Nucleic Acids Research

Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans.

Publication Date: 2010 Aug 30 PMID: 20805248
Authors: Barberan-Soler, S. - Medina, P. - Estella, J. - Williams, J. - Zahler, A. M.
Journal: Nucleic Acids Res

Regulation of alternative splicing is controlled by pre-mRNA sequences (cis-elements) and trans-acting protein factors that bind them. The combinatorial interactions of multiple protein factors with the cis-elements surrounding a given alternative splicing event lead to an integrated splicing decision. The mechanism of multifactorial splicing regulation is poorly understood. Using a splicing-sensitive DNA microarray, we assayed 352 Caenorhabditis elegans alternative cassette exons for changes in embryonic splicing patterns between wild-type and 12 different strains carrying mutations in a splicing factor. We identified many alternative splicing events that are regulated by multiple splicing factors. Many splicing factors have the ability to behave as splicing repressors for some alternative cassette exons and as splicing activators for others. Unexpectedly, we found that the ability of a given alternative splicing factor to behave as an enhancer or repressor of a specific splicing event can change during development. Our observations that splicing factors can change their effects on a substrate during development support a model in which combinatorial effects of multiple factors, both constitutive and developmentally regulated ones, contribute to the overall splicing decision.

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MEF/ELF4 transactivation by E2F1 is inhibited by p53.

Publication Date: 2010 Aug 30 PMID: 20805247
Authors: Taura, M. - Suico, M. A. - Fukuda, R. - Koga, T. - Shuto, T. - Sato, T. - Morino-Koga, S. - Okada, S. - Kai, H.
Journal: Nucleic Acids Res

Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unex-plored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

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Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity.

Publication Date: 2010 Aug 30 PMID: 20805246
Authors: Chan, S. H. - Stoddard, B. L. - Xu, S. Y.
Journal: Nucleic Acids Res

Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4-8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (;CCD; ; denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.

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Highly redundant function of multiple AT-rich sequences as core promoter elements in the TATA-less RPS5 promoter of Saccharomyces cerevisiae.

Publication Date: 2010 Aug 30 PMID: 20805245
Authors: Sugihara, F. - Kasahara, K. - Kokubo, T.
Journal: Nucleic Acids Res

In eukaryotes, protein-coding genes are transcribed by RNA polymerase II (pol II) together with general transcription factors (GTFs). TFIID, the largest GTF composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a critical role in transcription from TATA-less promoters. In metazoans, several core promoter elements other than the TATA element are thought to be recognition sites for TFIID. However, it is unclear whether functionally homologous elements also exist in TATA-less promoters in Saccharomyces cerevisiae. Here, we identify the cis-elements required to support normal levels of transcription and accurate initiation from sites within the TATA-less and TFIID-dependent RPS5 core promoter. Systematic mutational analyses show that multiple AT-rich sequences are required for these activities and appear to function as recognition sites for TFIID. A single copy of these sequences can support accurate initiation from the endogenous promoter, indicating that they carry highly redundant functions. These results show a novel architecture of yeast TATA-less promoters and support a model in which pol II scans DNA downstream from a recruited site, while searching for appropriate initiation site(s).

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Analysis of two human pre-ribosomal factors, bystin and hTsr1, highlights differences in evolution of ribosome biogenesis between yeast and mammals.

Publication Date: 2010 Aug 30 PMID: 20805244
Authors: Carron, C. - O'Donohue, M. F. - Choesmel, V. - Faubladier, M. - Gleizes, P. E.
Journal: Nucleic Acids Res

Recent studies reveal that maturation of the 40S ribosomal subunit precursors in mammals includes an additional step during processing of the internal transcribed spacer 1 (ITS1), when compared with yeast Saccharomyces cerevisiae, even though the protein content of the pre-40S particle appears to be the same. Here, we examine by depletion with siRNA treatment the function of human orthologs of two essential yeast pre-ribosomal factors, hEnp1/bystin and hTsr1. Like their yeast orthologs, bystin is required for efficient cleavage of the ITS1 and further processing of this domain within the pre-40S particles, whereas hTsr1 is necessary for the final maturation steps. However, bystin depletion leads to accumulation of an unusual 18S rRNA precursor, revealing a new step in ITS1 processing that potentially involves an exonuclease. In addition, pre-40S particles lacking hTsr1 are partially retained in the nucleus, whereas depletion of Tsr1p in yeast results in strong cytoplasmic accumulation of pre-40S particles. These data indicate that ITS1 processing in human cells may be more complex than currently envisioned and that coordination between maturation and nuclear export of pre-40S particles has evolved differently in yeast and mammalian cells.

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Structural insights into cis element recognition of non-polyadenylated RNAs by the Nab3-RRM.

Publication Date: 2010 Aug 30 PMID: 20805243
Authors: Lunde, B. M. - Horner, M. - Meinhart, A.
Journal: Nucleic Acids Res

Transcription termination of non-polyadenylated RNAs in Saccharomyces cerevisiae occurs through the action of the Nrd1-Nab3-Sen1 complex. Part of the decision to terminate via this pathway occurs via direct recognition of sequences within the nascent transcript by RNA recognition motifs (RRMs) within Nrd1 and Nab3. Here we present the 1.6 A structure of Nab3-RRM bound to its UCUU recognition sequence. The crystal structure reveals clear density for a UCU trinucleotide and a fourth putative U binding site. Nab3-RRM establishes a clear preference for the central cytidine of the UCUU motif, which forms pseudo-base pairing interactions primarily through hydrogen bonds to main chain atoms and one serine hydroxyl group. Specificity for the flanking uridines is less defined; however, binding experiments confirm that these residues are also important for high affinity binding. Comparison of the Nab3-RRM to other structures of RRMs bound to polypyrimidine RNAs showed that this mode of recognition is similar to what is observed for the polypyrimidine-tract binding RRMs, and that the serine residue involved in pseudo-base pairing is only found in RRMs that bind to polypyrimidine RNAs that contain a cytosine base, suggesting a possible mechanism for discriminating between cytosine and uracil bases in RRMs that bind to polypyrimidine-containing RNA.

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Efficient use of accessibility in microRNA target prediction.

Publication Date: 2010 Aug 30 PMID: 20805242
Authors: Marin, R. M. - Vanicek, J.
Journal: Nucleic Acids Res

Considering accessibility of the 3'UTR is believed to increase the precision of microRNA target predictions. We show that, contrary to common belief, ranking by the hybridization energy or by the sum of the opening and hybridization energies, used in currently available algorithms, is not an efficient way to rank predictions. Instead, we describe an algorithm which also considers only the accessible binding sites but which ranks predictions according to over-representation. When compared with experimentally validated and refuted targets in the fruit fly and human, our algorithm shows a remarkable improvement in precision while significantly reducing the computational cost in comparison with other free energy based methods. In the human genome, our algorithm has at least twice higher precision than other methods with their default parameters. In the fruit fly, we find five times more validated targets among the top 500 predictions than other methods with their default parameters. Furthermore, using a common statistical framework we demonstrate explicitly the advantages of using the canonical ensemble instead of using the minimum free energy structure alone. We also find that 'naive' global folding sometimes outperforms the local folding approach.

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Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase.

Publication Date: 2010 Aug 30 PMID: 20805241
Authors: Chen, X. - Ma, J. J. - Tan, M. - Yao, P. - Hu, Q. H. - Eriani, G. - Wang, E. D.
Journal: Nucleic Acids Res

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNA(Leu). It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNA(Leu) (hctRNA(Leu)) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit alpha-amino butyrate, although both amino acids can be charged to tRNA(Leu). Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution.

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FragGeneScan: predicting genes in short and error-prone reads.

Publication Date: 2010 Aug 30 PMID: 20805240
Authors: Rho, M. - Tang, H. - Ye, Y.
Journal: Nucleic Acids Res

The advances of next-generation sequencing technology have facilitated metagenomics research that attempts to determine directly the whole collection of genetic material within an environmental sample (i.e. the metagenome). Identification of genes directly from short reads has become an important yet challenging problem in annotating metagenomes, since the assembly of metagenomes is often not available. Gene predictors developed for whole genomes (e.g. Glimmer) and recently developed for metagenomic sequences (e.g. MetaGene) show a significant decrease in performance as the sequencing error rates increase, or as reads get shorter. We have developed a novel gene prediction method FragGeneScan, which combines sequencing error models and codon usages in a hidden Markov model to improve the prediction of protein-coding region in short reads. The performance of FragGeneScan was comparable to Glimmer and MetaGene for complete genomes. But for short reads, FragGeneScan consistently outperformed MetaGene (accuracy improved approximately 62% for reads of 400 bases with 1% sequencing errors, and approximately 18% for short reads of 100 bases that are error free). When applied to metagenomes, FragGeneScan recovered substantially more genes than MetaGene predicted (>90% of the genes identified by homology search), and many novel genes with no homologs in current protein sequence database.

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MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery.

