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Nucleic Acids Research: VOLUME 37 ISSUE 21 2009

Wed, 09 Dec 2009 04:36:18 PST

Nucleic Acids Research

Wed, 09 Dec 2009 04:36:18 PST

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Wed, 09 Dec 2009 04:36:18 PST

External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data

Wed, 09 Dec 2009 04:36:20 PST

The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.

HMMCONVERTER 1.0: a toolbox for hidden Markov models

Lam, T. Y., Meyer, I. M. — Wed, 09 Dec 2009 04:36:20 PST

Hidden Markov models (HMMs) and their variants are widely used in Bioinformatics applications that analyze and compare biological sequences. Designing a novel application requires the insight of a human expert to define the model's architecture. The implementation of prediction algorithms and algorithms to train the model's parameters, however, can be a time-consuming and error-prone task. We here present HMMConverter, a software package for setting up probabilistic HMMs, pair-HMMs as well as generalized HMMs and pair-HMMs. The user defines the model itself and the algorithms to be used via an XML file which is then directly translated into efficient C++ code. The software package provides linear-memory prediction algorithms, such as the Hirschberg algorithm, banding and the integration of prior probabilities and is the first to present computationally efficient linear-memory algorithms for automatic parameter training. Users of HMMConverter can thus set up complex applications with a minimum of effort and also perform parameter training and data analyses for large data sets.

An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

Wed, 09 Dec 2009 04:36:20 PST

We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ~21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

PiggyBac transgenic strategies in the developing chicken spinal cord

Lu, Y., Lin, C., Wang, X. — Wed, 09 Dec 2009 04:36:20 PST

The chicken spinal cord is an excellent model for the study of early neural development in vertebrates. However, the lack of robust, stable and versatile transgenic methods has limited the usefulness of chick embryos for the study of later neurodevelopmental events. Here we describe a new transgenic approach utilizing the PiggyBac (PB) transposon to facilitate analysis of late-stage neural development such as axon targeting and synaptic connection in the chicken embryo. Using PB transgenic approaches we achieved temporal and spatial regulation of transgene expression and performed stable RNA interference (RNAi). With these new capabilities, we mapped axon projection patterns of V2b subset of spinal interneurons and visualized maturation of the neuromuscular junction (NMJ). Furthermore, PB-mediated RNAi in the chick recapitulated the phenotype of loss of agrin function in the mouse NMJ. The simplicity and versatility of PB-mediated transgenic strategies hold great promise for large-scale genetic analysis of neuronal connectivity in the chick.

RNA-protein binding kinetics in an automated microfluidic reactor

Ridgeway, W. K., Seitaridou, E., Phillips, R., Williamson, J. R. — Wed, 09 Dec 2009 04:36:20 PST

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

MGEScan-non-LTR: computational identification and classification of autonomous non-LTR retrotransposons in eukaryotic genomes

Rho, M., Tang, H. — Wed, 09 Dec 2009 04:36:21 PST

Computational methods for genome-wide identification of mobile genetic elements (MGEs) have become increasingly necessary for both genome annotation and evolutionary studies. Non-long terminal repeat (non-LTR) retrotransposons are a class of MGEs that have been found in most eukaryotic genomes, sometimes in extremely high numbers. In this article, we present a computational tool, MGEScan-non-LTR, for the identification of non-LTR retrotransposons in genomic sequences, following a computational approach inspired by a generalized hidden Markov model (GHMM). Three different states represent two different protein domains and inter-domain linker regions encoded in the non-LTR retrotransposons, and their scores are evaluated by using profile hidden Markov models (for protein domains) and Gaussian Bayes classifiers (for linker regions), respectively. In order to classify the non-LTR retrotransposons into one of the 12 previously characterized clades using the same model, we defined separate states for different clades. MGEScan-non-LTR was tested on the genome sequences of four eukaryotic organisms, Drosophila melanogaster, Daphnia pulex, Ciona intestinalis and Strongylocentrotus purpuratus. For the D. melanogaster genome, MGEScan-non-LTR found all known ‘full-length’ elements and simultaneously classified them into the clades CR1, I, Jockey, LOA and R1. Notably, for the D. pulex genome, in which no non-LTR retrotransposon has been annotated, MGEScan-non-LTR found a significantly larger number of elements than did RepeatMasker, using the current version of the RepBase Update library. We also identified novel elements in the other two genomes, which have only been partially studied for non-LTR retrotransposons.

Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion

Yung, P. Y., Burke, C., Lewis, M., Egan, S., Kjelleberg, S., Thomas, T. — Wed, 09 Dec 2009 04:36:21 PST

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.

Impact of DNA-binding position variants on yeast gene expression

Swamy, K. B. S., Cho, C.-Y., Chiang, S., Tsai, Z. T.-Y., Tsai, H.-K. — Wed, 09 Dec 2009 04:36:18 PST

Transcription factors (TFs) regulate gene expression by binding to specific binding sites (TFBSs) in gene promoters. TFBS motifs may contain one or more variable positions. Although the prevailing assumption is that nucleotide variants at such positions are functionally equivalent, there is increasing evidence that such variants play a role in regulation of gene expression. In this article, we propose a method for studying the relationship between the expression of target genes and nucleotide variants in TFBS motifs at a genome-wide scale in Saccharomyces cerevisiae, especially the combinatorial effects of variants at two positions. Our analysis shows that nucleotide variations in more than one-third of variable positions and in 20% of dependent position pairs are highly correlated to gene expression. We define such positions as ‘functional’. However, some positions are only functional as dependent pairs, but not individually. In addition, a significant proportion of the functional positions have been well conserved across all yeast-related species studied. We also find that some positions require the presence of co-occurring TFs, while others maintain their functionality in the absence of a co-occurring TF. Our analysis supports the importance of nucleotide variants at variable positions of TFBSs in gene regulation.

Fine-grained annotation and classification of de novo predicted LTR retrotransposons

Steinbiss, S., Willhoeft, U., Gremme, G., Kurtz, S. — Wed, 09 Dec 2009 04:36:18 PST

Long terminal repeat (LTR) retrotransposons and endogenous retroviruses (ERVs) are transposable elements in eukaryotic genomes well suited for computational identification. De novo identification tools determine the position of potential LTR retrotransposon or ERV insertions in genomic sequences. For further analysis, it is desirable to obtain an annotation of the internal structure of such candidates. This article presents LTRdigest, a novel software tool for automated annotation of internal features of putative LTR retrotransposons. It uses local alignment and hidden Markov model-based algorithms to detect retrotransposon-associated protein domains as well as primer binding sites and polypurine tracts. As an example, we used LTRdigest results to identify 88 (near) full-length ERVs in the chromosome 4 sequence of Mus musculus, separating them from truncated insertions and other repeats. Furthermore, we propose a work flow for the use of LTRdigest in de novo LTR retrotransposon classification and perform an exemplary de novo analysis on the Drosophila melanogaster genome as a proof of concept. Using a new method solely based on the annotations generated by LTRdigest, 518 potential LTR retrotransposons were automatically assigned to 62 candidate groups. Representative sequences from 41 of these 62 groups were matched to reference sequences with >80% global sequence similarity.

Universal function-specificity of codon usage

Najafabadi, H. S., Goodarzi, H., Salavati, R. — Wed, 09 Dec 2009 04:36:18 PST

Synonymous codon usage has long been known as a factor that affects average expression level of proteins in fast-growing microorganisms, but neither its role in dynamic changes of expression in response to environmental changes nor selective factors shaping it in the genomes of higher eukaryotes have been fully understood. Here, we propose that codon usage is ubiquitously selected to synchronize the translation efficiency with the dynamic alteration of protein expression in response to environmental and physiological changes. Our analysis reveals that codon usage is universally correlated with gene function, suggesting its potential contribution to synchronized regulation of genes with similar functions. We directly show that coexpressed genes have similar synonymous codon usages within the genomes of human, yeast, Caenorhabditis elegans and Escherichia coli. We also demonstrate that perturbing the codon usage directly affects the level or even direction of changes in protein expression in response to environmental stimuli. Perturbing tRNA composition also has tangible phenotypic effects on the cell. By showing that codon usage is universally function-specific, our results expand, to almost all organisms, the notion that cells may need to dynamically alter their intracellular tRNA composition in order to adapt to their new environment or physiological role.

