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1.
Development ; 150(23)2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-38032088

RESUMEN

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.


Asunto(s)
Regulón , Pez Cebra , Animales , Humanos , Regiones no Traducidas 3' , Regulón/genética , Morfogénesis/genética , Válvulas Cardíacas , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo
2.
Nucleic Acids Res ; 50(4): e20, 2022 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-34850137

RESUMEN

The CRISPR-Cas9 genome editing tool is used to study genomic variants and gene knockouts, and can be combined with transcriptomic analyses to measure the effects of such alterations on gene expression. But how can one be sure that differential gene expression is due to a successful intended edit and not to an off-target event, without performing an often resource-demanding genome-wide sequencing of the edited cell or strain? To address this question we developed CRISPRroots: CRISPR-Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites. Our method combines Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells to discover potential off-targets. Applied on seven public datasets, CRISPRroots identified critical off-target candidates that were overlooked in all of the corresponding previous studies. CRISPRroots is available via https://rth.dk/resources/crispr.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes , ARN Guía de Kinetoplastida/genética , RNA-Seq
3.
Nucleic Acids Res ; 50(5): 2452-2463, 2022 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-35188540

RESUMEN

Accelerated evolution of any portion of the genome is of significant interest, potentially signaling positive selection of phenotypic traits and adaptation. Accelerated evolution remains understudied for structured RNAs, despite the fact that an RNA's structure is often key to its function. RNA structures are typically characterized by compensatory (structure-preserving) basepair changes that are unexpected given the underlying sequence variation, i.e., they have evolved through negative selection on structure. We address the question of how fast the primary sequence of an RNA can change through evolution while conserving its structure. Specifically, we consider predicted and known structures in vertebrate genomes. After careful control of false discovery rates, we obtain 13 de novo structures (and three known Rfam structures) that we predict to have rapidly evolving sequences-defined as structures where the primary sequences of human and mouse have diverged at least twice as fast (1.5 times for Rfam) as nearby neutrally evolving sequences. Two of the three known structures function in translation inhibition related to infection and immune response. We conclude that rapid sequence divergence does not preclude RNA structure conservation in vertebrates, although these events are relatively rare.


Asunto(s)
Genoma , ARN , Animales , Evolución Molecular , Ratones , Filogenia , ARN/química , ARN/genética , Vertebrados/genética
4.
Neurobiol Dis ; 178: 105980, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36572121

RESUMEN

Alzheimer's disease (AD) is a progressive and irreversible brain disorder, which can occur either sporadically, due to a complex combination of environmental, genetic, and epigenetic factors, or because of rare genetic variants in specific genes (familial AD, or fAD). A key hallmark of AD is the accumulation of amyloid beta (Aß) and Tau hyperphosphorylated tangles in the brain, but the underlying pathomechanisms and interdependencies remain poorly understood. Here, we identify and characterise gene expression changes related to two fAD mutations (A79V and L150P) in the Presenilin-1 (PSEN1) gene. We do this by comparing the transcriptomes of glutamatergic forebrain neurons derived from fAD-mutant human induced pluripotent stem cells (hiPSCs) and their individual isogenic controls generated via precision CRISPR/Cas9 genome editing. Our analysis of Poly(A) RNA-seq data detects 1111 differentially expressed coding and non-coding genes significantly altered in fAD. Functional characterisation and pathway analysis of these genes reveal profound expression changes in constituents of the extracellular matrix, important to maintain the morphology, structural integrity, and plasticity of neurons, and in genes involved in calcium homeostasis and mitochondrial oxidative stress. Furthermore, by analysing total RNA-seq data we reveal that 30 out of 31 differentially expressed circular RNA genes are significantly upregulated in the fAD lines, and that these may contribute to the observed protein-coding gene expression changes. The results presented in this study contribute to a better understanding of the cellular mechanisms impacted in AD neurons, ultimately leading to neuronal damage and death.


Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Péptidos beta-Amiloides/metabolismo , Transcriptoma , Presenilina-1/genética , Presenilina-1/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Mutación/genética , Neuronas/metabolismo , Precursor de Proteína beta-Amiloide/genética
5.
Bioinformatics ; 38(24): 5437-5439, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36271848

RESUMEN

SUMMARY: The effectiveness of CRISPR/Cas9-mediated genome editing experiments largely depends on the guide RNA (gRNA) used by the CRISPR/Cas9 system for target recognition and cleavage activation. Careful design is necessary to select a gRNA with high editing efficiency at the on-target site and with minimum off-target potential. Here, we present our webserver for gRNA design with a user-friendly graphical interface, which provides interoperability between our on- and off-target prediction tools, CRISPRon and CRISPRoff, for a complete and streamlined gRNA selection. AVAILABILITY AND IMPLEMENTATION: The graphical interface uses the Integrative Genomic Viewer (IGV) JavaScript plugin. The backend tools are implemented in Python and C. The CRISPRon and CRISPRoff webservers and command-line tools are freely available at https://rth.dk/resources/crispr.


Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Edición Génica
6.
Brain Behav Immun ; 113: 353-373, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37543250

RESUMEN

Frontotemporal dementia (FTD) is a common cause of early-onset dementia, with no current treatment options. FTD linked to chromosome 3 (FTD3) is a rare sub-form of the disease, caused by a point mutation in the Charged Multivesicular Body Protein 2B (CHMP2B). This mutation causes neuronal phenotypes, such as mitochondrial deficiencies, accompanied by metabolic changes and interrupted endosomal-lysosomal fusion. However, the contribution of glial cells to FTD3 pathogenesis has, until recently, been largely unexplored. Glial cells play an important role in most neurodegenerative disorders as drivers and facilitators of neuroinflammation. Microglia are at the center of current investigations as potential pro-inflammatory drivers. While gliosis has been observed in FTD3 patient brains, it has not yet been systematically analyzed. In the light of this, we investigated the role of microglia in FTD3 by implementing human induced pluripotent stem cells (hiPSC) with either a heterozygous or homozygous CHMP2B mutation, introduced into a healthy control hiPSC line via CRISPR-Cas9 precision gene editing. These hiPSC were differentiated into microglia to evaluate the pro-inflammatory profile and metabolic state. Moreover, hiPSC-derived neurons were cultured with conditioned microglia media to investigate disease specific interactions between the two cell populations. Interestingly, we identified two divergent inflammatory microglial phenotypes resulting from the underlying mutations: a severe pro-inflammatory profile in CHMP2B homozygous FTD3 microglia, and an "unresponsive" CHMP2B heterozygous FTD3 microglial state. These findings correlate with our observations of increased phagocytic activity in CHMP2B homozygous, and impaired protein degradation in CHMP2B heterozygous FTD3 microglia. Metabolic mapping confirmed these differences, revealing a metabolic reprogramming of the CHMP2B FTD3 microglia, displayed as a compensatory up-regulation of glutamine metabolism in the CHMP2B homozygous FTD3 microglia. Intriguingly, conditioned CHMP2B homozygous FTD3 microglia media caused neurotoxic effects, which was not evident for the heterozygous microglia. Strikingly, IFN-γ treatment initiated an immune boost of the CHMP2B heterozygous FTD3 microglia, and conditioned microglia media exposure promoted neural outgrowth. Our findings indicate that the microglial profile, activity, and behavior is highly dependent on the status of the CHMP2B mutation. Our results suggest that the heterozygous state of the mutation in FTD3 patients could potentially be exploited in form of immune-boosting intervention strategies to counteract neurodegeneration.


Asunto(s)
Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Humanos , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Células Madre Pluripotentes Inducidas/metabolismo , Microglía/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo
7.
Nucleic Acids Res ; 49(4): 1859-1871, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33524155

RESUMEN

Animal models are crucial for advancing our knowledge about the molecular pathways involved in human diseases. However, it remains unclear to what extent tissue expression of pathways in healthy individuals is conserved between species. In addition, organism-specific information on pathways in animal models is often lacking. Within these limitations, we explore the possibilities that arise from publicly available data for the animal models mouse, rat, and pig. We approximate the animal pathways activity by integrating the human counterparts of curated pathways with tissue expression data from the models. Specifically, we compare whether the animal orthologs of the human genes are expressed in the same tissue. This is complicated by the lower coverage and worse quality of data in rat and pig as compared to mouse. Despite that, from 203 human KEGG pathways and the seven tissues with best experimental coverage, we identify 95 distinct pathways, for which the tissue expression in one animal model agrees better with human than the others. Our systematic pathway-tissue comparison between human and three animal modes points to specific similarities with human and to distinct differences among the animal models, thereby suggesting the most suitable organism for modeling a human pathway or tissue.


