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Nucleic Acids Research: VOLUME 36 ISSUE 20 2008

2008-11-12

Nucleic Acids Research

2008-11-12

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2008-11-12

A sensitive array-based assay for identifying multiple TMPRSS2:ERG fusion gene variants

2008-11-12

Studies of gene fusions in solid tumors are not as extensive as in hematological malignancies due to several technical and analytical problems associated with tumor heterogeneity. Nevertheless, there is a growing interest in the role of fusion genes in common epithelial tumors after the discovery of recurrent TMPRSS2:ETS fusions in prostate cancer. Among all of the reported fusion partners in the ETS gene family, TMPRSS2:ERG is the most prevalent one. Here, we present a simple and sensitive microarray-based assay that is able to simultaneously determine multiple fusion variants with a single RT–PCR in impure RNA specimens. The assay detected TMPRSS2:ERG fusion transcripts with a detection sensitivity of <10 cells in the presence of more than 3000 times excess normal RNA, and in primary prostate tumors having no >1% of cancer cells. The ability to detect multiple transcript variants in a single assay is critically dependent on both the primer and probe designs. The assay should facilitate clinical and basic studies for fusion gene screening in clinical specimens, as it can be readily adapted to include multiple gene loci.

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

2008-11-12

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3'-terminal and 3'-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Poulsen, L., Soe, M. J., Snakenborg, D., Moller, L. B., Dufva, M. — 2008-11-12

Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 x 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 x SSC, while probes placed away from the surface required 0.35 x SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis.

Enhanced gene trapping in mouse embryonic stem cells

2008-11-12

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.

In vivo evaluation of PhiC31 recombinase activity using a self-excision cassette

Sangiorgi, E., Shuhua, Z., Capecchi, M. R. — 2008-11-12

Gene targeting allows precise tailoring of the mouse genome such that desired modifications can be introduced under precise temporal and spatial control. This can be achieved through the use of site-specific recombinases, which mediate deletion or inversion of genomic DNA flanked by recombinase-specific recognition sites, coupled with gene targeting to introduce the recombinase recognition sites at the desired genomic locations within the mouse genome. The introduction of multiple modifications at the same locus often requires use of multiple recombination systems. The most commonly used recombination system is Cre/lox. We here evaluated in vivo the ability of PhiC31 phage integrase to induce a genomic deletion in mouse. We engineered a self-excision cassette, modeled after one previously designed for Cre, containing a positive selection marker and PhiC31 driven by a testis-specific promoter, all flanked by PhiC31 specific attP/B sites. We found in vivo PhiC31 mediated self-excision in 38% of transmitted alleles, although 18% of these showed evidence of imprecise deletion. Furthermore, in the 69% of un-recombined cassettes, sequence analysis revealed that PhiC31 mediated an intra-molecular deletion of the attB site preventing any subsequent recombination. This study demonstrates that PhiC31 can be used to automatically remove Neo, in the male chimera germline, although it is not as efficient or as accurate as Cre.

Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay

Cheng, B., Price, D. H. — 2008-11-12

The elongation phase of transcription by RNA polymerase II (RNAP II) is controlled by a carefully orchestrated series of interactions with both negative and positive factors. However, due to the limitations of current methods and techniques, not much is known about whether and how these proteins physically associate with the engaged polymerases. To gain insight into the detailed mechanisms involved, we established an experimental system for analyzing direct factor interactions to RNAP II elongation complexes on native gels, namely elongation complex electrophoretic mobility shift assay (EC-EMSA). This new assay effectively allowed detection of interactions of TFIIF, TTF2, TFIIS, DSIF and P-TEFb with elongation complexes generated from a natural promoter using an immobilized template. As an application of this assay system, we characterized the association of transcription elongation factor DSIF with RNAP II elongation complexes and discovered that the nascent transcript facilitated recruitment of DSIF. Examples of how the system can be manipulated to address different questions are provided. EC-EMSA should be useful for further investigation of factor interactions with RNAP II elongation complexes.

