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Neoplasias , Humanos , Niño , Irlanda/epidemiología , Neoplasias/epidemiología , Neoplasias/terapia , Factores Socioeconómicos , Clase SocialRESUMEN
BACKGROUND: Conventional differential expression (DE) testing compares the grouped mean value of tumour samples to the grouped mean value of the normal samples, and may miss out dysregulated genes in small subgroup of patients. This is especially so for highly heterogeneous cancer like Hepatocellular Carcinoma (HCC). METHODS: Using multi-region sampled RNA-seq data of 90 patients, we performed patient-specific differential expression testing, together with the patients' matched adjacent normal samples. RESULTS: Comparing the results from conventional DE analysis and patient-specific DE analyses, we show that the conventional DE analysis omits some genes due to high inter-individual variability present in both tumour and normal tissues. Dysregulated genes shared in small subgroup of patients were useful in stratifying patients, and presented differential prognosis. We also showed that the target genes of some of the current targeted agents used in HCC exhibited highly individualistic dysregulation pattern, which may explain the poor response rate. DISCUSSION/CONCLUSION: Our results highlight the importance of identifying patient-specific DE genes, with its potential to provide clinically valuable insights into patient subgroups for applications in precision medicine.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Pronóstico , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: The transforming growth factor beta-1 (TGF ß-1) cytokine exerts both pro-tumor and anti-tumor effects in carcinogenesis. An increasing body of literature suggests that TGF ß-1 signaling outcome is partially dependent on the regulatory targets of downstream receptor-regulated Smad (R-Smad) proteins Smad2 and Smad3. However, the lack of Smad-specific antibodies for ChIP-seq hinders convenient identification of Smad-specific binding sites. RESULTS: In this study, we use localization and affinity purification (LAP) tags to identify Smad-specific binding sites in a cancer cell line. Using ChIP-seq data obtained from LAP-tagged Smad proteins, we develop a convolutional neural network with long-short term memory (CNN-LSTM) as a deep learning approach to classify a pool of Smad-bound sites as being Smad2- or Smad3-bound. Our data showed that this approach is able to accurately classify Smad2- versus Smad3-bound sites. We use our model to dissect the role of each R-Smad in the progression of breast cancer using a previously published dataset. CONCLUSIONS: Our results suggests that deep learning approaches can be used to dissect binding site specificity of closely related transcription factors.
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Aprendizaje Profundo , Sitios de Unión , Línea Celular , Transducción de Señal , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína smad3/química , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Multiple vaccines against Covid-19 have passed through phase-3 trials; however, concerns have been raised about alleged excessive similarity of efficacy across age groups for the Sputnik V vaccine. STUDY QUESTION: How likely are the observed efficacies for all age subgroups to fall within the range of by-age efficacies claimed for the AstraZeneca, Janssen, Moderna, Pfizer, and Sputnik V vaccines, assuming that there is no effect of age on efficacy? STUDY DESIGN: We performed a simulation study using R of 1000 and then 50,000 simulated trials for each vaccine, with random allocation to each arm but fixed enrollment numbers by age group. We used study-wide efficacy and infection rate for all age groups. We recorded the observed vaccine efficacies in each age group and summated how many simulations had all observed efficacies fall within the range of efficacies described in the relevant article. RESULTS: In the 1000-trial simulation for the AstraZeneca vaccine, in 23.8% of simulated trials, the observed efficacies of all age subgroups fell within the efficacy bounds for age subgroups in the published article. The J + J simulation showed 44.7%, Moderna 51.1%, Pfizer 30.5%, and 0.0% of the Sputnik simulated trials had all age subgroups fall within the limits of the efficacy estimates described by the published article. In 50,000 simulated trials of the Sputnik vaccine, 0.026% had all age subgroups fall within the limits of the efficacy estimates described by the published article, whereas 99.974% did not. CONCLUSIONS: The distribution of alleged vaccine efficacies of the Sputnik vaccine by age in the phase-III trial is very unlikely to occur in genuine experimental data, even if the number of patients recruited, vaccine efficacy, and overall infection rate are true and there is no underlying difference in vaccine efficacy by age.