Publication Date: 2010 Aug 27 PMID: 20802226
Authors: Wang, K. - Singh, D. - Zeng, Z. - Coleman, S. J. - Huang, Y. - Savich, G. L. - He, X. - Mieczkowski, P. - Grimm, S. A. - Perou, C. M. - Macleod, J. N. - Chiang, D. Y. - Prins, J. F. - Liu, J.
Journal: Nucleic Acids Res

The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (>/=75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice.

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Reference-unbiased copy number variant analysis using CGH microarrays.

Publication Date: 2010 Aug 27 PMID: 20802225
Authors: Ju, Y. S. - Hong, D. - Kim, S. - Park, S. S. - Kim, S. - Lee, S. - Park, H. - Kim, J. I. - Seo, J. S.
Journal: Nucleic Acids Res

Comparative genomic hybridization (CGH) microarrays have been used to determine copy number variations (CNVs) and their effects on complex diseases. Detection of absolute CNVs independent of genomic variants of an arbitrary reference sample has been a critical issue in CGH array experiments. Whole genome analysis using massively parallel sequencing with multiple ultra-high resolution CGH arrays provides an opportunity to catalog highly accurate genomic variants of the reference DNA (NA10851). Using information on variants, we developed a new method, the CGH array reference-free algorithm (CARA), which can determine reference-unbiased absolute CNVs from any CGH array platform. The algorithm enables the removal and rescue of false positive and false negative CNVs, respectively, which appear due to the effects of genomic variants of the reference sample in raw CGH array experiments. We found that the CARA remarkably enhanced the accuracy of CGH array in determining absolute CNVs. Our method thus provides a new approach to interpret CGH array data for personalized medicine.

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Verifying expressed transcript variants by detecting and assembling stretches of consecutive exons.

Publication Date: 2010 Aug 26 PMID: 20798177
Authors: Hsiao, T. H. - Lin, C. H. - Lee, T. T. - Cheng, J. Y. - Wei, P. K. - Chuang, E. Y. - Peck, K.
Journal: Nucleic Acids Res

We herein describe an integrated system for the high-throughput analysis of splicing events and the identification of transcript variants. The system resolves individual splicing events and elucidates transcript variants via a pipeline that combines aspects such as bioinformatic analysis, high-throughput transcript variant amplification, and high-resolution capillary electrophoresis. For the 14 369 human genes known to have transcript variants, minimal primer sets were designed to amplify all transcript variants and examine all splicing events; these have been archived in the ASprimerDB database, which is newly described herein. A high-throughput thermocycler, dubbed GenTank, was developed to simultaneously perform thousands of PCR amplifications. Following the resolution of the various amplicons by capillary gel electrophoresis, two new computer programs, AmpliconViewer and VariantAssembler, may be used to analyze the splicing events, assemble the consecutive exons embodied by the PCR amplicons, and distinguish expressed versus putative transcript variants. This novel system not only facilitates the validation of putative transcript variants and the detection of novel transcript variants, it also semi-quantitatively measures the transcript variant expression levels of each gene. To demonstrate the system's capability, we used it to resolve transcript variants yielded by single and multiple splicing events, and to decipher the exon connectivity of long transcripts.

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The NIP7 protein is required for accurate pre-rRNA processing in human cells.

Publication Date: 2010 Aug 26 PMID: 20798176
Authors: Morello, L. G. - Hesling, C. - Coltri, P. P. - Castilho, B. A. - Rimokh, R. - Zanchin, N. I.
Journal: Nucleic Acids Res

Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman-Bodian-Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S-80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.

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A yeast one-hybrid system to screen for methylated DNA-binding proteins.

Publication Date: 2010 Aug 26 PMID: 20798175
Authors: Feng, S. Y. - Ota, K. - Ito, T.
Journal: Nucleic Acids Res

We had previously exploited a method for targeted DNA methylation in budding yeast to succeed in one-hybrid detection of methylation-dependent DNA-protein interactions. Based on this finding, we developed a yeast one-hybrid system to screen cDNA libraries for clones encoding methylated DNA-binding proteins. Concurrent use of two independent bait sequences in the same cell, or dual-bait system, effectively reduced false positive clones, which were derived from methylation-insensitive sequence-specific DNA-binding proteins. We applied the dual-bait system to screen cDNA libraries and demonstrated efficient isolation of clones for methylated DNA-binding proteins. This system would serve as a unique research tool for epigenetics.

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Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNAfMet selection on the ribosomal P-site in Escherichia coli.

Publication Date: 2010 Aug 26 PMID: 20798174
Authors: Kapoor, S. - Das, G. - Varshney, U.
Journal: Nucleic Acids Res

The accuracy of the initiator tRNA (tRNA(fMet)) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G-C base pairs in the anticodon stem of tRNA(fMet) contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA(fMet) mutant wherein the three G-C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA(fMet) mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA(fMet) levels. Low cellular abundance of the chromosomally encoded tRNA(fMet) allows efficient initiation with the tRNA(fMet) mutant and an elongator tRNA(Gln), revealing that a high abundance of the cellular tRNA(fMet) is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA(fMet) abundance in proteome remodeling.

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A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage.

Publication Date: 2010 Aug 26 PMID: 20798173
Authors: Duss, O. - Maris, C. - von Schroetter, C. - Allain, F. H.
Journal: Nucleic Acids Res

Structural information on RNA, emerging more and more as a major regulator in gene expression, dramatically lags behind compared with information on proteins. Although NMR spectroscopy has proven to be an excellent tool to solve RNA structures, it is hampered by the severe spectral resonances overlap found in RNA, limiting its use for large RNA molecules. Segmental isotope labeling of RNA or ligation of a chemically synthesized RNA containing modified nucleotides with an unmodified RNA fragment have proven to have high potential in overcoming current limitations in obtaining structural information on RNA. However, low yields, cumbersome preparations and sequence requirements have limited its broader application in structural biology. Here we present a fast and efficient approach to generate multiple segmentally labeled RNAs with virtually no sequence requirements with very high yields (up to 10-fold higher than previously reported). We expect this approach to open new avenues in structural biology of RNA.

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Structural basis of microRNA length variety.

Publication Date: 2010 Aug 25 PMID: 20739353
Authors: Starega-Roslan, J. - Krol, J. - Koscianska, E. - Kozlowski, P. - Szlachcic, W. J. - Sobczak, K. - Krzyzosiak, W. J.
Journal: Nucleic Acids Res

The biogenesis of human microRNAs (miRNAs) includes two RNA cleavage steps in which the activities of the RNases Drosha and Dicer are involved. miRNAs of diverse lengths are generated from different genes, and miRNAs that are heterogeneous in length are produced from a single miRNA gene. We determined the solution structures of many miRNA precursors and analysed the structural basis of miRNA length diversity using a new measure: the weighted average length of diced RNA (WALDI). We found that asymmetrical structural motifs present in precursor hairpins are primarily responsible for the length diversity of miRNAs generated by Dicer. High-resolution northern blots of miRNAs and their precursors revealed that both Dicer and Drosha cleavages of imperfect specificity contributed to the miRNA length heterogeneity. The relevance of these findings to the dynamics of the dicing complex, mRNA regulation by miRNA, RNA interference and miRNA technologies are discussed.

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Improved deoxyribozymes for synthesis of covalently branched DNA and RNA.

Publication Date: 2010 Aug 25 PMID: 20739352
Authors: Lee, C. S. - Mui, T. P. - Silverman, S. K.
Journal: Nucleic Acids Res

A covalently branched nucleic acid can be synthesized by joining the 2'-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5'-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn(2+) as a cofactor, rather than Mg(2+) as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has k(obs) on the order of 0.1 min(-1), which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.

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The phosphate clamp: a small and independent motif for nucleic acid backbone recognition.

Publication Date: 2010 Aug 24 PMID: 20736180
Authors: Komeda, S. - Moulaei, T. - Chikuma, M. - Odani, A. - Kipping, R. - Farrell, N. P. - Williams, L. D.
Journal: Nucleic Acids Res

The 1.7 A X-ray crystal structure of the B-DNA dodecamer, [d(CGCGAATTCGCG)](2) (DDD)-bound non-covalently to a platinum(II) complex, [{Pt(NH(3))(3)}(2)-micro-{trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2)}](NO (3))(6) (1, TriplatinNC-A,) shows the trinuclear cation extended along the phosphate backbone and bridging the minor groove. The square planar tetra-am(m)ine Pt(II) units form bidentate N-O-N complexes with OP atoms, in a Phosphate Clamp motif. The geometry is conserved and the interaction prefers O2P over O1P atoms (frequency of interaction is O2P > O1P, base and sugar oxygens > N). The binding mode is very similar to that reported for the DDD and [{trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)(NH(3)(+))}(2)-micro-{trans-Pt(NH(3))( 2)(NH(2)(CH(2))(6)NH(2))(2)}](NO(3))(8) (3, TriplatinNC), which exhibits in vivo anti-tumour activity. In the present case, only three sets of Phosphate Clamps were found because one of the three Pt(II) coordination spheres was not clearly observed and was characterized as a bare Pt(2+) ion. Based on the electron density, the relative occupancy of DDD and the sum of three Pt(II) atoms in the DDD-1 complex was 1:1.69, whereas the ratio for DDD-2 was 1:2.85, almost the mixing ratio in the crystallization drop. The high repetition and geometric regularity of the motif suggests that it can be developed as a modular nucleic acid binding device with general utility.

