Nucleic Acids Research

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RNAlien - Unsupervised RNA family model construction

чт, 2016-09-29 11:06

Determining the function of a non-coding RNA requires costly and time-consuming wet-lab experiments. For this reason, computational methods which ascertain the homology of a sequence and thereby deduce functionality and family membership are often exploited. In this fashion, newly sequenced genomes can be annotated in a completely computational way. Covariance models are commonly used to assign novel RNA sequences to a known RNA family. However, to construct such models several examples of the family have to be already known. Moreover, model building is the work of experts who manually edit the necessary RNA alignment and consensus structure. Our method, RNAlien, starting from a single input sequence collects potential family member sequences by multiple iterations of homology search. RNA family models are fully automatically constructed for the found sequences. We have tested our method on a subset of the Rfam RNA family database. RNAlien models are a starting point to construct models of comparable sensitivity and specificity to manually curated ones from the Rfam database. RNAlien Tool and web server are available at http://rna.tbi.univie.ac.at/rnalien/.

Категории: Bioinformatics, Biology, Journals

YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit

чт, 2016-09-29 11:06

YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.

Категории: Bioinformatics, Biology, Journals

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

чт, 2016-09-29 11:06

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO2 stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.

Категории: Bioinformatics, Biology, Journals

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

чт, 2016-09-29 11:06

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3'-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.

Категории: Bioinformatics, Biology, Journals

FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing

пн, 2016-09-19 08:02

Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of ‘actionable’ mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel.

Категории: Bioinformatics, Biology, Journals

The exon quantification pipeline (EQP): a comprehensive approach to the quantification of gene, exon and junction expression from RNA-seq data

пн, 2016-09-19 08:02

The quantification of transcriptomic features is the basis of the analysis of RNA-seq data. We present an integrated alignment workflow and a simple counting-based approach to derive estimates for gene, exon and exon–exon junction expression. In contrast to previous counting-based approaches, EQP takes into account only reads whose alignment pattern agrees with the splicing pattern of the features of interest. This leads to improved gene expression estimates as well as to the generation of exon counts that allow disambiguating reads between overlapping exons. Unlike other methods that quantify skipped introns, EQP offers a novel way to compute junction counts based on the agreement of the read alignments with the exons on both sides of the junction, thus providing a uniformly derived set of counts. We evaluated the performance of EQP on both simulated and real Illumina RNA-seq data and compared it with other quantification tools. Our results suggest that EQP provides superior gene expression estimates and we illustrate the advantages of EQP's exon and junction counts. The provision of uniformly derived high-quality counts makes EQP an ideal quantification tool for differential expression and differential splicing studies. EQP is freely available for download at https://github.com/Novartis/EQP-cluster.

Категории: Bioinformatics, Biology, Journals

Boiler: lossy compression of RNA-seq alignments using coverage vectors

пн, 2016-09-19 08:02

We describe Boiler, a new software tool for compressing and querying large collections of RNA-seq alignments. Boiler discards most per-read data, keeping only a genomic coverage vector plus a few empirical distributions summarizing the alignments. Since most per-read data is discarded, storage footprint is often much smaller than that achieved by other compression tools. Despite this, the most relevant per-read data can be recovered; we show that Boiler compression has only a slight negative impact on results given by downstream tools for isoform assembly and quantification. Boiler also allows the user to pose fast and useful queries without decompressing the entire file. Boiler is free open source software available from github.com/jpritt/boiler.

