MAGIA2: from miRNA and genes expression data integrative analysis to microRNA-transcription factor mixed regulatory circuits (2012 update).

Nucleic Acids Res. 2012 May 21;

Authors: Bisognin A, Sales G, Coppe A, Bortoluzzi S, Romualdi C

Abstract
MAGIA(2) (http://gencomp.bio.unipd.it/magia2) is an update, extension and evolution of the MAGIA web tool. It is dedicated to the integrated analysis of in silico target prediction, microRNA (miRNA) and gene expression data for the reconstruction of post-transcriptional regulatory networks. miRNAs are fundamental post-transcriptional regulators of several key biological and pathological processes. As miRNAs act prevalently through target degradation, their expression profiles are expected to be inversely correlated to those of the target genes. Low specificity of target prediction algorithms makes integration approaches an interesting solution for target prediction refinement. MAGIA(2) performs this integrative approach supporting different association measures, multiple organisms and almost all target predictions algorithms. Nevertheless, miRNAs activity should be viewed as part of a more complex scenario where regulatory elements and their interactors generate a highly connected network and where gene expression profiles are the result of different levels of regulation. The updated MAGIA(2) tries to dissect this complexity by reconstructing mixed regulatory circuits involving either miRNA or transcription factor (TF) as regulators. Two types of circuits are identified: (i) a TF that regulates both a miRNA and its target and (ii) a miRNA that regulates both a TF and its target.

PMID: 22618880 [PubMed - as supplied by publisher]

A small ribozyme with dual-site kinase activity.

Nucleic Acids Res. 2012 May 21;

Authors: Biondi E, Maxwell AW, Burke DH

Abstract
Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-(32)P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5' target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5' mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation.

PMID: 22618879 [PubMed - as supplied by publisher]

PocketAnnotate: towards site-based function annotation.

Nucleic Acids Res. 2012 May 22;

Authors: Anand P, Yeturu K, Chandra N

Abstract
A computational pipeline PocketAnnotate for functional annotation of proteins at the level of binding sites has been proposed in this study. The pipeline integrates three in-house algorithms for site-based function annotation: PocketDepth, for prediction of binding sites in protein structures; PocketMatch, for rapid comparison of binding sites and PocketAlign, to obtain detailed alignment between pair of binding sites. A novel scheme has been developed to rapidly generate a database of non-redundant binding sites. For a given input protein structure, putative ligand-binding sites are identified, matched in real time against the database and the query substructure aligned with the promising hits, to obtain a set of possible ligands that the given protein could bind to. The input can be either whole protein structures or merely the substructures corresponding to possible binding sites. Structure-based function annotation at the level of binding sites thus achieved could prove very useful for cases where no obvious functional inference can be obtained based purely on sequence or fold-level analyses. An attempt has also been made to analyse proteins of no known function from Protein Data Bank. PocketAnnotate would be a valuable tool for the scientific community and contribute towards structure-based functional inference. The web server can be freely accessed at http://proline.biochem.iisc.ernet.in/pocketannotate/.

PMID: 22618878 [PubMed - as supplied by publisher]

TaxMan: a server to trim rRNA reference databases and inspect taxonomic coverage.

Nucleic Acids Res. 2012 May 22;

Authors: Brandt BW, Bonder MJ, Huse SM, Zaura E

Abstract
Amplicon sequencing of the hypervariable regions of the small subunit ribosomal RNA gene is a widely accepted method for identifying the members of complex bacterial communities. Several rRNA gene sequence reference databases can be used to assign taxonomic names to the sequencing reads using BLAST, USEARCH, GAST or the RDP classifier. Next-generation sequencing methods produce ample reads, but they are short, currently ∼100-450 nt (depending on the technology), as compared to the full rRNA gene of ∼1550 nt. It is important, therefore, to select the right rRNA gene region for sequencing. The primers should amplify the species of interest and the hypervariable regions should differentiate their taxonomy. Here, we introduce TaxMan: a web-based tool that trims reference sequences based on user-selected primer pairs and returns an assessment of the primer specificity by taxa. It allows interactive plotting of taxa, both amplified and missed in silico by the primers used. Additionally, using the trimmed sequences improves the speed of sequence matching algorithms. The smaller database greatly improves run times (up to 98%) and memory usage, not only of similarity searching (BLAST), but also of chimera checking (UCHIME) and of clustering the reads (UCLUST). TaxMan is available at http://www.ibi.vu.nl/programs/taxmanwww/.