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Ribonucleotide reductase is not limiting for mitochondrial DNA copy number in mice.

Publication Date: 2010 Aug 20 PMID: 20724444
Authors: Ylikallio, E. - Page, J. L. - Xu, X. - Lampinen, M. - Bepler, G. - Ide, T. - Tyynismaa, H. - Weiss, R. S. - Suomalainen, A.
Journal: Nucleic Acids Res

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in deoxyribonucleoside triphosphate (dNTP) biosynthesis, with important roles in nuclear genome maintenance. RNR is also essential for maintenance of mitochondrial DNA (mtDNA) in mammals. The mechanisms regulating mtDNA copy number in mammals are only being discovered. In budding yeast, RNR overexpression resulted in increased mtDNA levels, and rescued the disease phenotypes caused by a mutant mtDNA polymerase. This raised the question of whether mtDNA copy number increase by RNR induction could be a strategy for treating diseases with mtDNA mutations. We show here that high-level overexpression of RNR subunits (Rrm1, Rrm2 and p53R2; separately or in different combinations) in mice does not result in mtDNA copy number elevation. Instead, simultaneous expression of two RNR subunits leads to imbalanced dNTP pools and progressive mtDNA depletion in the skeletal muscle, without mtDNA mutagenesis. We also show that endogenous RNR transcripts are downregulated in response to large increases of mtDNA in mice, which is indicative of nuclear-mitochondrial crosstalk with regard to mtDNA copy number. Our results establish that RNR is not limiting for mtDNA copy number in mice, and provide new evidence for the importance of balanced dNTP pools in mtDNA maintenance in postmitotic tissues.

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Characterization of an interplay between a Mycobacterium tuberculosis MazF homolog, Rv1495 and its sole DNA topoisomerase I.

Publication Date: 2010 Aug 20 PMID: 20724443
Authors: Huang, F. - He, Z. G.
Journal: Nucleic Acids Res

The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative interplay between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and inhibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I.

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Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein.

Publication Date: 2010 Aug 19 PMID: 20724442
Authors: Davidson, A. - Patora-Komisarska, K. - Robinson, J. A. - Varani, G.
Journal: Nucleic Acids Res

The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained beta-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T(1) occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved >6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.

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Blank peak current-suppressed electrochemical aptameric sensing platform for highly sensitive signal-on detection of small molecule.

Publication Date: 2010 Aug 20 PMID: 20724441
Authors: Zhang, S. - Hu, R. - Hu, P. - Wu, Z. S. - Shen, G. L. - Yu, R. Q.
Journal: Nucleic Acids Res

In this contribution, an electrochemical aptameric sensing scheme for the sensitive detection of small molecules is proposed using adenosine as a target model. A ferrocene (Fc)-functionalized thiolated aptamer probe is adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only suppresses the peak current corresponding to blank sample but also provides a signal-on response mechanism. In the absence of adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc is capable of being removed from electrode surface by treatment with endonuclease, and almost no peak current is observed. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissociating the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, adenosine even at a low concentration can give a detectable current signal. Thus, a detection limit of 10(-)(1)(0) M and a linear response range from 3.74 x 10(-)(9) to 3.74 x 10(-)(5) M are achieved. The proposed proof-of-principle of a novel electrochemical sensing is expected to extend to establish various aptameric platforms for the analysis of a broad range of target molecules of interest.

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Building promoter aware transcriptional regulatory networks using siRNA perturbation and deepCAGE.

Publication Date: 2010 Aug 20 PMID: 20724440
Authors: Vitezic, M. - Lassmann, T. - Forrest, A. R. - Suzuki, M. - Tomaru, Y. - Kawai, J. - Carninci, P. - Suzuki, H. - Hayashizaki, Y. - Daub, C. O.
Journal: Nucleic Acids Res

Perturbation and time-course data sets, in combination with computational approaches, can be used to infer transcriptional regulatory networks which ultimately govern the developmental pathways and responses of cells. Here, we individually knocked down the four transcription factors PU.1, IRF8, MYB and SP1 in the human monocyte leukemia THP-1 cell line and profiled the genome-wide transcriptional response of individual transcription starting sites using deep sequencing based Cap Analysis of Gene Expression. From the proximal promoter regions of the responding transcription starting sites, we derived de novo binding-site motifs, characterized their biological function and constructed a network. We found a previously described composite motif for PU.1 and IRF8 that explains the overlapping set of transcriptional responses upon knockdown of either factor.

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Capturing, sharing and analysing biophysical data from protein engineering and protein characterization studies.

Publication Date: 2010 Aug 20 PMID: 20724439
Authors: Farrell, D. - O'Meara, F. - Johnston, M. - Bradley, J. - Sondergaard, C. R. - Georgi, N. - Webb, H. - Tynan-Connolly, B. M. - Bjarnadottir, U. - Carstensen, T. - Nielsen, J. E.
Journal: Nucleic Acids Res

Large amounts of data are being generated annually on the connection between the sequence, structure and function of proteins using site-directed mutagenesis, protein design and directed evolution techniques. These data provide the fundamental building blocks for our understanding of protein function, molecular biology and living organisms in general. However, much experimental data are never deposited in databases and is thus 'lost' in journal publications or in PhD theses. At the same time theoretical scientists are in need of large amounts of experimental data for benchmarking and calibrating novel predictive algorithms, and theoretical progress is therefore often hampered by the lack of suitable data to validate or disprove a theoretical assumption. We present PEAT (Protein Engineering Analysis Tool), an application that integrates data deposition, storage and analysis for researchers carrying out protein engineering projects or biophysical characterization of proteins. PEAT contains modules for DNA sequence manipulation, primer design, fitting of biophysical characterization data (enzyme kinetics, circular dichroism spectroscopy, NMR titration data, etc.), and facilitates sharing of experimental data and analyses for a typical university-based research group. PEAT is freely available to academic researchers at http://enzyme.ucd.ie/PEAT.

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Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1.

Publication Date: 2010 Aug 20 PMID: 20724438
Authors: Rappas, M. - Oliver, A. W. - Pearl, L. H.
Journal: Nucleic Acids Res

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR-ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2.

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PmiRKB: a plant microRNA knowledge base.

Publication Date: 2010 Aug 18 PMID: 20719744
Authors: Meng, Y. - Gou, L. - Chen, D. - Mao, C. - Jin, Y. - Wu, P. - Chen, M.
Journal: Nucleic Acids Res

MicroRNAs (miRNAs), one type of small RNAs (sRNAs) in plants, play an essential role in gene regulation. Several miRNA databases were established; however, successively generated new datasets need to be collected, organized and analyzed. To this end, we have constructed a plant miRNA knowledge base (PmiRKB) that provides four major functional modules. In the 'SNP' module, single nucleotide polymorphism (SNP) data of seven Arabidopsis (Arabidopsis thaliana) accessions and 21 rice (Oryza sativa) subspecies were collected to inspect the SNPs within pre-miRNAs (precursor microRNAs) and miRNA-target RNA duplexes. Depending on their locations, SNPs can affect the secondary structures of pre-miRNAs, or interactions between miRNAs and their targets. A second module, 'Pri-miR', can be used to investigate the tissue-specific, transcriptional contexts of pre- and pri-miRNAs (primary microRNAs), based on massively parallel signature sequencing data. The third module, 'MiR-Tar', was designed to validate thousands of miRNA-target pairs by using parallel analysis of RNA end (PARE) data. Correspondingly, the fourth module, 'Self-reg', also used PARE data to investigate the metabolism of miRNA precursors, including precursor processing and miRNA- or miRNA*-mediated self-regulation effects on their host precursors. PmiRKB can be freely accessed at http://bis.zju.edu.cn/pmirkb/.

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Anti-tumor activity of splice-switching oligonucleotides.

Publication Date: 2010 Aug 18 PMID: 20719743
Authors: Bauman, J. A. - Li, S. D. - Yang, A. - Huang, L. - Kole, R.
Journal: Nucleic Acids Res

Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-x(L) and pro-apoptotic Bcl-x(S). Bcl-x(L) is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-x(L) to -x(S) induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing.

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Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes.

Publication Date: 2010 Aug 17 PMID: 20719742
Authors: Ittig, D. - Gerber, A. B. - Leumann, C. J.
Journal: Nucleic Acids Res

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy DeltaH. Arranging the tc-residues in a continuous fashion rescues T(m) and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T(m) when paired to complementary DNA and leads to substantial increases in T(m) when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.

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A CUG codon adapted two-hybrid system for the pathogenic fungus Candida albicans.