Primary sequence and epigenetic determinants of in vivo occupancy of genomic DNA by GATA1

Wed, 09 Dec 2009 04:36:18 PST

DNA sequence motifs and epigenetic modifications contribute to specific binding by a transcription factor, but the extent to which each feature determines occupancy in vivo is poorly understood. We addressed this question in erythroid cells by identifying DNA segments occupied by GATA1 and measuring the level of trimethylation of histone H3 lysine 27 (H3K27me3) and monomethylation of H3 lysine 4 (H3K4me1) along a 66 Mb region of mouse chromosome 7. While 91% of the GATA1-occupied segments contain the consensus binding-site motif WGATAR, only ~0.7% of DNA segments with such a motif are occupied. Using a discriminative motif enumeration method, we identified additional motifs predictive of occupancy given the presence of WGATAR. The specific motif variant AGATAA and occurrence of multiple WGATAR motifs are both strong discriminators. Combining motifs to pair a WGATAR motif with a binding site motif for GATA1, EKLF or SP1 improves discriminative power. Epigenetic modifications are also strong determinants, with the factor-bound segments highly enriched for H3K4me1 and depleted of H3K27me3. Combining primary sequence and epigenetic determinants captures 52% of the GATA1-occupied DNA segments and substantially increases the specificity, to one out of seven segments with the required motif combination and epigenetic signals being bound.

Demonstration of all-or-none loss of imprinting in mRNA expression in single cells

Wed, 09 Dec 2009 04:36:18 PST

Loss of imprinting (LOI) is the reactivation of the silenced allele of an imprinted gene, leading to perturbation of monoallelic expression. We tested the hypothesis that LOI of PLAGL1, a representative maternally imprinted gene, occurs through an all-or-none process leading to a mixture of fully imprinted and nonimprinted cells. Herein using a quantitative RT-PCR-based experimental approach, we measured LOI at the single cell level in human trophoblasts and demonstrated a broad distribution of LOI among cells exhibiting LOI, with the mean centered at ~100% LOI. There was a significant (P < 0.01) increase in expression after 2 days of 5-aza-2'-deoxycytidine (AZA) treatment and a significant (P < 0.01) increase in LOI after both 1 and 2 days of AZA treatment, while the distribution remained broad and centered at ~100% LOI. We propose a transcriptional pulsing model to show that the broadness of the distribution reflects the stochastic nature of expression between the two alleles in each cell. The mean of the distribution of LOI in the cells is consistent with our hypothesis that LOI occurs by an all-or-none process. All-or-none LOI could lead to a second distinct cell population that may have a selective advantage, leading to variation of LOI in normal tissues, such as the placenta, or in neoplastic cells.

Two Dot1 isoforms in Saccharomyces cerevisiae as a result of leaky scanning by the ribosome

Wed, 09 Dec 2009 04:36:18 PST

Dot1 is a conserved histone methyltransferase that methylates histone H3 on lysine 79. We previously observed that in Saccharomyces cerevisiae, a single DOT1 gene encodes two Dot1 protein species. Here, we show that the relative abundance of the two isoforms changed under nutrient-limiting conditions. A mutagenesis approach showed that the two Dot1 isoforms are produced from two alternative translation start sites as a result of leaky scanning by the ribosome. The leaky scanning was not affected by the 5'- or 3'-untranslated regions of DOT1, indicating that translation initiation is determined by the DOT1 coding sequence. Construction of yeast strains expressing either one of the isoforms showed that both were sufficient for Dot1’s role in global H3K79 methylation and telomeric gene silencing. However, the absence of the long isoform of Dot1 altered the resistance of yeast cells to the chitin-binding drug Calcofluor White, suggesting that the two Dot1 isoforms have a differential function in cell wall biogenesis.

SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin

Wed, 09 Dec 2009 04:36:19 PST

SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.