Protein networks markedly improve prediction of subcellular localization in multiple eukaryotic species

Lee, K., Chuang, H.-Y., Beyer, A., Sung, M.-K., Huh, W.-K., Lee, B., Ideker, T. — 2008-11-12

The function of a protein is intimately tied to its subcellular localization. Although localizations have been measured for many yeast proteins through systematic GFP fusions, similar studies in other branches of life are still forthcoming. In the interim, various machine-learning methods have been proposed to predict localization using physical characteristics of a protein, such as amino acid content, hydrophobicity, side-chain mass and domain composition. However, there has been comparatively little work on predicting localization using protein networks. Here, we predict protein localizations by integrating an extensive set of protein physical characteristics over a protein's extended protein–protein interaction neighborhood, using a classification framework called ‘Divide and Conquer k-Nearest Neighbors’ (DC-kNN). These predictions achieve significantly higher accuracy than two well-known methods for predicting protein localization in yeast. Using new GFP imaging experiments, we show that the network-based approach can extend and revise previous annotations made from high-throughput studies. Finally, we show that our approach remains highly predictive in higher eukaryotes such as fly and human, in which most localizations are unknown and the protein network coverage is less substantial.

Gene expression module-based chemical function similarity search

2008-11-12

Investigation of biological processes using selective chemical interventions is generally applied in biomedical research and drug discovery. Many studies of this kind make use of gene expression experiments to explore cellular responses to chemical interventions. Recently, some research groups constructed libraries of chemical related expression profiles, and introduced similarity comparison into chemical induced transcriptome analysis. Resembling sequence similarity alignment, expression pattern comparison among chemical intervention related expression profiles provides a new way for chemical function prediction and chemical–gene relation investigation. However, existing methods place more emphasis on comparing profile patterns globally, which ignore noises and marginal effects. At the same time, though the whole information of expression profiles has been used, it is difficult to uncover the underlying mechanisms that lead to the functional similarity between two molecules. Here a new approach is presented to perform biological effects similarity comparison within small biologically meaningful gene categories. Regarding gene categories as units, a reduced similarity matrix is generated for measuring the biological distances between query and profiles in library and pointing out in which modules do chemical pairs resemble. Through the modularization of expression patterns, this method reduces experimental noises and marginal effects and directly correlates chemical molecules with gene function modules.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies

2008-11-12

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)₄ (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)₄–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.

Unifying evolutionary and thermodynamic information for RNA folding of multiple alignments

Seemann, S. E., Gorodkin, J., Backofen, R. — 2008-11-12

Computational methods for determining the secondary structure of RNA sequences from given alignments are currently either based on thermodynamic folding, compensatory base pair substitutions or both. However, there is currently no approach that combines both sources of information in a single optimization problem. Here, we present a model that formally integrates both the energy-based and evolution-based approaches to predict the folding of multiple aligned RNA sequences. We have implemented an extended version of Pfold that identifies base pairs that have high probabilities of being conserved and of being energetically favorable. The consensus structure is predicted using a maximum expected accuracy scoring scheme to smoothen the effect of incorrectly predicted base pairs. Parameter tuning revealed that the probability of base pairing has a higher impact on the RNA structure prediction than the corresponding probability of being single stranded. Furthermore, we found that structurally conserved RNA motifs are mostly supported by folding energies. Other problems (e.g. RNA-folding kinetics) may also benefit from employing the principles of the model we introduce. Our implementation, PETfold, was tested on a set of 46 well-curated Rfam families and its performance compared favorably to that of Pfold and RNAalifold.

Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids

Dash, C., Scarth, B. J., Badorrek, C., Gotte, M., Le Grice, S. F. J. — 2008-11-12

The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to an RNA/DNA hybrid reveals an extensive network of contacts with the phosphate backbone of the DNA strand ~4–9 bp downstream from the ribonuclease H (RNase H) catalytic center. Collectively designated as ‘the RNase H primer grip’, this motif contains a phosphate binding pocket analogous to the human and Bacillus halodurans RNases H. The notion that the RNase H primer grip mediates the trajectory of RNA/DNA hybrids accessing the RNase H active site suggests that locally neutralizing the phosphate backbone may be exploited to manipulate nucleic acid flexibility. To examine this, we introduced single and tandem methylphosphonate substitutions through the region of the DNA primer contacted by the RNase H primer grip and into the RNase H catalytic center. The ability of mutant hybrids to support RNase H and DNA polymerase activity was thereafter examined. In addition, site-specific chemical footprinting was used to evaluate movement of the DNA polymerase and RNase H domains. We show here that minor alteration to the RNase H primer can have a dramatic effect on enzyme positioning, and discuss these findings in light of recent crystallography of human RNase H containing an RNA/DNA hybrid.

Stra13/DEC1 and DEC2 inhibit sterol regulatory element binding protein-1c in a hypoxia-inducible factor-dependent mechanism

Choi, S. M., Cho, H.-J., Cho, H., Kim, K. H., Kim, J. B., Park, H. — 2008-11-12

Sterol regulatory element binding protein-1c (SREBP-1c) is a basic helix–loop–helix (bHLH) homodimeric transactivator, which induces itself and several lipogenic enzymes, notably fatty acid synthase (FAS). We demonstrated that hypoxia-inducible factor (HIF) represses the SREBP-1c gene by inducing Stimulated with retinoic acid (Stra)13/Differentiated embryo chondrocyte 1(DEC1) and its isoform, DEC2. Stra13/DEC1 and DEC2 are bHLH homodimeric transcription repressors. We found that both Stra13 and DEC2 inhibit SREBP-1c-induced transcription by competing with SREBP-1c for binding to the E-box in the SREBP-1c promoter and/or by interacting with SREBP-1c protein. DEC2 is instantly and temporarily induced in acute hypoxia, while Stra13 is induced in prolonged hypoxia. This expression profile reflects the finding that Stra13 represses DEC2, thus maintains low level of DEC2 in prolonged hypoxia. DEC2-siRNA restores the hypoxic repression but Stra13-siRNA fails to do so, suggesting that DEC2 is the major initiator of hypoxic repression of SREBP-1c, whereas Stra13 substitutes for DEC2 in prolonged hypoxia. Our findings imply that Stra13 and DEC2 are the mediators to repress SREBP-1c gene in response to hypoxia. By doing so, HIF and its targets, Stra13 and DEC2 reduce the ATP consuming anabolic lipogenesis prior to the actual decrease of ATP acting as a feed-forward mechanism.

Low conservation and species-specific evolution of alternative splicing in humans and mice: comparative genomics analysis using well-annotated full-length cDNAs

2008-11-12

Using full-length cDNA sequences, we compared alternative splicing (AS) in humans and mice. The alignment of the human and mouse genomes showed that 86% of 199 426 total exons in human AS variants were conserved in the mouse genome. Of the 20 392 total human AS variants, however, 59% consisted of all conserved exons. Comparing AS patterns between human and mouse transcripts revealed that only 431 transcripts from 189 loci were perfectly conserved AS variants. To exclude the possibility that the full-length human cDNAs used in the present study, especially those with retained introns, were cloning artefacts or prematurely spliced transcripts, we experimentally validated 34 such cases. Our results indicate that even retained-intron type transcripts are typically expressed in a highly controlled manner and interact with translating ribosomes. We found non-conserved AS exons to be predominantly outside the coding sequences (CDSs). This suggests that non-conserved exons in the CDSs of transcripts cause functional constraint. These findings should enhance our understanding of the relationship between AS and species specificity of human genes.