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COVID-19 , Vacunas , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19 , HumanosRESUMEN
Cholesterol metabolism operates autonomously within the central nervous system (CNS), where the majority of cholesterol resides in myelin. We demonstrate that TDP-43, the pathological signature protein for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), influences cholesterol metabolism in oligodendrocytes. TDP-43 binds directly to mRNA of SREBF2, the master transcription regulator for cholesterol metabolism, and multiple mRNAs encoding proteins responsible for cholesterol biosynthesis and uptake, including HMGCR, HMGCS1, and LDLR. TDP-43 depletion leads to reduced SREBF2 and LDLR expression, and cholesterol levels in vitro and in vivo. TDP-43-mediated changes in cholesterol levels can be restored by reintroducing SREBF2 or LDLR. Additionally, cholesterol supplementation rescues demyelination caused by TDP-43 deletion. Furthermore, oligodendrocytes harboring TDP-43 pathology from FTD patients show reduced HMGCR and HMGCS1, and coaggregation of LDLR and TDP-43. Collectively, our results indicate that TDP-43 plays a role in cholesterol homeostasis in oligodendrocytes, and cholesterol dysmetabolism may be implicated in TDP-43 proteinopathies-related diseases.
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Colesterol/metabolismo , Proteínas de Unión al ADN/genética , Demencia Frontotemporal/genética , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Animales , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Femenino , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vaina de Mielina/patología , Oligodendroglía/patología , Organoides/metabolismo , Organoides/patología , Cultivo Primario de Células , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción de Señal , Médula Espinal/metabolismo , Médula Espinal/patología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Lóbulo Temporal/metabolismo , Lóbulo Temporal/patologíaRESUMEN
TDP-43 is extensively studied in neurons in physiological and pathological contexts. However, emerging evidence indicates that glial cells are also reliant on TDP-43 function. We demonstrate that deletion of TDP-43 in Schwann cells results in a dramatic delay in peripheral nerve conduction causing significant motor deficits in mice, which is directly attributed to the absence of paranodal axoglial junctions. By contrast, paranodes in the central nervous system are unaltered in oligodendrocytes lacking TDP-43. Mechanistically, TDP-43 binds directly to Neurofascin mRNA, encoding the cell adhesion molecule essential for paranode assembly and maintenance. Loss of TDP-43 triggers the retention of a previously unidentified cryptic exon, which targets Neurofascin mRNA for nonsense-mediated decay. Thus, TDP-43 is required for neurofascin expression, proper assembly and maintenance of paranodes, and rapid saltatory conduction. Our findings provide a framework and mechanism for how Schwann cell-autonomous dysfunction in nerve conduction is directly caused by TDP-43 loss-of-function.
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Proteínas de Unión al ADN/genética , Exones , Uniones Intercelulares/metabolismo , Conducción Nerviosa , Células de Schwann/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Masculino , RatonesRESUMEN
The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping.
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Cromatina/metabolismo , Represión Epigenética , Histonas/genética , Neoplasias/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Línea Celular Tumoral , Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Histonas/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , RNA-Seq , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits.
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Esclerosis Amiotrófica Lateral/genética , Encéfalo/metabolismo , Disfunción Cognitiva/genética , Mitocondrias/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/genética , Proteostasis/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Encéfalo/fisiopatología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Neuroimagen Funcional , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Prueba del Laberinto Acuático de Morris , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiopatología , Prueba de Campo Abierto , Proteína FUS de Unión a ARN/genéticaRESUMEN
Laboratory pedagogy is moving away from step-by-step instructions and toward inquiry-based learning, but only now developing methods for integrating inquiry-based writing (IBW) practices into the laboratory course. Based on an earlier proposal (Science 2011;332:919), we designed and implemented an IBW sequence in a university bioinformatics course. We automatically generated unique, double-blinded, biologically plausible DNA sequences for each student. After guided instruction, students investigated sequences independently and responded through IBW writing assignments. IBW assignments were structured as condensed versions of a scientific research article, and because the sequences were double blinded, they were also assessed as authentic science and evaluated on clarity and persuasiveness. We piloted the approach in a seven-day workshop (35 students) at Perdana University in Malaysia. We observed dramatically improved student engagement and indirect evidence of improved learning outcomes over a similar workshop without IBW. Based on student feedback, initial discomfort with the writing component abated in favor of an overall positive response and increasing comfort with the high demands of student writing. Similarly, encouraging results were found in a semester length undergraduate module at the National University of Singapore (155 students).