The transcriptional co-activator PCAF regulates cdk2 activity

Wed, 09 Dec 2009 04:36:19 PST

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G₂/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G₂/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

Park, S. W., Huang, W.-H., Persaud, S. D., Wei, L.-N. — Wed, 09 Dec 2009 04:36:19 PST

Cellular retinoic acid binding protein 1 (Crabp1) gene is biphasically (proliferation versus differentiation) regulated by thyroid hormone (T3) in 3T3-L1 cells. This study examines T3-repression of Crabp1 gene during adipocyte differentiation. T3 repression of Crabp1 requires receptor interacting protein 140 (RIP140). During differentiation, the juxtaposed chromatin configuration of Crabp1 promoter with its upstream region is maintained, but the 6-nucleosomes spanning thyroid hormone response element to transcription initiation site slide bi-directionally, with the third nucleosome remaining at the same position throughout differentiation. On the basal promoter, RIP140 replaces coactivators GRIP1 and PCAF and forms a repressive complex with CtBP1, HDAC3 and G9a. Initially active chromatin marks on this promoter, histone modifications H3-Ac and H3K4-me3, are weakened whereas repressive chromatin marks, H3K9-me3 and H3K27-me3 modification and recruitment of G9a, HP1, HP1 and H1, are intensified. This is the first study to examine chromatin remodeling, during the phase of hormone repression, of a bi-directionally regulated hormone target gene, and provides evidence for a functional role of RIP140 in chromatin remodeling to repress hormone target gene expression.

Influence of local sequence context on damaged base conformation in human DNA polymerase {iota}: molecular dynamics studies of nucleotide incorporation opposite a benzo[a]pyrene-derived adenine lesion

Donny-Clark, K., Broyde, S. — Wed, 09 Dec 2009 04:36:19 PST

Human DNA polymerase is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase normally favors Hoogsteen base pairing. Polymerase can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5'-CAGA*TT-3' sequence. This facilitates correct incorporation of dT via a Watson–Crick pair. In a 5'-TTTA*GA-3' sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase and its lesion bypass functions in humans.

Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII

Wilson, G. W., Edgell, D. R. — Wed, 09 Dec 2009 04:36:19 PST

Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.

Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria

Wed, 09 Dec 2009 04:36:19 PST

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m¹A) and 3-methylcytosine (m³C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N⁶-ethenoadenine (A). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.

A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation

Korolev, N., Berezhnoy, N. V., Eom, K. D., Tam, J. P., Nordenskiold, L. — Wed, 09 Dec 2009 04:36:19 PST

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear -oligo(l-lysines), (denoted Kn, n = 3–10, 31; n is the number of lysines equal to the ligand charge) and branched -substituted homologues of K10: YK10, LK10 (Z = +10); RK10, YRK10 and LYRK10 (Z = +20). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, C_KCl). The dependence of EC₅₀ (ligand concentration at the midpoint of DNA condensation) on C_KCl shows the existence of a salt-independent regime at low C_KCl and a salt-dependent regime with a steep rise of EC₅₀ with increase of C_KCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher C_KCl. A novel and simple relationship describing the EC₅₀ dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the -oligolysines K3–K10, the experimental dependencies of EC₅₀ on C_KCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.

Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

Wed, 09 Dec 2009 04:36:19 PST

The conserved mRNA export receptor NXF1 (Mex67 in yeast) assembles with messenger ribonucleoproteins (mRNP) in the nucleus and guides them through the nuclear pore complex into the cytoplasm. The DEAD family RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage. However, the molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. Here we report that the human NXF1-binding protein RBM15 binds specifically to human DBP5 and facilitates its direct contact with mRNA in vivo. We found that RBM15 is targeted to the nuclear envelope, where it colocalizes extensively with DBP5 and NXF1. Gene silencing of RBM15 leads to cytoplasmic depletion and nuclear accumulation of general mRNA as well as individual endogenous transcripts, indicating that RBM15 is required for efficient mRNA export. We propose a model in which RBM15 acts locally at the nuclear pore complex, by facilitating the recognition of NXF1–mRNP complexes by DBP5 during translocation, thereby contributing to efficient mRNA export.