Regional mutagenesis of the gene encoding the phage Mu late gene activator C identifies two separate regions important for DNA binding

Jiang, Y., Howe, M. M. — 2008-11-12

Lytic development of bacteriophage Mu is controlled by a regulatory cascade and involves three phases of transcription: early, middle and late. Late transcription requires the host RNA polymerase holoenzyme and a 16.5-kDa Mu-encoded activator protein C. Consistent with these requirements, the four late promoters P_lys, P_I, P_P and P_mom have recognizable –10 hexamers but lack typical –35 hexamers. The C protein binds to a 16-bp imperfect dyad-symmetrical sequence element centered at –43.5 and overlapping the –35 region. Based on the crystal structure of the closely related Mor protein, the activator of Mu middle transcription, we predict that two regions of C are involved in DNA binding: a helix-turn-helix region and a β-strand region linking the dimerization and helix-turn-helix domains. To test this hypothesis, we carried out mutagenesis of the corresponding regions of the C gene by degenerate oligonucleotide-directed PCR and screened the resulting mutants for their ability to activate a P_lys-galK fusion. Analysis of the mutant proteins by gel mobility shift, β-galactosidase and polyacrylamide gel electrophoresis assays identified a number of amino acid residues important for C DNA binding in both regions.

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

2008-11-12

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.

Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle

2008-11-12

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)₄. We show that Pip–PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in ~3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)₄-PNADMD.

Evolution of Arabidopsis MIR genes generates novel microRNA classes

Vazquez, F., Blevins, T., Ailhas, J., Boller, T., Meins, F. — 2008-11-12

In Arabidopsis, canonical 21-nt miRNAs are generated by Dicer-like (DCL) 1 from hairpin precursors. We have identified a novel class of functional 23- to 25-nt long-miRNAs that is generated independently from the same miRNA precursors by DCL3. Long-miRNAs are developmentally regulated and in some cases have been conserved during evolution implying that they have biological functions. Plant microRNA genes (MIR) have been proposed to evolve by inverted duplication of the target gene. We found that recently evolved MIR genes consistently give rise to long-miRNAs, while ancient MIR genes give rise predominantly to canonical miRNAs. Transcripts from inverted repeats representing evolving proto-MIR genes were processed by DCL3 into long-miRNAs and also by DCL1, DCL2 or DCL4 depending on hairpin stem length to produce different sizes of miRNAs. Our results suggest that evolution of MIR genes is associated with gradual, overlapping changes in DCL usage resulting in specific size classes of miRNAs.

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells

2008-11-12

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large ~3 MDa complex in the cytoplasm and a 20-fold smaller complex of ~158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

The Cohesin loading factor NIPBL recruits histone deacetylases to mediate local chromatin modifications

2008-11-12

Cornelia de Lange Syndrome (CdLS) is a rare congenital malformation disorder. About half of the patients with CdLS carry mutations in the NIPBL gene encoding the NIPBL protein, a subunit of the Cohesin loading complex. Recent studies show association of Cohesin with chromatin-remodeling complexes, either by establishing cohesion or by recruiting Cohesin to specific chromosome locations. In yeast two-hybrid assays, we identified an interaction of NIPBL with the histone deacetylases -1 and -3. These interactions were confirmed in mammalian cells by coimmunoprecipitation and a critical region for interaction was defined to a stretch of 163 amino acids of a highly conserved region of NIPBL, which is mutated in patients with CdLS. Utilizing reporter gene assays, we could show that NIPBL fused to the GAL4-DNA-binding domain (GAL4-DBD) represses promoter activity via the recruitment of histone deacetylases. Interestingly, this effect is dramatically reduced by both NIPBL missense mutations identified in CdLS and by chemical inhibition of the histone deacetylases. Our data are the first to indicate a molecular and functional connection of NIPBL with chromatin-remodeling processes via the direct interaction with histone deacetylases.