Publication Date: 2010 Aug 17 PMID: 20719741
Authors: Stynen, B. - Van Dijck, P. - Tournu, H.
Journal: Nucleic Acids Res

The genetics of the most common human pathogenic fungus Candida albicans has several unique characteristics. Most notably, C. albicans does not follow the universal genetic code, by translating the CUG codon into serine instead of leucine. Consequently, the use of Saccharomyces cerevisiae as a host for yeast two-hybrid experiments with C. albicans proteins is limited due to erroneous translation caused by the aberrant codon usage of C. albicans. To circumvent the need for heterologous expression and codon optimalization of C. albicans genes we constructed a two-hybrid system with C. albicans itself as the host with components that are compatible for use in this organism. The functionality of this two-hybrid system was shown by successful interaction assays with the protein pairs Kis1-Snf4 and Ino4-Ino2. We further confirmed interactions between components of the filamentation/mating MAP kinase pathway, including the unsuspected interaction between the MAP kinases Cek2 and Cek1. We conclude that this system can be used to enhance our knowledge of protein-protein interactions in C. albicans.

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AUF1 p42 isoform selectively controls both steady-state and PGE2-induced FGF9 mRNA decay.

Publication Date: 2010 Aug 16 PMID: 20716519
Authors: Chen, T. M. - Hsu, C. H. - Tsai, S. J. - Sun, H. S.
Journal: Nucleic Acids Res

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays vital roles in many physiologic processes including embryonic development. Aberrant expression of FGF9 causes human diseases and thus it highlights the importance of controlling FGF9 expression; however, the mechanism responsible for regulation of FGF9 expression is largely unknown. Here, we show the crucial role of an AU-rich element (ARE) in FGF9 3'-untranslated region (UTR) on controlling FGF9 expression. Our data demonstrated that AUF1 binds to this ARE to regulate FGF9 mRNA stability. Overexpression of each isoform of AUF1 (p37, p40, p42 and p45) showed that only the p42 isoform reduced the steady-state FGF9 mRNA. Also, knockdown of p42(AUF1) prolonged the half-life of FGF9 mRNA. The induction of FGF9 mRNA in prostaglandin (PG) E(2)-treated human endometrial stromal cells was accompanied with declined cytoplasmic AUF1. Nevertheless, ablation of AUF1 led to sustained elevation of FGF9 expression in these cells. Our study demonstrated that p42(AUF1) regulates both steady-state and PGE(2)-induced FGF9 mRNA stability through ARE-mediated mRNA degradation. Since almost half of the FGF family members are ARE-containing genes, our findings also suggest that ARE-mediated mRNA decay is a common pathway to control FGFs expression, and it represents a novel RNA regulon to coordinate FGFs homeostasis in various physiological conditions.

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Diversity and strength of internal outward-oriented promoters in group IIC-attC introns.

Publication Date: 2010 Aug 16 PMID: 20716518
Authors: Leon, G. - Quiroga, C. - Centron, D. - Roy, P. H.
Journal: Nucleic Acids Res

Integrons are genetic elements that incorporate mobile gene cassettes by site-specific recombination and express them as an operon from a promoter (Pc) located upstream of the cassette insertion site. Most gene cassettes found in integrons contain only one gene followed by an attC recombination site. We have recently shown that a specific lineage of group IIC introns, named group IIC-attC introns, inserts into the bottom strand sequence of attC sites. Here, we show that S.ma.I2, a group IIC-attC intron inserted in an integron cassette array of Serratia marcescens, impedes transcription from Pc while allowing expression of the following antibiotic resistance cassette using an internal outward-oriented promoter (P(out)). Bioinformatic analyses indicate that one or two putative P(out), which have sequence similarities with the Escherichia coli consensus promoters, are conserved in most group IIC-attC intron sequences. We show that P(out) with different versions of the -35 and -10 sequences are functionally active in expressing a promoterless chloramphenicol acetyltransferase (cat) reporter gene in E. coli. P(out) in group IIC-attC introns may therefore play a role in the expression of one or more gene cassettes whose transcription from Pc would otherwise be impeded by insertion of the intron.

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Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion.

Publication Date: 2010 Aug 16 PMID: 20716517
Authors: Sollu, C. - Pars, K. - Cornu, T. I. - Thibodeau-Beganny, S. - Maeder, M. L. - Joung, J. K. - Heilbronn, R. - Cathomen, T.
Journal: Nucleic Acids Res

Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in approximately 10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.

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Archaeal/Eukaryal RNase P: subunits, functions and RNA diversification.

Publication Date: 2010 Aug 16 PMID: 20716516
Authors: Jarrous, N. - Gopalan, V.
Journal: Nucleic Acids Res

RNase P, a catalytic ribonucleoprotein (RNP), is best known for its role in precursor tRNA processing. Recent discoveries have revealed that eukaryal RNase P is also required for transcription and processing of select non-coding RNAs, thus enmeshing RNase P in an intricate network of machineries required for gene expression. Moreover, the RNase P RNA seems to have been subject to gene duplication, selection and divergence to generate two new catalytic RNPs, RNase MRP and MRP-TERT, which perform novel functions encompassing cell cycle control and stem cell biology. We present new evidence and perspectives on the functional diversification of the RNase P RNA to highlight it as a paradigm for the evolutionary plasticity that underlies the extant broad repertoire of catalytic and unexpected regulatory roles played by RNA-driven RNPs.

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MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor {beta}1: a novel role of miR-382.

Publication Date: 2010 Aug 16 PMID: 20716515
Authors: Kriegel, A. J. - Fang, Y. - Liu, Y. - Tian, Z. - Mladinov, D. - Matus, I. R. - Ding, X. - Greene, A. S. - Liang, M.
Journal: Nucleic Acids Res

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor beta1 (TGFbeta1), a model of epithelial-mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFbeta1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA-target interactions. Knockdown of miR-382, which was up-regulated by TGFbeta1, attenuated TGFbeta1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3'-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFbeta1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFbeta1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFbeta1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFbeta1.

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A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding.

Publication Date: 2010 Aug 12 PMID: 20705654
Authors: Rhodin, M. H. - Dinman, J. D.
Journal: Nucleic Acids Res

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 'P-site loop'. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.

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RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription.

Publication Date: 2010 Aug 12 PMID: 20705653
Authors: Kanagaraj, R. - Huehn, D. - Mackellar, A. - Menigatti, M. - Zheng, L. - Urban, V. - Shevelev, I. - Greenleaf, A. L. - Janscak, P.
Journal: Nucleic Acids Res

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2-Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcription.

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Functional microRNA generated from a cytoplasmic RNA virus.

Publication Date: 2010 Aug 12 PMID: 20705652
Authors: Rouha, H. - Thurner, C. - Mandl, C. W.
Journal: Nucleic Acids Res

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play a pivotal role in the regulation of posttranscriptional gene expression in a wide range of eukaryotic organisms. Although DNA viruses have been shown to encode miRNAs and exploit the cellular RNA silencing machinery as a convenient way to regulate viral and host gene expression, it is generally believed that this pathway is not available to RNA viruses that replicate in the cytoplasm of the cell because miRNA biogenesis is initiated in the nucleus. In fact, among the >200 viral miRNAs that have been experimentally verified so far, none is derived from an RNA virus. Here, we show that a cytoplasmic RNA virus can indeed encode and produce a functional miRNA. We introduced a heterologous miRNA-precursor stem-loop sequence element into the RNA genome of the flavivirus tick-borne encephalitis virus, and this led to the production of a functional miRNA during viral infection without impairing viral RNA replication. These findings demonstrate that miRNA biogenesis can be used by cytoplasmic RNA viruses to produce regulatory molecules for the modulation of the transcriptome.

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Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure.

Publication Date: 2010 Aug 12 PMID: 20705651
Authors: Gavrilov, A. A. - Zukher, I. S. - Philonenko, E. S. - Razin, S. V. - Iarovaia, O. V.
Journal: Nucleic Acids Res

We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken alpha-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the alpha-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a 'guest' compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited.

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Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins.

Publication Date: 2010 Aug 12 PMID: 20705650
Authors: Cridge, A. G. - Castelli, L. M. - Smirnova, J. B. - Selley, J. N. - Rowe, W. - Hubbard, S. J. - McCarthy, J. E. - Ashe, M. P. - Grant, C. M. - Pavitt, G. D.
Journal: Nucleic Acids Res

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Delta and caf20Delta cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

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Objective sequence-based subfamily classifications of mouse homeodomains reflect their in vitro DNA-binding preferences.