A SRS2 homolog from Arabidopsis thaliana disrupts recombinogenic DNA intermediates and facilitates single strand annealing

Blanck, S., Kobbe, D., Hartung, F., Fengler, K., Focke, M., Puchta, H. — Wed, 09 Dec 2009 04:36:19 PST

Genetic and biochemical analyses of SRS2 homologs in fungi indicate a function in the processing of homologous recombination (HR) intermediates. To date, no SRS2 homologs have been described and analyzed in higher eukaryotes. Here, we report the first biochemical characterization of an SRS2 homolog from a multicellular eukaryote, the plant Arabidopsis thaliana. We studied the basic properties of AtSRS2 and were able to show that it is a functional 3'- to 5'-helicase. Furthermore, we characterized its biochemical function on recombinogenic intermediates and were able to show the unwinding of nicked Holliday junctions (HJs) and partial HJs (PX junctions). For the first time, we demonstrated strand annealing activity for an SRS2 homolog and characterized its strand pairing activity in detail. Our results indicate that AtSRS2 has properties that enable it to be involved in different steps during the processing of recombination intermediates. On the one hand, it could be involved in the unwinding of an elongating invading strand from a donor strand, while on the other hand, it could be involved in the annealing of the elongated strand at a later step.

Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions

Wed, 09 Dec 2009 04:36:19 PST

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE•GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE•GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.

A trimeric DNA polymerase complex increases the native replication processivity

Mikheikin, A. L., Lin, H.-K., Mehta, P., Jen-Jacobson, L., Trakselis, M. A. — Wed, 09 Dec 2009 04:36:19 PST

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.

DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI

Smith, R. M., Diffin, F. M., Savery, N. J., Josephsen, J., Szczelkun, M. D. — Wed, 09 Dec 2009 04:36:19 PST

LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a -family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5'-CTnGAyG-3' (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5'-CrTCnAG-3' being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).

The single polypeptide restriction-modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops

Smith, R. M., Josephsen, J., Szczelkun, M. D. — Wed, 09 Dec 2009 04:36:19 PST

To cleave DNA, the single polypeptide restriction–modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5'-CTnGAyG-3' as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.

An Mrr-family nuclease motif in the single polypeptide restriction-modification enzyme LlaGI

Smith, R. M., Josephsen, J., Szczelkun, M. D. — Wed, 09 Dec 2009 04:36:19 PST

Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Wed, 09 Dec 2009 04:36:20 PST

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA-K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE-mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C–rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism.

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

Pastor, T., Talotti, G., Lewandowska, M. A., Pagani, F. — Wed, 09 Dec 2009 04:36:20 PST

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

tRNA over-expression in breast cancer and functional consequences

Pavon-Eternod, M., Gomes, S., Geslain, R., Dai, Q., Rosner, M. R., Pan, T. — Wed, 09 Dec 2009 04:36:20 PST

Increased proliferation and elevated levels of protein synthesis are characteristics of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, what role tRNA plays in cancer cells has not been explored. We compare genome-wide tRNA expression in cancer-derived versus non-cancer-derived breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues. In cancer-derived versus non-cancer-derived cell lines, nuclear-encoded tRNAs increase by up to 3-fold and mitochondrial-encoded tRNAs increase by up to 5-fold. In tumors versus normal breast tissues, both nuclear- and mitochondrial-encoded tRNAs increase up to 10-fold. This tRNA over-expression is selective and coordinates with the properties of cognate amino acids. Nuclear- and mitochondrial-encoded tRNAs exhibit distinct expression patterns, indicating that tRNAs can be used as biomarkers for breast cancer. We also performed association analysis for codon usage-tRNA expression for the cell lines. tRNA isoacceptor expression levels are not geared towards optimal translation of house-keeping or cell line specific genes. Instead, tRNA isoacceptor expression levels may favor the translation of cancer-related genes having regulatory roles. Our results suggest a functional consequence of tRNA over-expression in tumor cells. tRNA isoacceptor over-expression may increase the translational efficiency of genes relevant to cancer development and progression.

Ion-induced folding of a kink turn that departs from the conventional sequence

Schroeder, K. T., Lilley, D. M. J. — Wed, 09 Dec 2009 04:36:20 PST

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg²⁺ or Na⁺ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other.