Asunto(s)
Biología Computacional/educación , Laboratorios , Pensamiento , Escritura , Secuencia de Bases , Curriculum , ADN/genética , Humanos , Aprendizaje , Estudiantes , UniversidadesRESUMEN
BACKGROUND: Bivalent promoters marked with both H3K27me3 and H3K4me3 histone modifications are characteristic of poised promoters in embryonic stem (ES) cells. The model of poised promoters postulates that bivalent chromatin in ES cells is resolved to monovalency upon differntiation. With the availability of single-cell RNA sequencing (scRNA-seq) data, subsequent switches in transcriptional state at bivalent promoters can be studied more closely. RESULTS: We develop an approach for capturing genes undergoing transcriptional switching by detecting 'bimodal' gene expression patterns from scRNA-seq data. We integrate the identification of bimodal genes in ES cell differentiation with analysis of chromatin state, and identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudotime approximation and use a hidden Markov model to infer gene activity switching pseudotimes, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets. CONCLUSIONS: Genes with expression levels too low to be informative in conventional scRNA analysis can be used to infer transcriptional switching networks that connect transcriptional activity to chromatin state. Since chromatin bivalency is a hallmark of gene promoters poised for activity, this approach provides an alternative that complements conventional scRNA-seq analysis while focusing on genes near the ON/OFF boundary of activity. This offers a novel and productive means of inferring regulatory networks from scRNA-seq data.
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Cromatina , Células Madre Embrionarias , Diferenciación Celular/genética , Cromatina/genética , Código de Histonas , Regiones Promotoras GenéticasRESUMEN
Patients with amyotrophic lateral sclerosis (ALS) can have abnormal TDP-43 aggregates in the nucleus and cytosol of their surviving neurons and glia. Although accumulating evidence indicates that astroglial dysfunction contributes to motor neuron degeneration in ALS, the normal function of TDP-43 in astrocytes are largely unknown, and the role of astroglial TDP-43 loss to ALS pathobiology remains to be clarified. Herein, we show that TDP-43-deleted astrocytes exhibit a cell-autonomous increase in GFAP immunoreactivity without affecting astrocyte or microglia proliferation. At the transcriptomic level, TDP-43-deleted astrocytes resemble A1-reactive astrocytes and induce microglia to increase C1q expression. These astrocytic changes do not cause loss of motor neurons in the spinal cord or denervation at the neuromuscular junction. In contrast, there is a selective reduction of mature oligodendrocytes, but not oligodendrocyte precursor cells, suggesting triglial dysfunction mediated by TDP-43 loss in astrocytes. Moreover, mice with astroglial TDP-43 deletion develop motor, but not sensory, deficits. Taken together, our results demonstrate that TDP-43 is required to maintain the protective functions of astrocytes relevant to the development of motor deficits in mice.
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Astrocitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Fenotipo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proliferación Celular , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , Oligodendroglía/metabolismo , TranscriptomaRESUMEN
Understanding macromolecular structures is essential for biology education. Augmented reality (AR) applications have shown promise in science, technology, engineering, and mathematics (STEM) education, but are not widely used for protein visualization. While there are some tools for AR protein visualization, none of them are accessible to the layperson who possesses neither specialized AR hardware nor the technical skill to comfortably navigate three-dimensional (3D) rendering and file conversions. Here, we describe Palantir, an open source mobile Android application easily installable on compatible devices from the Google Play Store. Palantir does not require specialized hardware, printed image, manual 3D rendering, or file format conversion. Palantir makes AR macromolecular visualization accessible to anyone with a compatible mobile device, and we hope it finds widespread application in STEM education.