Asunto(s)
Modelos Animales , Animales , Expresión Génica , Genoma , Humanos , Ratones , Especificidad de Órganos , Mapeo de Interacción de Proteínas , Ratas , Porcinos
8.
J Ind Microbiol Biotechnol ; 50(1)2023 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-36564025

RESUMEN

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.


Asunto(s)
Bacillus subtilis , alfa-Amilasas , alfa-Amilasas/biosíntesis , alfa-Amilasas/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , ARN/genética , Análisis de Secuencia de ARN
9.
Microb Cell Fact ; 21(1): 131, 2022 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-35780132

RESUMEN

BACKGROUND: Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients. RESULTS: In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2-threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar. CONCLUSIONS: We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.


Asunto(s)
Amilasas , Bacillus subtilis , Flagelos , alfa-Amilasas , Amilasas/genética , Bacillus subtilis/genética , Eritromicina , Flagelos/genética , alfa-Amilasas/genética , alfa-Amilasas/metabolismo
10.
Nucleic Acids Res ; 48(6): 3228-3243, 2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-31989168

RESUMEN

Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Reparación del ADN/genética , Endodesoxirribonucleasas/genética , Edición Génica , Proteína 9 Asociada a CRISPR/genética , Escherichia coli/genética , Proteínas Luminiscentes/genética , Plásmidos/genética , ARN Guía de Kinetoplastida/genética
11.
Genome Res ; 27(8): 1371-1383, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28487280

RESUMEN

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.


Asunto(s)
Regulación de la Expresión Génica , Conformación de Ácido Nucleico , ARN/química , ARN/genética , Elementos Reguladores de la Transcripción , Vertebrados/genética , Animales , Secuencia de Bases , Secuencia Conservada , Genoma Humano , Humanos , Ratones , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia , Transcripción Genética
12.
Bioinformatics ; 35(9): 1494-1502, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30295698

RESUMEN

MOTIVATION: Long non-coding RNAs (lncRNAs) are important regulators in wide variety of biological processes, which are linked to many diseases. Compared to protein-coding genes (PCGs), the association between diseases and lncRNAs is still not well studied. Thus, inferring disease-associated lncRNAs on a genome-wide scale has become imperative. RESULTS: In this study, we propose a machine learning-based method, DislncRF, which infers disease-associated lncRNAs on a genome-wide scale based on tissue expression profiles. DislncRF uses random forest models trained on expression profiles of known disease-associated PCGs across human tissues to extract general patterns between expression profiles and diseases. These models are then applied to score associations between lncRNAs and diseases. DislncRF was benchmarked against a gold standard dataset and compared to other methods. The results show that DislncRF yields promising performance and outperforms the existing methods. The utility of DislncRF is further substantiated on two diseases in which we find that top scoring candidates are supported by literature or independent datasets. AVAILABILITY AND IMPLEMENTATION: https://github.com/xypan1232/DislncRF. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
ARN Largo no Codificante/genética , Genoma , Humanos , Aprendizaje Automático
13.
Nucleic Acids Res ; 46(1): 104-119, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29059375

RESUMEN

In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3'UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3'UTR, the lft1 and lft2 3'UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a 'translational regulon'.