Publication Date: 2010 Aug 12 PMID: 20705649
Authors: Santos, M. A. - Turinsky, A. L. - Ong, S. - Tsai, J. - Berger, M. F. - Badis, G. - Talukder, S. - Gehrke, A. R. - Bulyk, M. L. - Hughes, T. R. - Wodak, S. J.
Journal: Nucleic Acids Res

Classifying proteins into subgroups with similar molecular function on the basis of sequence is an important step in deriving reliable functional annotations computationally. So far, however, available classification procedures have been evaluated against protein subgroups that are defined by experts using mainly qualitative descriptions of molecular function. Recently, in vitro DNA-binding preferences to all possible 8-nt DNA sequences have been measured for 178 mouse homeodomains using protein-binding microarrays, offering the unprecedented opportunity of evaluating the classification methods against quantitative measures of molecular function. To this end, we automatically derive homeodomain subtypes from the DNA-binding data and independently group the same domains using sequence information alone. We test five sequence-based methods, which use different sequence-similarity measures and algorithms to group sequences. Results show that methods that optimize the classification robustness reflect well the detailed functional specificity revealed by the experimental data. In some of these classifications, 73-83% of the subfamilies exactly correspond to, or are completely contained in, the function-based subtypes. Our findings demonstrate that certain sequence-based classifications are capable of yielding very specific molecular function annotations. The availability of quantitative descriptions of molecular function, such as DNA-binding data, will be a key factor in exploiting this potential in the future.

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Incorporation of dUTP does not mediate mutation of A:T base pairs in Ig genes in vivo.

Publication Date: 2010 Aug 12 PMID: 20705648
Authors: Sharbeen, G. - Cook, A. J. - Lau, K. K. - Raftery, J. - Yee, C. W. - Jolly, C. J.
Journal: Nucleic Acids Res

Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP(+) cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.

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Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex.

Publication Date: 2010 Aug 12 PMID: 20705647
Authors: Chen, W. Y. - Pulukkunat, D. K. - Cho, I. M. - Tsai, H. Y. - Gopalan, V.
Journal: Nucleic Acids Res

RNase P catalyzes the Mg(2)(+)-dependent 5'-maturation of precursor tRNAs. Biochemical studies on the bacterial holoenzyme, composed of one catalytic RNase P RNA (RPR) and one RNase P protein (RPP), have helped understand the pleiotropic roles (including substrate/Mg(2+) binding) by which a protein could facilitate RNA catalysis. As a model for uncovering the functional coordination among multiple proteins that aid an RNA catalyst, we use archaeal RNase P, which comprises one catalytic RPR and at least four RPPs. Exploiting our previous finding that these archaeal RPPs function as two binary RPP complexes (POP5*RPP30 and RPP21*RPP29), we prepared recombinant RPP pairs from three archaea and established interchangeability of subunits through homologous/heterologous assemblies. Our finding that archaeal POP5*RPP30 reconstituted with bacterial and organellar RPRs suggests functional overlap of this binary complex with the bacterial RPP and highlights their shared recognition of a phylogenetically-conserved RPR catalytic core, whose minimal attributes we further defined through deletion mutagenesis. Moreover, single-turnover kinetic studies revealed that while POP5*RPP30 is solely responsible for enhancing the RPR's rate of precursor tRNA cleavage (by 60-fold), RPP21*RPP29 contributes to increased substrate affinity (by 16-fold). Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein.

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Codon reassignment in the Escherichia coli genetic code.

Publication Date: 2010 Aug 11 PMID: 20702426
Authors: Mukai, T. - Hayashi, A. - Iraha, F. - Sato, A. - Ohtake, K. - Yokoyama, S. - Sakamoto, K.
Journal: Nucleic Acids Res

Most organisms, from Escherichia coli to humans, use the 'universal' genetic code, which have been unchanged or 'frozen' for billions of years. It has been argued that codon reassignment causes mistranslation of genetic information, and must be lethal. In this study, we successfully reassigned the UAG triplet from a stop to a sense codon in the E. coli genome, by eliminating the UAG-recognizing release factor, an essential cellular component, from the bacterium. Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary. Thus, UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA. The result reveals the unexpected flexibility of the genetic code.

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Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction.

Publication Date: 2010 Aug 11 PMID: 20702425
Authors: Westblade, L. F. - Campbell, E. A. - Pukhrambam, C. - Padovan, J. C. - Nickels, B. E. - Lamour, V. - Darst, S. A.
Journal: Nucleic Acids Res

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the beta-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP beta-subunit. We determined the 2.9-A resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-beta1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.

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O6-Methylguanine induces altered proteins at the level of transcription in human cells.

Publication Date: 2010 Aug 11 PMID: 20702424
Authors: Burns, J. A. - Dreij, K. - Cartularo, L. - Scicchitano, D. A.
Journal: Nucleic Acids Res

O(6)-Methylguanine (O(6)-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O(6)-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O(6)-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs O(6)-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O(6)-benzylguanine, approximately 58% of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O(6)-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.

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A network of conserved co-occurring motifs for the regulation of alternative splicing.

Publication Date: 2010 Aug 11 PMID: 20702423
Authors: Suyama, M. - Harrington, E. D. - Vinokourova, S. - von Knebel Doeberitz, M. - Ohara, O. - Bork, P.
Journal: Nucleic Acids Res

Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT-PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages.

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Sequence-specific cleavage of RNA by Type II restriction enzymes.

Publication Date: 2010 Aug 11 PMID: 20702422
Authors: Murray, I. A. - Stickel, S. K. - Roberts, R. J.
Journal: Nucleic Acids Res

The ability of 223 Type II restriction endonucleases to hydrolyze RNA-DNA heteroduplex oligonucleotide substrates was assessed. Despite the significant topological and sequence asymmetry introduced when one strand of a DNA duplex is substituted by RNA we find that six restriction enzymes (AvaII, AvrII, BanI, HaeIII, HinfI and TaqI), exclusively of the Type IIP class that recognize palindromic or interrupted-palindromic DNA sequences, catalyze robust and specific cleavage of both RNA and DNA strands of such a substrate. Time-course analyses indicate that some endonucleases hydrolyze phosphodiester bonds in both strands simultaneously whereas others appear to catalyze sequential reactions in which either the DNA or RNA product accumulates more rapidly. Such strand-specific variation in cleavage susceptibility is both significant (up to orders of magnitude difference) and somewhat sequence dependent, notably in relation to the presence or absence of uracil residues in the RNA strand. Hybridization to DNA oligonucleotides that contain endonuclease recognition sites can be used to achieve targeted hydrolysis of extended RNA substrates produced by in vitro transcription. The ability to 'restrict' an RNA-DNA hybrid, albeit with a limited number of restriction endonucleases, provides a method whereby individual RNA molecules can be targeted for site-specific cleavage in vitro.

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Selection systems based on dominant-negative transcription factors for precise genetic engineering.

Publication Date: 2010 Aug 11 PMID: 20702421
Authors: Dutoit, R. - Dubois, E. - Jacobs, E.
Journal: Nucleic Acids Res

Diverse tools are available for performing genetic modifications of microorganisms. However, new methods still need to be developed for performing precise genomic engineering without introducing any undesirable side-alteration. Indeed for functional analyses of genomic elements, as well as for some industrial applications, only the desired mutation should be introduced at the locus considered. This article describes a new approach fulfilling these requirements, based on the use of selection systems consisting in truncated genes encoding dominant-negative transcription factors. We have demonstrated dominant-negative effects mediated by truncated Gal4p and Arg81p proteins in Saccharomyces cerevisiae, interfering with galactose and arginine metabolic pathways, respectively. These genes can be used as positive and negative markers, since they provoke both growth inhibition on substrates and resistance to specific drugs. These selection markers have been successfully used for precisely deleting HO and URA3 in wild yeasts. This genetic engineering approach could be extended to other microorganisms.

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Atomic resolution structure of CAG RNA repeats: structural insights and implications for the trinucleotide repeat expansion diseases.

Publication Date: 2010 Aug 11 PMID: 20702420
Authors: Kiliszek, A. - Kierzek, R. - Krzyzosiak, W. J. - Rypniewski, W.
Journal: Nucleic Acids Res

CAG repeats occur predominantly in the coding regions of human genes, which suggests their functional importance. In some genes, these sequences can undergo pathogenic expansions leading to neurodegenerative polyglutamine (poly-Q) diseases. The mutant transcripts containing expanded CAG repeats possibly contribute to pathogenesis in addition to the well-known pathogenic effects of mutant proteins. We have analysed two crystal forms of RNA duplexes containing CAG repeats: (GGCAGCAGCC)(2). One of the structures has been determined at atomic resolution (0.95 A) and the other at 1.9 A. The duplexes include non-canonical A-A pairs that fit remarkably well within a regular A-helix. All the adenosines are in the anti-conformation and the only interaction within each A-A pair is a single C2-H2...N1 hydrogen bond. Both adenosines in each A-A pair are shifted towards the major groove, although to different extents; the A which is the H-bond donor stands out more (the 'thumbs-up' conformation). The main effect on the helix conformation is a local unwinding. The CAG repeats and the previously examined CUG structures share a similar pattern of electrostatic charge distribution in the minor groove, which could explain their affinity for the pathogenesis-related MBNL1 protein.

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Foxm1 transcription factor is required for maintenance of pluripotency of P19 embryonal carcinoma cells.

Publication Date: 2010 Aug 11 PMID: 20702419
Authors: Xie, Z. - Tan, G. - Ding, M. - Dong, D. - Chen, T. - Meng, X. - Huang, X. - Tan, Y.
Journal: Nucleic Acids Res

Transcription factor Foxm1 plays a critical role during embryonic development and its expression is repressed during retinoic acid (RA)-induced differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, correlated with downregulation of expression of pluripotency markers. To study whether Foxm1 participates in the maintenance of pluripotency of stem cells, we knock down Foxm1 expression in P19 cells and identify that Oct4 are regulated directly by Foxm1. Knockdown of Foxm1 also results in spontaneous differentiation of P19 cells to mesodermal derivatives, such as muscle and adipose tissues. Maintaining Foxm1 expression prevents the downregulation of pluripotency-related transcription factors such as Oct4 and Nanog during P19 cell differentiation. Furthermore, overexpression of FOXM1 alone in RA-differentiated P19 cells (4 days) or human newborn fibroblasts restarts the expression of pluripotent genes Oct4, Nanog and Sox2. Together, our results suggest a critical involvement of Foxm1 in maintenance of stem cell pluripotency.

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Design of a minimal silencer for the silent mating-type locus HML of Saccharomyces cerevisiae.

Publication Date: 2010 Aug 10 PMID: 20699276
Authors: Weber, J. M. - Ehrenhofer-Murray, A. E.
Journal: Nucleic Acids Res

The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS DeltaI) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Delta. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS DeltaI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing.

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On the power and limits of evolutionary conservation--unraveling bacterial gene regulatory networks.

Publication Date: 2010 Aug 10 PMID: 20699275
Authors: Baumbach, J.
Journal: Nucleic Acids Res

The National Center for Biotechnology Information (NCBI) recently announced '1000 prokaryotic genomes are now completed and available in the Genome database'. The increasing trend will provide us with thousands of sequenced microbial organisms over the next years. However, this is only the first step in understanding how cells survive, reproduce and adapt their behavior while being exposed to changing environmental conditions. One major control mechanism is transcriptional gene regulation. Here, striking is the direct juxtaposition of the handful of bacterial model organisms to the 1000 prokaryotic genomes. Next-generation sequencing technologies will further widen this gap drastically. However, several computational approaches have proven to be helpful. The main idea is to use the known transcriptional regulatory network of reference organisms as template in order to unravel evolutionarily conserved gene regulations in newly sequenced species. This transfer essentially depends on the reliable identification of several types of conserved DNA sequences. We decompose this problem into three computational processes, review the state of the art and illustrate future perspectives.

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TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.

Publication Date: 2010 Aug 10 PMID: 20699274
Authors: Li, T. - Huang, S. - Jiang, W. Z. - Wright, D. - Spalding, M. H. - Weeks, D. P. - Yang, B.
Journal: Nucleic Acids Res

DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.

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A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination.

Publication Date: 2010 Aug 10 PMID: 20699273
Authors: de Almeida, S. F. - Garcia-Sacristan, A. - Custodio, N. - Carmo-Fonseca, M.
Journal: Nucleic Acids Res

In eukaryotes, the production of mature messenger RNA that exits the nucleus to be translated into protein in the cytoplasm requires precise and extensive modification of the nascent transcript. Any failure that compromises the integrity of an mRNA may cause its retention in the nucleus and trigger its degradation. Multiple studies indicate that mRNAs with processing defects accumulate in nuclear foci or 'dots' located near the site of transcription, but how exactly are defective RNAs recognized and tethered is still unknown. Here, we present evidence suggesting that unprocessed beta-globin transcripts render RNA polymerase II (Pol II) incompetent for termination and that this quality control process requires the integrity of the nuclear exosome. Our results show that unprocessed pre-mRNAs remain tethered to the DNA template in association with Pol II, in an Rrp6-dependent manner. This reveals an unprecedented link between nuclear RNA surveillance, the exosome and Pol II transcriptional termination.

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Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes.

Publication Date: 2010 Aug 10 PMID: 20699272
Authors: Das, S. K. - Austin, M. D. - Akana, M. C. - Deshpande, P. - Cao, H. - Xiao, M.
Journal: Nucleic Acids Res

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.

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Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R.

Publication Date: 2010 Aug 10 PMID: 20699271
Authors: Izquierdo, J. M.
Journal: Nucleic Acids Res

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

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Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions.

Publication Date: 2010 Aug 10 PMID: 20699270
Authors: Fantini, D. - Vascotto, C. - Marasco, D. - D'Ambrosio, C. - Romanello, M. - Vitagliano, L. - Pedone, C. - Poletto, M. - Cesaratto, L. - Quadrifoglio, F. - Scaloni, A. - Radicella, J. P. - Tell, G.
Journal: Nucleic Acids Res

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

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Identification of rare alleles and their carriers using compressed se(que)nsing.

Publication Date: 2010 Aug 10 PMID: 20699269
Authors: Shental, N. - Amir, A. - Zuk, O.
Journal: Nucleic Acids Res

Identification of rare variants by resequencing is important both for detecting novel variations and for screening individuals for known disease alleles. New technologies enable low-cost resequencing of target regions, although it is still prohibitive to test more than a few individuals. We propose a novel pooling design that enables the recovery of novel or known rare alleles and their carriers in groups of individuals. The method is based on a Compressed Sensing (CS) approach, which is general, simple and efficient. CS allows the use of generic algorithmic tools for simultaneous identification of multiple variants and their carriers. We model the experimental procedure and show via computer simulations that it enables the recovery of rare alleles and their carriers in larger groups than were possible before. Our approach can also be combined with barcoding techniques to provide a feasible solution based on current resequencing costs. For example, when targeting a small enough genomic region ( approximately 100 bp) and using only approximately 10 sequencing lanes and approximately 10 distinct barcodes per lane, one recovers the identity of 4 rare allele carriers out of a population of over 4000 individuals. We demonstrate the performance of our approach over several publicly available experimental data sets.

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RNAMotifScan: automatic identification of RNA structural motifs using secondary structural alignment.

Publication Date: 2010 Aug 8 PMID: 20696653
Authors: Zhong, C. - Tang, H. - Zhang, S.
Journal: Nucleic Acids Res

Recent studies have shown that RNA structural motifs play essential roles in RNA folding and interaction with other molecules. Computational identification and analysis of RNA structural motifs remains a challenging task. Existing motif identification methods based on 3D structure may not properly compare motifs with high structural variations. Other structural motif identification methods consider only nested canonical base-pairing structures and cannot be used to identify complex RNA structural motifs that often consist of various non-canonical base pairs due to uncommon hydrogen bond interactions. In this article, we present a novel RNA structural alignment method for RNA structural motif identification, RNAMotifScan, which takes into consideration the isosteric (both canonical and non-canonical) base pairs and multi-pairings in RNA structural motifs. The utility and accuracy of RNAMotifScan is demonstrated by searching for kink-turn, C-loop, sarcin-ricin, reverse kink-turn and E-loop motifs against a 23S rRNA (PDBid: 1S72), which is well characterized for the occurrences of these motifs. Finally, we search these motifs against the RNA structures in the entire Protein Data Bank and the abundances of them are estimated. RNAMotifScan is freely available at our supplementary website (http://genome.ucf.edu/RNAMotifScan).

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Selection of RNA aptamers that bind HIV-1 LTR DNA duplexes: strand invaders.

Publication Date: 2010 Aug 6 PMID: 20693539
Authors: Srisawat, C. - Engelke, D. R.
Journal: Nucleic Acids Res

RNA that can specifically bind to double-stranded DNA is of interest because it might be used as a means to regulate transcription of the target genes. To explore possible interactions between RNA and duplex DNA, we selected for RNA aptamers that can bind to the long terminal repeats (LTRs) of human immunodeficiency virus type 1 DNA. The selected aptamers were classified into four major groups based on the consensus sequences, which were found to locate in the non-stem regions of the predicted RNA secondary structures, consistent with roles in target binding. Analysis of the aptamer consensus sequences suggested that the conserved segments could form duplexes via Watson-Crick base-pairing with preferred sequences in one strand of the DNA, assuming the aptamer invaded the duplex. The aptamer binding sites on the LTR were experimentally determined to be located preferentially at these sites near the termini of double-stranded target DNA, despite selection schemes that were designed to minimize preferences for termini. The results presented here show that aptamer RNAs can be selected in vitro that strand-invade at preferred DNA duplex sequences to form stable complexes.

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Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair.

Publication Date: 2010 Aug 6 PMID: 20693538
Authors: Krasikova, Y. S. - Rechkunova, N. I. - Maltseva, E. A. - Petruseva, I. O. - Lavrik, O. I.
Journal: Nucleic Acids Res

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA-protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5'-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5'-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5'-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG.

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Biophysical and atomic force microscopy characterization of the RNA from satellite tobacco mosaic virus.