The HSV-1 ICP27 RGG box specifically binds flexible, GC-rich sequences but not G-quartet structures

Corbin-Lickfett, K. A., Chen, I-H. B., Cocco, M. J., Sandri-Goldin, R. M. — Wed, 09 Dec 2009 04:36:20 PST

Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures.

FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots

Liao, P.-Y., Choi, Y. S., Lee, K. H. — Wed, 09 Dec 2009 04:36:20 PST

In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3'-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt window along a gene sequence according to different effects of the stimulatory signals, and ribosome E-, P- and A-site interactions. FSscan successfully identified the +1 PRF site in prfB and predicted yehP, pepP, nuoE and cheA as +1 frameshift candidates in the E. coli genome. Empirical results demonstrated that potential +1 frameshift sequences identified promoted significant levels of +1 frameshifting in vivo. Mass spectrometry analysis confirmed the presence of the frameshifted proteins expressed from a yehP-egfp fusion construct. FSscan allows a genome-wide and systematic search for +1 frameshift sites in E. coli. The results have implications for bioinformatic identification of novel frameshift proteins, ribosomal frameshifting, coding sequence detection and the application of mass spectrometry on studying frameshift proteins.

Crystal structure of the EndoG/EndoGI complex: mechanism of EndoG inhibition

Loll, B., Gebhardt, M., Wahle, E., Meinhart, A. — Wed, 09 Dec 2009 04:36:20 PST

EndoG is a ubiquitous nuclease that is translocated into the nucleus during apoptosis to participate in DNA degradation. The enzyme cleaves double- and single-stranded DNA and RNA. Related nucleases are found in eukaryotes and prokaryotes, which have evolved sophisticated mechanisms for genome protection against self-antagonizing nuclease activity. Common mechanisms of inhibition are secretion, sequestration into a separate cellular compartment or by binding to protein inhibitors. Although EndoG is silenced by compartmentalization into the mitochondrial intermembrane space, a nucleus-localized protein inhibitor protects cellular polynucleotides from degradation by stray EndoG under non-apoptotic conditions in Drosophila. Here, we report the first three-dimensional structure of EndoG in complex with its inhibitor EndoGI. Although the mechanism of inhibition is reminiscent of bacterial protein inhibitors, EndoGI has evolved independently from a generic protein-protein interaction module. EndoGI is a two-domain protein that binds the active sites of two monomers of EndoG, with EndoG being sandwiched between EndoGI. Since the amino acid sequences of eukaryotic EndoG homologues are highly conserved, this model is valid for eukaryotic dimeric EndoG in general. The structure indicates that the two active sites of EndoG occupy the most remote spatial position possible at the molecular surface and a concerted substrate processing is unlikely.

Revisiting the planarity of nucleic acid bases: Pyramidilization at glycosidic nitrogen in purine bases is modulated by orientation of glycosidic torsion

Wed, 09 Dec 2009 04:36:20 PST

We describe a novel, fundamental property of nucleobase structure, namely, pyramidilization at the N1/9 sites of purine and pyrimidine bases. Through a combined analyses of ultra-high-resolution X-ray structures of both oligonucleotides extracted from the Nucleic Acid Database and isolated nucleotides and nucleosides from the Cambridge Structural Database, together with a series of quantum chemical calculations, molecular dynamics (MD) simulations, and published solution nuclear magnetic resonance (NMR) data, we show that pyramidilization at the glycosidic nitrogen is an intrinsic property. This property is common to isolated nucleosides and nucleotides as well as oligonucleotides—it is also common to both RNA and DNA. Our analysis suggests that pyramidilization at N1/9 sites depends in a systematic way on the local structure of the nucleoside. Of note, the pyramidilization undergoes stereo-inversion upon reorientation of the glycosidic bond. The extent of the pyramidilization is further modulated by the conformation of the sugar ring. The observed pyramidilization is more pronounced for purine bases, while for pyrimidines it is negligible. We discuss how the assumption of nucleic acid base planarity can lead to systematic errors in determining the conformation of nucleotides from experimental data and from unconstrained MD simulations.

A directed evolution design of a GCG-specific DNA hemimethylase

Gerasimaite, R., Vilkaitis, G., Klimasauskas, S. — Wed, 09 Dec 2009 04:36:20 PST

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases.