expansions of short sequences have been classified as coding and noncoding and are thought to be caused by protein gain-of-function and RNA gain-of-function mechanisms, respectively. The boundary between such classifications has recently been blurred by the discovery of repeat-associated non-AUG (RAN) translation reported in spinocerebellar ataxia type 8, myotonic dystrophy type 1, fragile X tremor/ataxia syndrome and C9ORF72 amyotrophic lateral sclerosis and frontotemporal dementia. This noncanonical translation requires no AUG start codon and can initiate in multiple frames of CAG, CGG and GGGGCC repeats of the sense and antisense strands of disease-relevant transcripts. RNA structures formed by the repeats have been suggested as possible triggers; however, the precise mechanism of the translation initiation remains elusive. Templates containing expansions of microsatellites have also been shown to challenge translation elongation, as frameshifting has been recognized across CAG repeats in spinocerebellar ataxia type 3 and Huntington's disease. Determining the critical requirements for RAN translation and frameshifting is essential to decipher the mechanisms that govern these processes. The contribution of unusual translation products to pathogenesis needs to be better understood. In this review, we present current knowledge regarding RAN translation and frameshifting and discuss the proposed mechanisms of translational challenges imposed by simple repeat expansions.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: In eukaryotes, the location of a gene on the chromosome is known to affect its expression, but such position effects are poorly understood in bacteria. Here, using Escherichia coli K-12, we demonstrate that expression of a reporter gene cassette, comprised of the model E. coli lac promoter driving expression of gfp, varies by ∼300-fold depending on its precise position on the chromosome. At some positions, expression was more than 3-fold higher than at the natural lac promoter locus, whereas at several other locations, the reporter cassette was completely silenced: effectively overriding local lac promoter control. These effects were not due to differences in gene copy number, caused by partially replicated genomes. Rather, the differences in gene expression occur predominantly at the level of transcription and are mediated by several different features that are involved in chromosome organization. Taken together, our findings identify a tier of gene regulation above local promoter control and highlight the importance of chromosome position effects on gene expression profiles in bacteria.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far; two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n=382), survival data (n=530), and lymph node metastases (n=100) in lung cancer patients identified non-metastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling. Importantly, we detected binding of NME2 in promoter of focal adhesion factor, vinculin. Reduced expression of NME2 led to enhanced transcription of vinculin. In comparison, NME1, a close homolog of NME2 did not bind to vinculin promoter nor regulate its expression. In line, enhanced metastasis of NME2-depleted lung cancer cells was found in zebrafish and nude mice tumor models. The metastatic potential of NME2-depleted cells was remarkably diminished upon selective RNA-i mediated silencing of vinculin. Together, we demonstrate that reduced NME2 levels lead to transcriptional de-repression of vinculin and regulate lung cancer metastasis.
    Nucleic Acids Research 09/2014; In Press.
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    ABSTRACT: Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high-throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3' major domain of the 20S pre-ribosomal RNA.
    Nucleic Acids Research 09/2014;
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    ABSTRACT: This study unveils Mycobacterium smegmatis DinB2 as the founder of a clade of Y-family DNA polymerase that is naturally adept at incorporating ribonucleotides by virtue of a leucine in lieu of a canonical aromatic steric gate. DinB2 efficiently scavenges limiting dNTP and rNTP substrates in the presence of manganese. DinB2's sugar selectivity factor, gauged by rates of manganese-dependent dNMP versus rNMP addition, is 2.7- to 3.8-fold. DinB2 embeds ribonucleotides during DNA synthesis when rCTP and dCTP are at equimolar concentration. DinB2 can incorporate at least 16 consecutive ribonucleotides. In magnesium, DinB2 has a 26- to 78-fold lower affinity for rNTPs than dNTPs, but only a 2.6- to 6-fold differential in rates of deoxy versus ribo addition (kpol). Two other M. smegmatis Y-family polymerases, DinB1 and DinB3, are characterized here as template-dependent DNA polymerases that discriminate strongly against ribonucleotides, a property that, in the case of DinB1, correlates with its aromatic steric gate side chain. We speculate that the unique ability of DinB2 to utilize rNTPs might allow for DNA repair with a 'ribo patch' when dNTPs are limiting. Phylogenetic analysis reveals DinB2-like polymerases, with leucine, isoleucine or valine steric gates, in many taxa of the phylum Actinobacteria.
    Nucleic Acids Research 09/2014;

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