PMID: 22618877 [PubMed - as supplied by publisher]

Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H37Rv and H37Ra reveal novel and interesting differences with implications in virulence.

Nucleic Acids Res. 2012 May 22;

Authors: Kohli S, Singh Y, Sharma K, Mittal A, Ehtesham NZ, Hasnain SE

Abstract
Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H(37)Rv and H(37)Ra, derived from the same parental strain M. tuberculosis H(37), show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H(37)Ra and virulent H(37)Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with 5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H(37)Rv and H(37)Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H(37)Rv and H(37)Ra can be correlated to differences in pathogenesis and virulence of the two strains.

PMID: 22618876 [PubMed - as supplied by publisher]

The evolutionary dynamics of functional modules and the extraordinary plasticity of regulons: the Escherichia coli perspective.

Nucleic Acids Res. 2012 May 22;

Authors: Moreno-Hagelsieb G, Jokic P

Abstract
Using profiles of phylogenetic profiles (P-cubic) we compared the evolutionary dynamics of different kinds of functional associations. Ordered from most to least evolutionarily stable, these associations were genes in the same operons, genes whose products participate in the same biochemical pathway, genes coding for physically interacting proteins and genes in the same regulons. Regulons showed the most plastic functional interactions with evolutionary stabilities barely better than those of unrelated genes. Further regulon analyses showed that global regulators contain less evolutionarily stable associations than local regulators. Genes co-repressed by global regulators had a higher evolutionary conservation than genes co-activated by global regulators. However, the reverse was true for genes co-repressed and co-activated by local regulators. Of all the regulon-related associations, the relationship between regulators and their target genes showed the most evolutionary stability. Different negative data sets built to contrast against each of the analysed kinds of modules also differed in evolutionary conservation revealing further underlying genome organization. Applying P-cubic analyses to other genomes might help visualize genome organization, understand the evolutionary importance and plasticity of functional associations and compare the quality of data sets expected to reflect functional interactions, such as those coming from high-throughput experiments.

PMID: 22618875 [PubMed - as supplied by publisher]

Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes.

Nucleic Acids Res. 2012 May 22;

Authors: Wahlgren J, Karlson TD, Brisslert M, Vaziri Sani F, Telemo E, Sunnerhagen P, Valadi H

Abstract
Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.

PMID: 22618874 [PubMed - as supplied by publisher]

Direct comparison of small RNA and transcription factor signaling.

Nucleic Acids Res. 2012 May 22;

Authors: Hussein R, Lim HN

Abstract
Small RNAs (sRNAs) and proteins acting as transcription factors (TFs) are the principal components of gene networks. These two classes of signaling molecules have distinct mechanisms of action; sRNAs control mRNA translation, whereas TFs control mRNA transcription. Here, we directly compare the properties of sRNA and TF signaling using mathematical models and synthetic gene circuits in Escherichia coli. We show the abilities of sRNAs to act on existing target mRNAs (as opposed to TFs, which alter the production of future target mRNAs) and, without needing to be first translated, have surprisingly little impact on the dynamics. Instead, the dynamics are primarily determined by the clearance rates, steady-state concentrations and response curves of the sRNAs and TFs; these factors determine the time delay before a target gene's expression can maximally respond to changes in sRNA and TF transcription. The findings are broadly applicable to the analysis of signaling in gene networks, and we demonstrate that they can be used to rationally reprogram the dynamics of synthetic circuits.

PMID: 22618873 [PubMed - as supplied by publisher]

A new method for evaluating the specificity of indirect readout in protein-DNA recognition.