Asunto(s)
Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Regiones no Traducidas 3'/genética , Animales , Embrión no Mamífero/embriología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Determinación Derecha-Izquierda/genética , Factores de Determinación Derecha-Izquierda/metabolismo , Ligandos , Ligandos de Señalización Nodal/genética , Ligandos de Señalización Nodal/metabolismo , ARN/genética , ARN/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
14.
J Proteome Res ; 18(2): 623-632, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30450911

RESUMEN

Protein networks have become a popular tool for analyzing and visualizing the often long lists of proteins or genes obtained from proteomics and other high-throughput technologies. One of the most popular sources of such networks is the STRING database, which provides protein networks for more than 2000 organisms, including both physical interactions from experimental data and functional associations from curated pathways, automatic text mining, and prediction methods. However, its web interface is mainly intended for inspection of small networks and their underlying evidence. The Cytoscape software, on the other hand, is much better suited for working with large networks and offers greater flexibility in terms of network analysis, import, and visualization of additional data. To include both resources in the same workflow, we created stringApp, a Cytoscape app that makes it easy to import STRING networks into Cytoscape, retains the appearance and many of the features of STRING, and integrates data from associated databases. Here, we introduce many of the stringApp features and show how they can be used to carry out complex network analysis and visualization tasks on a typical proteomics data set, all through the Cytoscape user interface. stringApp is freely available from the Cytoscape app store: http://apps.cytoscape.org/apps/stringapp .


Asunto(s)
Análisis de Datos , Proteómica/métodos , Programas Informáticos , Biología Computacional/métodos , Internet , Mapas de Interacción de Proteínas , Interfaz Usuario-Computador
16.
Mol Vis ; 25: 1-11, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30820140

RESUMEN

Purpose: To identify the mutation for Volkmann cataract (CTRCT8) at 1p36.33. Methods: The genes in the candidate region 1p36.33 were Sanger and parallel deep sequenced, and informative single nucleotide polymorphisms (SNPs) were identified for linkage analysis. Expression analysis with reverse transcription polymerase chain reaction (RT-PCR) of the candidate gene was performed using RNA from different human tissues. Quantitative transcription polymerase chain reaction (qRT-PCR) analysis of the GNB1 gene was performed in affected and healthy individuals. Bioinformatic analysis of the linkage regions including the candidate gene was performed. Results: Linkage analysis of the 1p36.33 CCV locus applying new marker systems obtained with Sanger and deep sequencing reduced the candidate locus from 2.1 Mb to 0.389 Mb flanked by the markers STS-22AC and rs549772338 and resulted in an logarithm of the odds (LOD) score of Z = 21.67. The identified mutation, rs763295804, affects the donor splice site in the long non-coding RNA gene RP1-140A9.1 (ENSG00000231050). The gene including splice-site junctions is conserved in primates but not in other mammalian genomes, and two alternative transcripts were shown with RT-PCR. One of these transcripts represented a lens cell-specific transcript. Meta-analysis of the Cross-Linking-Immuno-Precipitation sequencing (CLIP-Seq) data suggested the RNA binding protein (RBP) eIF4AIII is an active counterpart for RP1-140A9.1, and several miRNA and transcription factors binding sites were predicted in the proximity of the mutation. ENCODE DNase I hypersensitivity and histone methylation and acetylation data suggest the genomic region may have regulatory functions. Conclusions: The mutation in RP1-140A9.1 suggests the long non-coding RNA as the candidate cataract gene associated with the autosomal dominant inherited congenital cataract from CCV. The mutation has the potential to destroy exon/intron splicing of both transcripts of RP1-140A9.1. Sanger and massive deep resequencing of the linkage region failed to identify alternative candidates suggesting the mutation in RP1-140A9.1 is causative for the CCV phenotype.


Asunto(s)
Catarata/congénito , Cromosomas Humanos Par 1/química , Mutación , ARN Largo no Codificante/genética , ARN Mensajero/genética , Acetilación , Adulto , Secuencia de Bases , Sitios de Unión , Catarata/diagnóstico , Catarata/genética , Catarata/patología , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Exones , Familia , Femenino , Genes Dominantes , Sitios Genéticos , Marcadores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Histonas/metabolismo , Humanos , Intrones , Masculino , Metilación , Persona de Mediana Edad , Linaje , Sitios de Empalme de ARN , Empalme del ARN , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo
17.
Nucleic Acids Res ; 45(8): e60, 2017 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-28108657

