RESUMO
To-date systematic testing and comparison of the accuracy of available primer-dimer prediction software has never been conducted, due in part to a lack of tools able to measure the efficacy of Gibbs free energy (ΔG) calculations at predicting dimer formation in PCR. To address this we have developed a novel online tool called PrimerROC ( www.primer-dimer.com/roc/ ), which uses epidemiologically-based Receiver Operating Characteristic (ROC) curves to assess dimer prediction accuracy. Moreover, by integrating PrimerROC with our PrimerDimer prediction software we can determine a ΔG-based dimer-free threshold above which dimer formation is predicted unlikely to occur. Notably, PrimerROC determines this cut-off without any additional information such as salt concentration or annealing temperature, meaning that our PrimerROC method is an assay and condition independent prediction tool. To demonstrate the broad utility of PrimerROC we assessed the performance of seven publically available primer design and dimer analysis tools using a dataset of over 300 primer pairs. We found that our PrimerROC/PrimerDimer software consistently outperforms these other tools and can achieve predictive accuracies greater than 92%. To illustrate its predictive power this method was used in multiplex PCR design to successfully generate four resequencing assays containing up to 126 primers with no observable primer-primer amplification artefacts.
RESUMO
MicroRNAs (miRNAs) are highly conserved â¼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (CNVK), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNVK-containing miR-29b (CNVK-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNVK-miR-29b with ultra-low-input RNA sequencing, we recover â¼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNVK-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNVK-containing small RNA mimics may find applicability in other experimental models.
Assuntos
Carbazóis/química , MicroRNAs/metabolismo , Nitrilas/química , RNA Antissenso/genética , Compostos de Vinila/química , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteínas Argonautas/química , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Adesões Focais/metabolismo , GTP Fosfo-Hidrolases/química , Proteínas Homeobox A10 , Proteínas de Homeodomínio/química , Humanos , Proteínas de Membrana/química , Camundongos , MicroRNAs/química , Proteínas Repressoras/químicaRESUMO
Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.
Assuntos
Ilhas de CpG , Metilação de DNA , Reação em Cadeia da Polimerase Multiplex , Sítios de Ligação , Biologia Computacional/métodos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Técnicas de Amplificação de Ácido NucleicoRESUMO
The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).
Assuntos
Primers do DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Internet , Reação em Cadeia da Polimerase Multiplex/métodos , Sulfitos/química , Algoritmos , Dimerização , Genoma , Humanos , Software , Estatística como AssuntoRESUMO
Protein phosphorylation is one of the most prominent post-translational mechanisms for protein regulation, which is frequently impaired in cancer. Through the covalent addition of phosphate groups to certain amino-acids, the interactions of former residues with nearby amino-acids are drastically altered, resulting in major changes of protein conformation that impacts its biological function. Herein, we report that these conformational changes can also disturb the protein's ability to interact with and adsorb onto bare gold surfaces. We exploited this feature to develop a simple electrochemical method for detecting the aberrant phosphorylation of EGFR protein in several lung cancer cell lines. This method, which required as low as 10ng/µL (i.e., 50ng) of purified EGFR protein, also enabled monitoring cell sensitivity to tyrosine kinase inhibitors (TKI) - a common drug used for restoring the function of aberrantly phosphorylated proteins in lung cancer. The reported strategy based on direct gold-protein affinity interactions avoids the conventional paradigm of requiring a phospho-specific antibody for detection and could be a potential alternative of widely used mass spectrometry.
Assuntos
Técnicas Biossensoriais/métodos , Receptores ErbB/análise , Neoplasias Pulmonares/metabolismo , Técnicas Biossensoriais/instrumentação , Linhagem Celular Tumoral , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Desenho de Equipamento , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Ouro/química , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Modelos Moleculares , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.
Assuntos
Padronização Corporal/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Embrião de Mamíferos/embriologia , Gastrulação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/biossíntese , RNA Longo não Codificante/biossíntese , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Proteínas de Homeodomínio/genética , Camundongos , RNA Longo não Codificante/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMO
Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031-1037.