Publication Date: 2010 Aug 6 PMID: 20693537
Authors: Kuznetsov, Y. G. - Dowell, J. J. - Gavira, J. A. - Ng, J. D. - McPherson, A.
Journal: Nucleic Acids Res

Agarose gel electrophoresis, circular dichroism and differential scanning calorimetry showed that single-stranded RNA from satellite tobacco mosaic virus transforms from a conformationally 'closed state' at 4 degrees C to a more conformationally 'open state' at 65 degrees C. The transition is reversible and shows no hysteresis. Atomic force microscopy (AFM) allowed visualization of the two states and indicated that the conformationally 'closed state' probably corresponds to the native encapsidated conformation, and that the 'open state' represents a conformation, characterized as short, thick chains of domains, as a consequence of the loss of tertiary interactions. Heating from 75 degrees C to 85 degrees C in the presence of EDTA was necessary to further unravel the 'open' conformation RNA into extended chains of lengths >280 nm. Virus exposed to low concentrations of phenol at 65 degrees C, extruded RNA as distinctive 'pigtails' in a synchronous fashion, and these 'pigtails' then elongated, as the RNA was further discharged by the particles. Moderate concentrations of phenol at 65 degrees C produced complete disruption of virions and only remains of decomposed particles and disordered RNA were evident. AFM images of RNA emerging from disrupted virions appear most consistent with linear arrangements of structural domains.

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Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes.

Publication Date: 2010 Aug 6 PMID: 20693536
Authors: Singh, P. - Cho, J. - Tsai, S. Y. - Rivas, G. E. - Larson, G. P. - Szabo, P. E.
Journal: Nucleic Acids Res

Genomic imprinting is an epigenetic inheritance system characterized by parental allele-specific gene expression. Allele-specific DNA methylation and chromatin composition are two epigenetic modification systems that control imprinted gene expression. To get a general assessment of histone lysine acetylation at imprinted genes we measured allele-specific acetylation of a wide range of lysine residues, H3K4, H3K18, H3K27, H3K36, H3K79, H3K64, H4K5, H4K8, H4K12, H2AK5, H2BK12, H2BK16 and H2BK46 at 11 differentially methylated regions (DMRs) in reciprocal mouse crosses using multiplex chromatin immunoprecipitation SNuPE assays. Histone acetylation marks generally distinguished the methylation-free alleles from methylated alleles at DMRs in mouse embryo fibroblasts and embryos. Acetylated lysines that are typically found at transcription start sites exhibited stronger allelic bias than acetylated histone residues in general. Maternally methylated DMRs, that usually overlap with promoters exhibited higher levels of acetylation and a 10% stronger allele-specific bias than paternally methylated DMRs that reside in intergenic regions. Along the H19/Igf2 imprinted domain, allele-specific acetylation at each lysine residue depended on functional CTCF binding sites in the imprinting control region. Our results suggest that many different histone acetyltransferase and histone deacetylase enzymes must act in concert in setting up and maintaining reciprocal parental allelic histone acetylation at DMRs.

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Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates.

Publication Date: 2010 Aug 6 PMID: 20693535
Authors: Matovina, M. - Seah, N. - Hamilton, T. - Warren, D. - Landy, A.
Journal: Nucleic Acids Res

Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Forster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the lambda integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.

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Dissecting the role of conserved box C/D sRNA sequences in di-sRNP assembly and function.

Publication Date: 2010 Aug 6 PMID: 20693534
Authors: Bleichert, F. - Baserga, S. J.
Journal: Nucleic Acids Res

In all three kingdoms of life, nucleotides in ribosomal RNA (rRNA) are post-transcriptionally modified. One type of chemical modification is 2'-O-ribose methylation, which is, in eukaryotes and archaea, performed by box C/D small ribonucleoproteins (box C/D sRNPs in archaea) and box C/D small nucleolar ribonucleoproteins (box C/D snoRNPs in eukaryotes), respectively. Recently, the first structure of any catalytically active box C/D s(no)RNP determined by electron microscopy and single particle analysis surprisingly demonstrated that they are dimeric RNPs. Mutational analyses of the Nop5 protein interface suggested that di-sRNP formation is also required for the in vitro catalytic activity. We have now analyzed the functional relevance of the second interface, the sRNA interface, within the box C/D di-sRNP. Mutations in conserved sequence elements of the sRNA, which allow sRNP assembly but which severely interfere with the catalytic activity of box C/D sRNPs, prevent formation of the di-sRNP. In addition, we can observe the dimeric box C/D sRNP architecture with a different box C/D sRNP, suggesting that this architecture is conserved. Together, these results provide further support for the functional relevance of the di-sRNP architecture and also provide a structural explanation for the observed defects in catalysis of 2'-O-ribose methylation.

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Homeotic proteins participate in the function of human-DNA replication origins.

Publication Date: 2010 Aug 6 PMID: 20693533
Authors: Marchetti, L. - Comelli, L. - D'Innocenzo, B. - Puzzi, L. - Luin, S. - Arosio, D. - Calvello, M. - Mendoza-Maldonado, R. - Peverali, F. - Trovato, F. - Riva, S. - Biamonti, G. - Abdurashidova, G. - Beltram, F. - Falaschi, A.
Journal: Nucleic Acids Res

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13-origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.

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Identification of functional clock-controlled elements involved in differential timing of Per1 and Per2 transcription.

Publication Date: 2010 Aug 6 PMID: 20693532
Authors: Yamajuku, D. - Shibata, Y. - Kitazawa, M. - Katakura, T. - Urata, H. - Kojima, T. - Nakata, O. - Hashimoto, S.
Journal: Nucleic Acids Res

It has been proposed that robust rhythmic gene expression requires clock-controlled elements (CCEs). Transcription of Per1 was reported to be regulated by the E-box and D-box in conventional reporter assays. However, such experiments are inconclusive in terms of how the CCEs and their combinations determine the phase of the Per1 gene. Whereas the phase of Per2 oscillation was found to be the most delayed among the three Period genes, the phase-delaying regions of the Per2 promoter remain to be determined. We therefore investigated the regulatory mechanism of circadian Per1 and Per2 transcription using an in vitro rhythm oscillation-monitoring system. We found that the copy number of the E-box might play an important role in determining the phase of Per1 oscillation. Based on real-time bioluminescence assays with various promoter constructs, we provide evidence that the non-canonical E-box is involved in the phase delay of Per2 oscillation. Transfection experiments confirmed that the non-canonical E-box could be activated by CLOCK/BMAL1. We also show that the D-box in the third conserved segment of the Per2 promoter generated high amplitude. Our experiments demonstrate that the copy number and various combinations of functional CCEs ultimately led to different circadian phases and amplitudes.

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High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides.

Publication Date: 2010 Aug 6 PMID: 20693531
Authors: Borovkov, A. Y. - Loskutov, A. V. - Robida, M. D. - Day, K. M. - Cano, J. A. - Le Olson, T. - Patel, H. - Brown, K. - Hunter, P. D. - Sykes, K. F.
Journal: Nucleic Acids Res

To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5 cent per base, making gene synthesis more affordable than traditional cloning.

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An engineered mammalian band-pass network.

Publication Date: 2010 Aug 6 PMID: 20693530
Authors: Greber, D. - Fussenegger, M.
Journal: Nucleic Acids Res

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of 'low-threshold' morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a 'high-threshold' concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing applications.

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The Type II restriction endonuclease MvaI has dual specificity.

Publication Date: 2010 Aug 6 PMID: 20693529
Authors: Stier, I. - Kiss, A.
Journal: Nucleic Acids Res

The MvaI restriction endonuclease cuts 5'-CC downward arrowAGG-3'/5'-CC upward arrowTGG-3' sites as indicated by the arrows. N4-methylation of the inner cytosines (C(m4)CAGG/C(m4)CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C(m5)C downward arrowGGG/CC(m5)CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC(m5)C downward arrowGGG/CC(m5)C upward arrowGGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the 'normal' activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.

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Deep sequencing analysis of mutations resulting from the incorporation of dNTP analogs.

Publication Date: 2010 Aug 6 PMID: 20693528
Authors: Petrie, K. L. - Joyce, G. F.
Journal: Nucleic Acids Res

Next-generation DNA sequencing technology was used to score >100 000 mutations resulting from exposure of a nucleic acid template to a mutagenic dNTP analog during a single pass of a DNA polymerase. An RNA template of known secondary structure was reverse transcribed in the presence of 8-oxo-dGTP, dPTP or both, followed by forward transcription in the presence of standard NTPs. Each mutagen, whether used alone or in combination, resulted in a highly characteristic mutation profile. Mutations were generated at a mean frequency of 1-2% per eligible nucleotide position, but there was substantial variation in the frequency of mutation at different positions, with a SD close to the mean. This variation was partly due to the identity of the immediately surrounding nucleotides and was not significantly influenced by the secondary structure of the RNA template. Most of the variation appears to result from idiosyncratic features that derive from local sequence context, demonstrating how different genetic sequences have different chemical phenotypes.

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Stimulation of ribosomal frameshifting by antisense LNA.