Категории: Bioinformatics, Biology, Journals

NEpiC: a network-assisted algorithm for epigenetic studies using mean and variance combined signals

пн, 2016-09-19 08:02

DNA methylation plays an important role in many biological processes. Existing epigenome-wide association studies (EWAS) have successfully identified aberrantly methylated genes in many diseases and disorders with most studies focusing on analysing methylation sites one at a time. Incorporating prior biological information such as biological networks has been proven to be powerful in identifying disease-associated genes in both gene expression studies and genome-wide association studies (GWAS) but has been under studied in EWAS. Although recent studies have noticed that there are differences in methylation variation in different groups, only a few existing methods consider variance signals in DNA methylation studies. Here, we present a network-assisted algorithm, NEpiC, that combines both mean and variance signals in searching for differentially methylated sub-networks using the protein–protein interaction (PPI) network. In simulation studies, we demonstrate the power gain from using both the prior biological information and variance signals compared to using either of the two or neither information. Applications to several DNA methylation datasets from the Cancer Genome Atlas (TCGA) project and DNA methylation data on hepatocellular carcinoma (HCC) from the Columbia University Medical Center (CUMC) suggest that the proposed NEpiC algorithm identifies more cancer-related genes and generates better replication results.

Категории: Bioinformatics, Biology, Journals

Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA

пн, 2016-09-19 08:02

RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly used ligation approach, and Illumina sequencing. We describe a methodology to detect 2'-O-methylations with high sensitivity and reproducibility even with limited amount of starting material (1 ng of total RNA). The method provides a quantification of the 2'-O-methylation occupancy of a given site, allowing to detect relatively small changes (>10%) in 2'-O-methylation profiles. Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in pathologies.

Категории: Bioinformatics, Biology, Journals

PABP enhances release factor recruitment and stop codon recognition during translation termination

пн, 2016-09-19 08:02

Poly(A)-binding protein (PABP) is a major component of the messenger RNA–protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl–tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1–eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.

Категории: Bioinformatics, Biology, Journals

The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

пн, 2016-09-19 08:02

The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D.

Категории: Bioinformatics, Biology, Journals

Efficiency of integron cassette insertion in correct orientation is ensured by the interplay of the three unpaired features of attC recombination sites

пн, 2016-09-19 08:02

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins. Here, we investigated the basis of such strand selectivity by comparing recombination of wild-type and mutated attC sites with different lengths, sequences and structures. We show that all three unpaired structural features that distinguish the bottom and top strands contribute to strand selectivity. The localization of Extra-Helical Bases (EHBs) directly favors integrase binding to the bottom strand. The Unpaired Central Spacer (UCS) and the Variable Terminal Structure (VTS) influence strand selectivity indirectly, probably through the stabilization of the bottom strand and the resulting synapse due to the nucleotide skew between the two strands. These results underscore the importance of the single-stranded nature of the attC site that allows such tight control over integron cassette orientation.

Категории: Bioinformatics, Biology, Journals

Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs

пн, 2016-09-19 08:02

Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.

Категории: Bioinformatics, Biology, Journals

Length heterogeneity at conserved sequence block 2 in human mitochondrial DNA acts as a rheostat for RNA polymerase POLRMT activity

пн, 2016-09-19 08:02

The guanine (G)-tract of conserved sequence block 2 (CSB 2) in human mitochondrial DNA can result in transcription termination due to formation of a hybrid G-quadruplex between the nascent RNA and the nontemplate DNA strand. This structure can then influence genome replication, stability and localization. Here we surveyed the frequency of variation in sequence identity and length at CSB 2 amongst human mitochondrial genomes and used in vitro transcription to assess the effects of this length heterogeneity on the activity of the mitochondrial RNA polymerase, POLRMT. In general, increased G-tract length correlated with increased termination levels. However, variation in the population favoured CSB 2 sequences which produced efficient termination while particularly weak or strong signals were avoided. For all variants examined, the 3' end of the transcripts mapped to the same downstream sequences and were prevented from terminating by addition of the transcription factor TEFM. We propose that CSB 2 length heterogeneity allows variation in the efficiency of transcription termination without affecting the position of the products or the capacity for regulation by TEFM.