Transcription elongation past O6-methylguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase

Dimitri, A., Burns, J. A., Broyde, S., Scicchitano, D. A. — 2008-11-12

O⁶-Methylguanine (O⁶-meG) is a major mutagenic, carcinogenic and cytotoxic DNA adduct produced by various endogenous and exogenous methylating agents. We report the results of transcription past a site-specifically modified O⁶-meG DNA template by bacteriophage T7 RNA polymerase and human RNA polymerase II. These data show that O⁶-meG partially blocks T7 RNA polymerase and human RNA polymerase II elongation. In both cases, the sequences of the truncated transcripts indicate that both polymerases stop precisely at the damaged site without nucleotide incorporation opposite the lesion, while extensive misincorporation of uracil is observed in the full-length RNA. For both polymerases, computer models suggest that bypass occurs only when O⁶-meG adopts an anti conformation around its glycosidic bond, with the methyl group in the proximal orientation; in contrast, blockage requires the methyl group to adopt a distal conformation. Furthermore, the selection of cytosine and uracil partners opposite O⁶-meG is rationalized with modeled hydrogen-bonding patterns that agree with experimentally observed O⁶-meG:C and O⁶-meG:U pairing schemes. Thus, in vitro, O⁶-meG contributes substantially to transcriptional mutagenesis. In addition, the partial blockage of RNA polymerase II suggests that transcription-coupled DNA repair could play an auxiliary role in the clearance of this lesion.

Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

Christensen-Dalsgaard, M., Gerdes, K. — 2008-11-12

Prokaryotic toxin–antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but does not cleave pure RNA in vitro. RelE exhibits an incomplete RNase fold that may explain why RelE requires its substrate mRNA to presented by the ribosome. In contrast, RelE homologue YoeB has a complete RNase fold and cleaves RNA independently of ribosomes in vitro. Here, we show that YoeB cleavage of mRNA is strictly dependent on translation of the mRNA in vivo. Non-translated model mRNAs were not cleaved whereas the corresponding wild-type mRNAs were cleaved efficiently. Model mRNAs carrying frameshift mutations exhibited a YoeB-mediated cleavage pattern consistent with the reading frameshift thus giving strong evidence that YoeB cleavage specificity was determined by the translational reading frame. In contrast, site-specific mRNA cleavage by MazF occurred independently of translation. In one case, translation seriously influenced MazF cleavage efficiency, thus solving a previous apparent paradox. We propose that translation enhances MazF-mediated cleavage of mRNA by destabilization of the mRNA secondary structure.

The assembly of a spliceosomal small nuclear ribonucleoprotein particle

Patel, S. B., Bellini, M. — 2008-11-12

The U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) are essential elements of the spliceosome, the enzyme that catalyzes the excision of introns and the ligation of exons to form a mature mRNA. Since their discovery over a quarter century ago, the structure, assembly and function of spliceosomal snRNPs have been extensively studied. Accordingly, the functions of splicing snRNPs and the role of various nuclear organelles, such as Cajal bodies (CBs), in their nuclear maturation phase have already been excellently reviewed elsewhere. The aim of this review is, then, to briefly outline the structure of snRNPs and to synthesize new and exciting developments in the snRNP biogenesis pathways.

Analysis of regulatory network topology reveals functionally distinct classes of microRNAs

Yu, X., Lin, J., Zack, D. J., Mendell, J. T., Qian, J. — 2008-11-12

MicroRNAs (miRNAs) negatively regulate the expression of target genes at the post-transcriptional level. Little is known about the crosstalk between miRNAs and transcription factors (TFs). Here we provide data suggesting that the interaction patterns between TFs and miRNAs can influence the biological functions of miRNAs. From this global survey, we find that a regulated feedback loop, in which two TFs regulate each other and one miRNA regulates both of the factors, is the most significantly overrepresented network motif. Mathematical modeling shows that the miRNA in this motif stabilizes the feedback loop to resist environmental perturbation, providing one mechanism to explain the robustness of developmental programs that is contributed by miRNAs. Furthermore, on the basis of a network motif profile analysis, we demonstrate the existence of two classes of miRNAs with distinct network topological properties. The first class of miRNAs is regulated by a large number of TFs, whereas the second is regulated by only a few TFs. The differential expression level of the two classes of miRNAs in embryonic developmental stages versus adult tissues suggests that the two classes may have fundamentally different biological functions. Our results demonstrate that the TFs and miRNAs extensively interact with each other and the biological functions of miRNAs may be wired in the regulatory network topology.