Publication Date: 2010 Aug 6 PMID: 20693527
Authors: Yu, C. H. - Noteborn, M. H. - Olsthoorn, R. C.
Journal: Nucleic Acids Res

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12-18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

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ChIPing the cistrome of PXR in mouse liver.

Publication Date: 2010 Aug 6 PMID: 20693526
Authors: Cui, J. Y. - Gunewardena, S. S. - Rockwell, C. E. - Klaassen, C. D.
Journal: Nucleic Acids Res

The pregnane X receptor (PXR) is a key regulator of xenobiotic metabolism and disposition in liver. However, little is known about the PXR DNA-binding signatures in vivo, or how PXR regulates novel direct targets on a genome-wide scale. Therefore, we generated a roadmap of hepatic PXR bindings in the entire mouse genome [chromatin immunoprecipitation (ChIP)-Seq]. The most frequent PXR DNA-binding motif is the AGTTCA-like direct repeat with a 4bp spacer [direct repeat (DR)-4)]. Surprisingly, there are also high motif occurrences with spacers of a periodicity of 5 bp, forming a novel DR-(5n + 4) pattern for PXR binding. PXR-binding overlaps with the epigenetic mark for gene activation (histone-H3K4-di-methylation), but not with epigenetic marks for gene suppression (DNA methylation or histone-H3K27-tri-methylation) (ChIP-on-chip). After administering a PXR agonist, changes in mRNA of most PXR-direct target genes correlate with increased PXR binding. Specifically, increased PXR binding triggers the trans-activation of critical drug-metabolizing enzymes and transporters. The mRNA induction of these genes is absent in PXR-null mice. The current work provides the first in vivo evidence of PXR DNA-binding signatures in the mouse genome, paving the path for predicting and further understanding the multifaceted roles of PXR in liver.

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SEWAL: an open-source platform for next-generation sequence analysis and visualization.

Publication Date: 2010 Aug 6 PMID: 20693400
Authors: Pitt, J. N. - Rajapakse, I. - Ferre-D'Amare, A. R.
Journal: Nucleic Acids Res

Next-generation DNA sequencing platforms provide exciting new possibilities for in vitro genetic analysis of functional nucleic acids. However, the size of the resulting data sets presents computational and analytical challenges. We present an open-source software package that employs a locality-sensitive hashing algorithm to enumerate all unique sequences in an entire Illumina sequencing run ( approximately 10(8) sequences). The algorithm results in quasilinear time processing of entire Illumina lanes ( approximately 10(7) sequences) on a desktop computer in minutes. To facilitate visual analysis of sequencing data, the software produces three-dimensional scatter plots similar in concept to Sewall Wright and John Maynard Smith's adaptive or fitness landscape. The software also contains functions that are particularly useful for doped selections such as mutation frequency analysis, information content calculation, multivariate statistical functions (including principal component analysis), sequence distance metrics, sequence searches and sequence comparisons across multiple Illumina data sets. Source code, executable files and links to sample data sets are available at http://www.sourceforge.net/projects/sewal.

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Slingshot: a PiggyBac based transposon system for tamoxifen-inducible 'self-inactivating' insertional mutagenesis.

Publication Date: 2010 Aug 5 PMID: 20688953
Authors: Kong, J. - Wang, F. - Brenton, J. D. - Adams, D. J.
Journal: Nucleic Acids Res

We have developed a self-inactivating PiggyBac transposon system for tamoxifen inducible insertional mutagenesis from a stably integrated chromosomal donor. This system, which we have named 'Slingshot', utilizes a transposon carrying elements for both gain- and loss-of-function screens in vitro. We show that the Slingshot transposon can be efficiently mobilized from a range of chromosomal loci with high inducibility and low background generating insertions that are randomly dispersed throughout the genome. Furthermore, we show that once the Slingshot transposon has been mobilized it is not remobilized producing stable clonal integrants in all daughter cells. To illustrate the efficacy of Slingshot as a screening tool we set out to identify mediators of resistance to puromycin and the chemotherapeutic drug vincristine by performing genetrap screens in mouse embryonic stem cells. From these genome-wide screens we identified multiple independent insertions in the multidrug resistance transporter genes Abcb1a/b and Abcg2 conferring resistance to drug treatment. Importantly, we also show that the Slingshot transposon system is functional in other mammalian cell lines such as human HEK293, OVCAR-3 and PE01 cells suggesting that it may be used in a range of cell culture systems. Slingshot represents a flexible and potent system for genome-wide transposon-mediated mutagenesis with many potential applications.

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Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.

Publication Date: 2010 Aug 4 PMID: 20685817
Authors: Szwagierczak, A. - Bultmann, S. - Schmidt, C. S. - Spada, F. - Leonhardt, H.
Journal: Nucleic Acids Res

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.

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Using protein design algorithms to understand the molecular basis of disease caused by protein-DNA interactions: the Pax6 example.

Publication Date: 2010 Aug 4 PMID: 20685816
Authors: Alibes, A. - Nadra, A. D. - De Masi, F. - Bulyk, M. L. - Serrano, L. - Stricher, F.
Journal: Nucleic Acids Res

Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein's function is not trivial. Protein design tools are commonly used to explain mutations that affect protein stability, or protein-protein interaction, but not for mutations that could affect protein-DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several diseases such as aniridia. The validity of FoldX to deal with protein-DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos. We conclude that 88% of the Pax6 mutations can be linked to changes in intrinsic stability (77%) and/or to its capabilities to bind DNA (30%). Our study emphasizes the importance of structure-based analysis to understand the molecular basis of diseases and shows that protein-DNA interactions can be analyzed to the same level of accuracy as protein stability, or protein-protein interactions.

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A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding.

Publication Date: 2010 Aug 4 PMID: 20685815
Authors: Bermudez, I. - Garcia-Martinez, J. - Perez-Ortin, J. E. - Roca, J.
Journal: Nucleic Acids Res

The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA arrays. The entire procedure was robust. Comparison of PB obtained in vivo with that obtained in vitro with naked DNA revealed that numerous chromosomal regions had deviated PB values. Similar analyses in yeast topoisomerase mutants uncovered further PB alterations across specific chromosomal domains. These results suggest that distinct chromosome compartments might confine different levels of DNA helical tension in yeast. Genome-wide analysis of psoralen-DNA PB can be, therefore, a useful approach to uncover a trait of the chromosome architecture not amenable to other techniques.

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Computational identification of tissue-specific alternative splicing elements in mouse genes from RNA-Seq.

Publication Date: 2010 Aug 4 PMID: 20685814
Authors: Wen, J. - Chiba, A. - Cai, X.
Journal: Nucleic Acids Res

Tissue-specific alternative splicing is a key mechanism for generating tissue-specific proteomic diversity in eukaryotes. Splicing regulatory elements (SREs) in pre-mature messenger RNA play a very important role in regulating alternative splicing. In this article, we use mouse RNA-Seq data to determine a positive data set where SREs are over-represented and a reliable negative data set where the same SREs are most likely under-represented for a specific tissue and then employ a powerful discriminative approach to identify SREs. We identified 456 putative splicing enhancers or silencers, of which 221 were predicted to be tissue-specific. Most of our tissue-specific SREs are likely different from constitutive SREs, since only 18% of our exonic splicing enhancers (ESEs) are contained in constitutive RESCUE-ESEs. A relatively small portion (20%) of our SREs is included in tissue-specific SREs in human identified in two recent studies. In the analysis of position distribution of SREs, we found that a dozen of SREs were biased to a specific region. We also identified two very interesting SREs that can function as an enhancer in one tissue but a silencer in another tissue from the same intronic region. These findings provide insight into the mechanism of tissue-specific alternative splicing and give a set of valuable putative SREs for further experimental investigations.

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A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing.

Publication Date: 2010 Aug 3 PMID: 20682560
Authors: Cantacessi, C. - Jex, A. R. - Hall, R. S. - Young, N. D. - Campbell, B. E. - Joachim, A. - Nolan, M. J. - Abubucker, S. - Sternberg, P. W. - Ranganathan, S. - Mitreva, M. - Gasser, R. B.
Journal: Nucleic Acids Res

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

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Microarray-based STR genotyping using RecA-mediated ligation.

Publication Date: 2010 Aug 3 PMID: 20682559
Authors: Herrmann, D. - Rose, E. - Muller, U. - Wagner, R.
Journal: Nucleic Acids Res

We describe a novel assay capable of accurately determining the length of short tandem repeat (STR) alleles. STR genotyping is achieved utilizing RecA-mediated ligation (RML), which combines the high fidelity of RecA-mediated homology searching with allele-specific ligation. RecA catalyzes the pairing of synthetic oligonucleotides with one strand of a double-stranded DNA target, in this case a PCR amplicon. Ligation occurs only when two adjacent oligonucleotides are base paired to the STR region without any overlap or gap. RecA activity is required to overcome the inherent difficulty of annealing repeated sequences in register. This assay is capable of determining STR genotypes of human samples, is easily adapted to high throughput or automated systems and can have widespread utility in diagnostic and forensic applications.

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