Assuntos
Análise Mutacional de DNA , Exoma/genética , Leucoencefalopatias/diagnóstico , Leucoencefalopatias/genética , Substância Branca/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Leucoencefalopatias/patologia , Masculino , Mutação , Adulto JovemRESUMO
Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.
Assuntos
Epilepsia/genética , Canais de Potássio Éter-A-Go-Go/genética , Hallux/anormalidades , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Unhas Malformadas/genética , Polegar/anormalidades , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Sequência Conservada , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/fisiologia , Éxons/genética , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Mosaicismo , Oócitos , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Inherited white-matter disorders are a broad class of diseases for which treatment and classification are both challenging. Indeed, nearly half of the children presenting with a leukoencephalopathy remain without a specific diagnosis. Here, we report on the application of high-throughput genome and exome sequencing to a cohort of ten individuals with a leukoencephalopathy of unknown etiology and clinically characterized by hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL), as well as the identification of compound-heterozygous and homozygous mutations in cytoplasmic aspartyl-tRNA synthetase (DARS). These mutations cause nonsynonymous changes to seven highly conserved amino acids, five of which are unchanged between yeast and man, in the DARS C-terminal lobe adjacent to, or within, the active-site pocket. Intriguingly, HBSL bears a striking resemblance to leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate (LBSL), which is caused by mutations in the mitochondria-specific DARS2, suggesting that these two diseases might share a common underlying molecular pathology. These findings add to the growing body of evidence that mutations in tRNA synthetases can cause a broad range of neurologic disorders.
Assuntos
Aspartato-tRNA Ligase/genética , Leucoencefalopatias/genética , Modelos Moleculares , Espasticidade Muscular/genética , Conformação Proteica , Aspartato-tRNA Ligase/química , Tronco Encefálico/patologia , Cristalografia por Raios X , Humanos , Perna (Membro)/patologia , Leucoencefalopatias/patologia , Mutação/genética , Medula Espinal/patologiaRESUMO
Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and ß-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABC's clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC.
Assuntos
Gânglios da Base/patologia , Cerebelo/patologia , Leucoencefalopatias/genética , Modelos Moleculares , Conformação Proteica , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Exoma/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Humanos , Leucoencefalopatias/patologia , Imageamento por Ressonância Magnética , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Neurônios/metabolismo , Análise de Sequência de DNA , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 5' end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Processamento Alternativo , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Camundongos , RNA Nucleolar Pequeno/genética , RNA não Traduzido , Transcrição Gênica , beta Catenina/metabolismoRESUMO
The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , RNA não Traduzido/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem da Célula , Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , RNA não Traduzido/metabolismoRESUMO
Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific "transcriptional noise." Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.
Assuntos
Biblioteca Gênica , Genoma/genética , RNA não Traduzido/genética , Animais , Northern Blotting , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.
Assuntos
Proteínas de Bactérias/fisiologia , Fímbrias Bacterianas/fisiologia , Proteínas de Membrana/fisiologia , Pseudomonas aeruginosa/fisiologia , Elementos de DNA Transponíveis , MovimentoRESUMO
BACKGROUND: The nucleolus is the site of rRNA synthesis and processing in eukaryotic cells, but its composition remains poorly understood. RESULTS: We have identified a novel nucleolar RNA-associated protein (Nrap) which is highly conserved from yeast (Saccharomyces cerevisiae) to human, with homologues in mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and other species. In the mouse, we have found that Nrap is ubiquitously expressed and is specifically localized in the nucleolus. We have also identified splice variants in human and mouse, and defined the intron-exon structure of the human Nrap gene. Nrap is inherited into daughter nuclei by associating with the condensed chromosomes during mitosis. RNase treatment of permeabilized cells indicated that the nucleolar localization of Nrap is RNA dependent. The effects of actinomycin D, cycloheximide and 5,6-dichloro-beta-d-ribofuranosyl-benzimidazole on Nrap expression and distribution in cultured cells suggest that Nrap is associated with the pre-rRNA transcript. CONCLUSIONS: Nrap is a large nucleolar protein (of more than 1000 amino acids), and is a new class of protein with new structural and functional motifs. Nrap appears to be associated with ribosome biogenesis by interacting with pre-rRNA primary transcript.