Rad51 gain-of-function mutants that exhibit high affinity DNA binding cause DNA damage sensitivity in the absence of Srs2

Malik, P. S., Symington, L. S. — 2008-11-12

We previously identified several rad51 gain-of-function alleles that partially suppress the requirement for RAD55 and RAD57 in DNA repair. To gain further insight into the mechanism of action of these alleles, we compared the activities of Rad51-V328A, Rad51-P339S and Rad51-I345T with wild-type Rad51, for DNA binding, filament stability, strand exchange and interaction with the antirecombinase helicase, Srs2. These alleles were chosen because they show the highest activity in suppression of ionizing radiation sensitivity of the rad57 mutant, and Val 328 and Ile 345 are conserved in the human Rad51 protein. All three mutant proteins exhibited higher affinity for single-stranded DNA (ssDNA) and showed more robust strand exchange activity with oligonucleotide substrates than wild-type Rad51, with the Rad51-I345T and Rad51-V328A proteins displaying higher activity than Rad51-P339S. However, the Srs2 antirecombinase was able to disrupt Rad51–ssDNA complexes formed with all the mutant proteins. In vivo, the rad51-I345T mutant strain exhibited high resistance to methyl methane sulfonate that was dependent on functional SRS2. These results suggest the Srs2 translocase is able to disrupt Rad51–ssDNA complexes at stalled replication forks, but in the absence of Srs2 the enhanced DNA binding of the Rad51-I345T protein is detrimental to cell survival.

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

2008-11-12

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.

CRX controls retinal expression of the X-linked juvenile retinoschisis (RS1) gene

2008-11-12

X-linked juvenile retinoschisis is a heritable condition of the retina in males caused by mutations in the RS1 gene. Still, the cellular function and retina-specific expression of RS1 are poorly understood. To address the latter issue, we characterized the minimal promoter driving expression of RS1 in the retina. Binding site prediction, site-directed mutagenesis, and reporter assays suggest an essential role of two nearby cone-rod homeobox (CRX)-responsive elements (CRE) in the proximal –177/+32 RS1 promoter. Chromatin immunoprecipitation associates the RS1 promoter in vivo with CRX, the coactivators CBP, P300, GCN5 and acetylated histone H3. Transgenic Xenopus laevis expressing a green fluorescent protein (GFP) reporter under the control of RS1 promoter sequences show that the –177/+32 fragment drives GFP expression in photoreceptors and bipolar cells. Mutating either of the two conserved CRX binding sites results in strongly decreased RS1 expression. Despite the presence of sequence motifs in the promoter, NRL and NR2E3 appear not to be essential for RS1 expression. Together, our in vitro and in vivo results indicate that two CRE sites in the minimal RS1 promoter region control retinal RS1 expression and establish CRX as a key factor driving this expression.

Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer

2008-11-12

Alternative processing of pre-mRNA transcripts is a major source of protein diversity in eukaryotes and has been implicated in several disease processes including cancer. In this study we have performed a genome wide analysis of alternative splicing events in lung adenocarcinoma. We found that 2369 of the 17 800 core Refseq genes appear to have alternative transcripts that are differentially expressed in lung adenocarcinoma versus normal. According to their known functions the largest subset of these genes (30.8%) is believed to be cancer related. Detailed analysis was performed for several genes using PCR, quantitative RT-PCR and DNA sequencing. We found overexpression of ERG variant 2 but not variant 1 in lung tumors and overexpression of CEACAM1 variant 1 but not variant 2 in lung tumors but not in breast or colon tumors. We also identified a novel, overexpressed variant of CDH3 and verified the existence and overexpression of a novel variant of P16 transcribed from the CDKN2A locus. These findings demonstrate how analysis of alternative pre-mRNA processing can shed additional light on differences between tumors and normal tissues as well as between different tumor types. Such studies may lead to the development of additional tools for tumor diagnosis, prognosis and therapy.

Synthesis and investigation of the 5-formylcytidine modified, anticodon stem and loop of the human mitochondrial tRNAMet

2008-11-12

Human mitochondrial methionine transfer RNA (hmtRNA^Met_CAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f⁵C₃₄). The role of this modification in (hmtRNA^Met_CAU) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

Morgan, R. D., Bhatia, T. K., Lovasco, L., Davis, T. B. — 2008-11-12

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5'-TCCRAC-3'. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5'-GTYGGA-3'. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.

The N-clasp of human DNA polymerase {kappa} promotes blockage or error-free bypass of adenine- or guanine-benzo[a]pyrenyl lesions

Jia, L., Geacintov, N. E., Broyde, S. — 2008-11-12

DNA bypass polymerases are utilized to transit bulky DNA lesions during replication, but the process frequently causes mutations. The structural origins of mutagenic versus high fidelity replication in lesion bypass is therefore of fundamental interest. As model systems, we investigated the molecular basis of the experimentally observed essentially faithful bypass of the guanine 10S-(+)-trans-anti-benzo[a]pyrene-N²-dG adduct by the Y-family human DNA polymerase , and the observed blockage of pol produced by the adenine 10S-(+)-trans-anti-benzo[a]pyrene-N²-dA adduct. These lesions are derived from the most tumorigenic metabolite of the ubiquitous cancer-causing pollutant, benzo[a]pyrene. We compare our results for the dG adduct with our earlier studies for the pol archaeal homolog Dpo4, which processes the same lesion in an error-prone manner. Molecular modeling, molecular mechanics calculations and molecular dynamics simulations were utilized. Our results show that the pol N-clasp is a key structural feature that accounts for the dA adduct blockage and the near-error-free bypass of the dG lesion. Absence of the N-clasp in Dpo4 explains the error-prone processing of the same lesion by this enzyme. Thus, our studies elucidate structure-function relationships in the fidelity of lesion bypass.

A competitive hybridization model predicts probe signal intensity on high density DNA microarrays

Li, S., Pozhitkov, A., Brouwer, M. — 2008-11-12

A central, unresolved problem of DNA microarray technology is the interpretation of different signal intensities from multiple probes targeting the same transcript. We propose a competitive hybridization model for DNA microarray hybridization. Our model uses a probe-specific dissociation constant that is computed with current nearest neighbor model and existing parameters, and only four global parameters that are fitted to Affymetrix Latin Square data. This model can successfully predict signal intensities of individual probes, therefore makes it possible to quantify the absolute concentration of targets. Our results offer critical insights into the design and data interpretation of DNA microarrays.

NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations

Gatta, R., Mantovani, R. — 2008-11-12

The CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis, and it is bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone methylations and the association of enzymatic activities. Two novel results emerged: (i) H3-H4 are present on core promoters under active conditions, with the expected cohort of ‘positive’ modifications; H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show that NF-Y is important for H3K36me3 deposition and for elongation, not recruitment of Pol II; (ii) H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Functional siRNA inactivation and treatment with Tranylcypromine determined that KDM1 (LSD1) plays a positive role in transcription, specifically of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. These data are the first in vivo indication of a crucial interplay between core histones and ‘deviant’ histone-fold such as NF-Y, leading to fine-tuning of histone methylations.

Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-{kappa}B pathway

Gatto, G., Rossi, A., Rossi, D., Kroening, S., Bonatti, S., Mallardo, M. — 2008-11-12

The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-B (NF-B). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-B binding sites in the miR-155 promoter; both sites recruited NF-B complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-B sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-B binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target.

Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging

Xiao, F., Zhang, H., Guo, P. — 2008-11-12

Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.

Structural explanation for the role of Mn2+ in the activity of {phi}6 RNA-dependent RNA polymerase

2008-11-12

The biological role of manganese (Mn²⁺) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn²⁺ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn²⁺-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn²⁺, reduced RNA binding and a compromised elongation rate. Loss of Mn²⁺ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn²⁺ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn²⁺ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.