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Realidad Aumentada , Imagenología Tridimensional , Aplicaciones Móviles , Sitio Alostérico , Aminoácidos/química , Ingeniería/educación , Humanos , Matemática/educación , Estructura Secundaria de Proteína , Ciencia/educación , Estudiantes , Enseñanza , Tecnología/educación , UniversidadesRESUMEN
Pathological fused in sarcoma (FUS) inclusions are found in 10% of patients with frontotemporal dementia and those with amyotrophic lateral sclerosis (ALS) carrying FUS mutations. Current work indicates that FUS mutations may incur gain-of-toxic functions to drive ALS pathogenesis. However, how FUS dysfunction may affect cognition remains elusive. Using a mouse model expressing wild-type human FUS mimicking the endogenous expression pattern and level within the central nervous system, we found that they developed hippocampus-mediated cognitive deficits accompanied by an age-dependent reduction in spine density and long-term potentiation in their hippocampus. However, there were no apparent FUS aggregates, nuclear envelope defects and cytosolic FUS accumulation. These suggest that these proposed pathogenic mechanisms may not be the underlying causes for the observed cognitive deficits. Unbiased transcriptomic analysis identified expression changes in a small set of genes with preferential expression in the neurons and oligodendrocyte lineage cells. Of these, we focused on Sema5a, a gene involved in axon guidance, spine dynamics, Parkinson's disease and autism spectrum disorders. Critically, FUS binds directly to Sema5a mRNA and regulates Sema5a expression in a FUS-dose-dependent manner. Taken together, our data suggest that FUS-driven Sema5a deregulation may underlie the cognitive deficits in FUS transgenic mice.
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Disfunción Cognitiva/genética , Proteína FUS de Unión a ARN/genética , Semaforinas/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Línea Celular Tumoral , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Neuronas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Semaforinas/metabolismoRESUMEN
Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.
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Autofagia , Homeostasis , Lisosomas/metabolismo , Proteína FUS de Unión a ARN/biosíntesis , Proteína FUS de Unión a ARN/toxicidad , ARN/metabolismo , Animales , Perfilación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Proteínas Mutantes/toxicidad , Proteína FUS de Unión a ARN/genéticaRESUMEN
TDP-43 aggregates in neurons and glia are the defining pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), raising the possibility of glial damage in the disease pathogenesis. However, the normal physiological functions of TDP-43 in glia are largely unknown. To address how TDP-43 may be required for oligodendroglial functions we selectively deleted TDP-43 in mature oligodendrocytes in mice. Although mice with TDP-43 deleted in oligodendrocytes are born in an expected Mendelian ratio, they develop progressive neurological phenotypes leading to early lethality accompanied by a progressive reduction in myelination. The progressive myelin reduction is likely due to a combination of the cell-autonomous RIPK1-mediated necroptosis of mature oligodendrocytes and the TDP-43-dependent reduction in the expression of myelin genes. Strikingly, enhanced proliferation of NG2-positive oligodendrocyte precursor cells within the white matter, but not the gray matter, was able to replenish the loss of mature oligodendrocytes, indicating an intrinsic regeneration difference between the gray and white matter oligodendrocytes. By contrast, there was no loss of spinal cord motor neurons and no sign of denervation at the neuromuscular synapses. Taken together, our data demonstrate that TDP-43 is indispensable for oligodendrocyte survival and myelination, and loss of TDP-43 in oligodendrocytes exerts no apparent toxicity on motor neurons.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Demencia Frontotemporal/patología , Sustancia Gris/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Vaina de Mielina/genética , Fibras Nerviosas Mielínicas/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Oligodendroglía/citología , Médula Espinal/metabolismo , Sustancia Blanca/metabolismoRESUMEN
Mature microRNAs (miRNAs) are processed from primary transcripts (pri-miRNAs), and their expression is controlled at transcriptional and post-transcriptional levels. However, how regulation at multiple levels achieves precise control remains elusive. Using published and new datasets, we profile a time course of mature and pri-miRNAs in Drosophila embryos and reveal the dynamics of miRNA production and degradation as well as dynamic changes in pri-miRNA isoform selection. We found that 5' nucleotides influence stability of mature miRNAs. Furthermore, distinct half-lives of miRNAs from the mir-309 cluster shape their temporal expression patterns, and the importance of rapid degradation of the miRNAs in gene regulation is detected as distinct evolutionary signatures at the target sites in the transcriptome. Finally, we show that rapid degradation of miR-3/-309 may be important for regulation of the planar cell polarity pathway component Vang. Altogether, the results suggest that complex mechanisms regulate miRNA expression to support normal development.