Nucleic Acids Res. 2012 May 22;

Authors: Yamasaki S, Terada T, Kono H, Shimizu K, Sarai A

Abstract
Proteins recognize a specific DNA sequence not only through direct contact (direct readout) with base pairs but also through sequence-dependent conformation and/or flexibility of DNA (indirect readout). However, it is difficult to assess the contribution of indirect readout to the sequence specificity. What is needed is a straightforward method for quantifying its contributions to specificity. Using Bayesian statistics, we derived the probability of a particular sequence for a given DNA structure from the trajectories of molecular dynamics (MD) simulations of DNAs containing all possible tetramer sequences. Then, we quantified the specificity of indirect readout based on the information entropy associated with the probability. We tested this method with known structures of protein-DNA complexes. This method enabled us to correctly predict those regions where experiments suggested the involvement of indirect readout. The results also indicated new regions where the indirect readout mechanism makes major contributions to the recognition. The present method can be used to estimate the contribution of indirect readout without approximations to the distributions in the conformational ensembles of DNA, and would serve as a powerful tool to study the mechanism of protein-DNA recognition.

PMID: 22618872 [PubMed - as supplied by publisher]

NetAligner--a network alignment server to compare complexes, pathways and whole interactomes.

Nucleic Acids Res. 2012 May 22;

Authors: Pache RA, Céol A, Aloy P

Abstract
The many ongoing genome sequencing initiatives are delivering comprehensive lists of the individual molecular components present in an organism, but these reveal little about how they work together. Follow-up initiatives are revealing thousands of interrelationships between gene products that need to be analyzed with novel bioinformatics approaches able to capture their complex emerging properties. Recently, we developed NetAligner, a novel network alignment tool that allows the identification of conserved protein complexes and pathways across organisms, providing valuable hints as to how those interaction networks evolved. NetAligner includes the prediction of likely conserved interactions, based on evolutionary distances, to counter the high number of missing interactions in current interactome networks, and a fast assessment of the statistical significance of individual alignment solutions, which increases its performance with respect to existing tools. The web server implementation of the NetAligner algorithm presented here features complex, pathway and interactome to interactome alignments for seven model organisms, namely Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae and Escherichia coli. The user can query complexes and pathways of arbitrary topology against a target species interactome, or directly compare two complete interactomes to identify conserved complexes and subnetworks. Alignment solutions can be downloaded or directly visualized in the browser. The NetAligner web server is publicly available at http://netaligner.irbbarcelona.org/.

PMID: 22618871 [PubMed - as supplied by publisher]

Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function.

Nucleic Acids Res. 2012 May 22;

Authors: Lemp NA, Hiraoka K, Kasahara N, Logg CR

Abstract
A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

PMID: 22618870 [PubMed - as supplied by publisher]

VarioWatch: providing large-scale and comprehensive annotations on human genomic variants in the next generation sequencing era.

Nucleic Acids Res. 2012 May 22;

Authors: Cheng YC, Hsiao FC, Yeh EC, Lin WJ, Tang CY, Tseng HC, Wu HT, Liu CK, Chen CC, Chen YT, Yao A

Abstract
VarioWatch (http://genepipe.ncgm.sinica.edu.tw/variowatch/) has been vastly improved since its former publication GenoWatch in the 2008 Web Server Issue. It is now at least 10 000-times faster in annotating a variant. Drastic speed increase, through complete re-design of its working mechanism, makes VarioWatch capable of annotating millions of human genomic variants generated from next generation sequencing in minutes, if not seconds. While using MegaQuery of VarioWatch to quickly annotate variants, users can apply various filters to retrieve a subgroup of variants according to the risk levels, interested regions, etc. that satisfy users' requirements. In addition to performance leap, many new features have also been added, such as annotation on novel variants, functional analyses on splice sites and in/dels, detailed variant information in tabulated form, plus a risk level decision tree regarding the analyzed variant. Up to 1000 target variants can be visualized with our carefully designed Genome View, Gene View, Transcript View and Variation View. Two commonly used reference versions, NCBI build 36.3 and NCBI build 37.2, are supported. VarioWatch is unique in its ability to annotate comprehensively and efficiently millions of variants online, immediately delivering the results in real time, plus visualizes up to 1000 annotated variants.

PMID: 22618869 [PubMed - as supplied by publisher]