RESUMEN

Intermolecular interactions of ncRNAs are at the core of gene regulation events, and identifying the full map of these interactions bears crucial importance for ncRNA functional studies. It is known that RNA-RNA interactions are built up by complementary base pairings between interacting RNAs and high level of complementarity between two RNA sequences is a powerful predictor of such interactions. Here, we present RIsearch2, a large-scale RNA-RNA interaction prediction tool that enables quick localization of potential near-complementary RNA-RNA interactions between given query and target sequences. In contrast to previous heuristics which either search for exact matches while including G-U wobble pairs or employ simplified energy models, we present a novel approach using a single integrated seed-and-extend framework based on suffix arrays. RIsearch2 enables fast discovery of candidate RNA-RNA interactions on genome/transcriptome-wide scale. We furthermore present an siRNA off-target discovery pipeline that not only predicts the off-target transcripts but also computes the off-targeting potential of a given siRNA. This is achieved by combining genome-wide RIsearch2 predictions with target site accessibilities and transcript abundance estimates. We show that this pipeline accurately predicts siRNA off-target interactions and enables off-targeting potential comparisons between different siRNA designs. RIsearch2 and the siRNA off-target discovery pipeline are available as stand-alone software packages from http://rth.dk/resources/risearch.


Asunto(s)
Modelos Estadísticos , ARN Interferente Pequeño/genética , ARN no Traducido/genética , Programas Informáticos , Transcriptoma , Algoritmos , Emparejamiento Base , Secuencia de Bases , Línea Celular Tumoral , Humanos , Modelos Genéticos , ARN Interferente Pequeño/metabolismo , ARN no Traducido/metabolismo
18.
BMC Genomics ; 19(1): 899, 2018 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-30537930

RESUMEN

BACKGROUND: Comparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). The increasing availability of related genomes now makes it possible to systematically search for compensatory base changes - and thus for conserved secondary structures - even in genomic regions that are poorly alignable in the primary sequence. The wealth of available transcriptome data can add valuable insight into expression and possible function for new ncRNA candidates. Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome. RESULTS: To search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, we conducted a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster and integrated this with an extensive set of tiling array data. The structural alignment screen revealed ∼30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, we found that most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units. CONCLUSIONS: This study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.


Asunto(s)
Secuencia Conservada , Drosophila melanogaster/genética , ARN/química , Animales , Composición de Base/genética , Secuencia de Bases , Drosophila melanogaster/crecimiento & desarrollo , Regulación de la Expresión Génica , Anotación de Secuencia Molecular , ARN no Traducido/genética , Programas Informáticos
19.
Bioinformatics ; 33(14): 2089-2096, 2017 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-28334186

RESUMEN

MOTIVATION: Clustering RNA sequences with common secondary structure is an essential step towards studying RNA function. Whereas structural RNA alignment strategies typically identify common structure for orthologous structured RNAs, clustering seeks to group paralogous RNAs based on structural similarities. However, existing approaches for clustering paralogous RNAs, do not take the compensatory base pair changes obtained from structure conservation in orthologous sequences into account. RESULTS: Here, we present RNAscClust , the implementation of a new algorithm to cluster a set of structured RNAs taking their respective structural conservation into account. For a set of multiple structural alignments of RNA sequences, each containing a paralog sequence included in a structural alignment of its orthologs, RNAscClust computes minimum free-energy structures for each sequence using conserved base pairs as prior information for the folding. The paralogs are then clustered using a graph kernel-based strategy, which identifies common structural features. We show that the clustering accuracy clearly benefits from an increasing degree of compensatory base pair changes in the alignments. AVAILABILITY AND IMPLEMENTATION: RNAscClust is available at http://www.bioinf.uni-freiburg.de/Software/RNAscClust . CONTACT: gorodkin@rth.dk or backofen@informatik.uni-freiburg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
ARN/química , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Algoritmos , Análisis por Conglomerados , Humanos , Conformación de Ácido Nucleico
20.
PLoS Genet ; 11(6): e1005333, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26125550

RESUMEN

Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3' to 5' direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems.


Asunto(s)
Mitocondrias/genética , Mutación , Neoplasias/genética , Alelos , ADN Mitocondrial , ADN de Neoplasias/genética , Genoma Mitocondrial , Humanos , ARN Neoplásico , ARN de Transferencia/genética , Análisis de Secuencia de ARN
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