Категории: Bioinformatics, Biology, Journals

DNA minicircles clarify the specific role of DNA structure on retroviral integration

пн, 2016-09-19 08:02

Chromatin regulates the selectivity of retroviral integration into the genome of infected cells. At the nucleosome level, both histones and DNA structure are involved in this regulation. We propose a strategy that allows to specifically study a single factor: the DNA distortion induced by the nucleosome. This strategy relies on mimicking this distortion using DNA minicircles (MCs) having a fixed rotational orientation of DNA curvature, coupled with atomic-resolution modeling. Contrasting MCs with linear DNA fragments having identical sequences enabled us to analyze the impact of DNA distortion on the efficiency and selectivity of integration. We observed a global enhancement of HIV-1 integration in MCs and an enrichment of integration sites in the outward-facing DNA major grooves. Both of these changes are favored by LEDGF/p75, revealing a new, histone-independent role of this integration cofactor. PFV integration is also enhanced in MCs, but is not associated with a periodic redistribution of integration sites, thus highlighting its distinct catalytic properties. MCs help to separate the roles of target DNA structure, histone modifications and integrase (IN) cofactors during retroviral integration and to reveal IN-specific regulation mechanisms.

Категории: Bioinformatics, Biology, Journals

APOBEC3 proteins can copackage and comutate HIV-1 genomes

пн, 2016-09-19 08:02

Although APOBEC3 cytidine deaminases A3G, A3F, A3D and A3H are packaged into virions and inhibit viral replication by inducing G-to-A hypermutation, it is not known whether they are copackaged and whether they can act additively or synergistically to inhibit HIV-1 replication. Here, we showed that APOBEC3 proteins can be copackaged by visualization of fluorescently-tagged APOBEC3 proteins using single-virion fluorescence microscopy. We further determined that viruses produced in the presence of A3G + A3F and A3G + A3H, exhibited extensive comutation of viral cDNA, as determined by the frequency of G-to-A mutations in the proviral genomes in the contexts of A3G (GG-to-AG) and A3D, A3F or A3H (GA-to-AA) edited sites. The copackaging of A3G + A3F and A3G + A3H resulted in an additive increase and a modest synergistic increase (1.8-fold) in the frequency of GA-to-AA mutations, respectively. We also identified distinct editing site trinucleotide sequence contexts for each APOBEC3 protein and used them to show that hypermutation of proviral DNAs from seven patients was induced by A3G, A3F (or A3H), A3D and A3G + A3F (or A3H). These results indicate that APOBEC3 proteins can be copackaged and can comutate the same genomes, and can cooperate to inhibit HIV replication.

Категории: Bioinformatics, Biology, Journals

Single strand transposition at the host replication fork

пн, 2016-09-19 08:02

Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the ‘peel and paste’ pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the β sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork.

Категории: Bioinformatics, Biology, Journals

Profiling of 2'-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity

пн, 2016-09-19 08:02

Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2'-O-Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2'-O-Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2'-O-Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings.

Категории: Bioinformatics, Biology, Journals

Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins

пн, 2016-09-19 08:02

The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIAGlc of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3' segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source.

Категории: Bioinformatics, Biology, Journals

The catalytic efficiency of yeast ribonuclease III depends on substrate specific product release rate

пн, 2016-09-19 08:02

Members of the ribonuclease III (RNase III) family regulate gene expression by triggering the degradation of double stranded RNA (dsRNA). Hundreds of RNase III cleavage targets have been identified and their impact on RNA maturation and stability is now established. However, the mechanism defining substrates’ reactivity remains unclear. In this study, we developed a real-time FRET assay for the detection of dsRNA degradation by yeast RNase III (Rnt1p) and characterized the kinetic bottlenecks controlling the reactivity of different substrates. Surprisingly, the results indicate that Rnt1p cleavage reaction is not only limited by the rate of catalysis but can also depend on base-pairing of product termini. Cleavage products terminating with paired nucleotides, like the degradation signals found in coding mRNA sequence, were less reactive and more prone to inhibition than products having unpaired nucleotides found in non-coding RNA substrates. Mutational analysis of U5 snRNA and Mig2 mRNA confirms the pairing of the cleavage site as a major determinant for the difference between cleavage rates of coding and non-coding RNA. Together the data indicate that the base-pairing of Rnt1p substrates encodes reactivity determinants that permit both constitutive processing of non-coding RNA while limiting the rate of mRNA degradation.

Категории: Bioinformatics, Biology, Journals