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Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , MicroARNs/biosíntesis , Estabilidad del ARN , Animales , Perfilación de la Expresión GénicaRESUMEN
We first constructed small RNA libraries of TMV- and TMV-43A-infected N. benthamiana for high throughput sequencing. A total number of 181 novel microRNAs (miRNAs) were identified through an improved miRNAs analysis pipeline. We were able to identify consistent miRNA expression changes induced in TMV and TMV-43A-infected plants, as well as differences associated with the UPD substitution in the TMV-43A viral genome. Virally induced miRNAs are associated with distinct processes and functions of predicted mRNA targets, including relation to host target defense. This study suggests an approach for functional genomics miRNAs in incompletely assembled genomes. These findings provide valuable information for further characterization of miRNAs by two genomic similar viruses, and provide clues to the study of TMV-UPD to find potential defense-related host genes targeted by miRNAs (126 words).
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Regulación de la Expresión Génica de las Plantas , Genoma Viral , Interacciones Huésped-Patógeno/genética , MicroARNs/genética , Mutación , Nicotiana/genética , Nicotiana/virología , Poli A , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Familia de Multigenes , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , ARN Viral , Reproducibilidad de los Resultados , Análisis de Secuencia de ARNRESUMEN
In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing, but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs-null embryos and found that only â¼40% of miRNAs showed clear Loqs dependence. Genome-wide comparison of the hairpin structure and Loqs dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , MicroARNs/química , MicroARNs/genética , Conformación de Ácido Nucleico , ARN Helicasas/metabolismo , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo , Animales , Emparejamiento Base/genética , Secuencia de Bases , Línea Celular , Evolución Molecular , Técnicas de Inactivación de Genes , Biblioteca de Genes , MicroARNs/metabolismo , Mutación/genética , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
MicroRNAs (miRNAs) are 22- to 24-nucleotide, small, non-coding RNAs that bind to the 3'UTR of target genes to control gene expression. Consequently, their dysregulation contributes to many diseases, including diabetes and cancer. miR-22 is up-regulated in numerous metastatic cancers and recent studies have suggested a role for miR-22 in promoting stemness and metastasis. TIP60 is a lysine acetyl-transferase reported to be down-regulated in cancer but the molecular mechanism of this reduction is still unclear. In this study, we identify TIP60 as a target of miR-22. We show a negative correlation in the expression of TIP60 and miR-22 in breast cancer patients, and show that low levels of TIP60 and high levels of miR-22 are associated with poor overall survival. Furthermore, pathway analysis using high miR-22/low TIP60 and low miR-22/high TIP60 breast cancer patient datasets suggests association of TIP60/miR-22 with epithelial-mesenchymal transition (EMT), a key alteration in progression of cancer cells. We show that blocking endogenous miR-22 can restore TIP60 levels, which in turn decreases the migration and invasion capacity of metastatic breast cancer cell line. These results provide mechanistic insight into TIP60 regulation and evidence for the utility of the combination of TIP60 and miR-22 as prognostic indicator of breast cancer progression.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Histona Acetiltransferasas/genética , MicroARNs/genética , Regiones no Traducidas 3'/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Histona Acetiltransferasas/metabolismo , Humanos , Estimación de Kaplan-Meier , Lisina Acetiltransferasa 5 , Células MCF-7 , Microscopía Confocal , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the pre-rRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5' and 3' ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha-Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor â¼ 100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms.