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Sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes throughout the COVID-19 pandemic has generated a wealth of data on viral evolution across populations, but only a few studies have so far explored SARS-CoV-2 evolution across large connected transmission networks. Here, we couple data from SARS-CoV-2 sequencing with contact tracing data from an outbreak with a single origin in a rural Norwegian community where samples from all exposed persons were collected prospectively. A total of 134 nasopharyngeal samples were positive by PCR. Among the 121 retrievable genomes, 81 were identical to the genome of the introductor, thus demonstrating that genomics beyond clustering genotypically similar viral genomes to confirm relatedness offers limited additional value to manual contact tracing. In the cases where mutations were discovered, five small genetic clusters were identified. We observed a household secondary attack rate of 77%, with 92% of household members infected among households with secondary transmission, suggesting that SARS-CoV-2 introduction into large families is likely to affect all household members. IMPORTANCE: In outbreak investigations, obtaining a full overview of infected individuals within a population is seldom achieved. We here present an example where a single introduction of B1.1.7 SARS-CoV-2 within a rural community allowed for tracing of the virus from an introductor via dissemination through larger gatherings into households. The outbreak occurred before widespread vaccination, allowing for a "natural" outbreak development with community lockdown. We show through sequencing that the virus can infect up to five consecutive persons without gaining mutations, thereby showing that contact tracing seems more important than sequencing for local outbreak investigations in settings with few alternative introductory transmission pathways. We also show how larger households where a child introduced transmission appeared more likely to promote further spread of the virus compared to households with an adult as the primary introductor.
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COVID-19 , Trazado de Contacto , Brotes de Enfermedades , Composición Familiar , Genoma Viral , Población Rural , SARS-CoV-2 , Humanos , COVID-19/transmisión , COVID-19/epidemiología , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/clasificación , Adulto , Femenino , Persona de Mediana Edad , Masculino , Noruega/epidemiología , Anciano , Niño , Adolescente , Adulto Joven , Preescolar , Lactante , Variación Genética , Mutación , Anciano de 80 o más AñosRESUMEN
Terminal nucleotidyl transferases add nucleotides to the 3' end of RNA to modify their stability and function. In Caenorhabditis elegans, the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, expression of PUP-2, PUP-3, and especially PUP-4 limit uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that addition of U-tails to non-adenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development.
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The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, within both annotated protein-coding and noncoding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term ribosome decision graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the latter "translons." Nondeterministic events, such as initiation, reinitiation, selenocysteine insertion, or ribosomal frameshifting, are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions and for analyzing genetic variation and quantitative genome-wide data on translation for characterization of regulatory modulators of translation.
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Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Ribosomas/metabolismo , Ribosomas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Sistemas de Lectura Abierta , Eucariontes/genéticaRESUMEN
The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, both within annotated protein-coding and non-coding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term Ribosome Decision Graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the later 'translons'. Non-deterministic events, such as initiation, re-initiation, selenocysteine insertion or ribosomal frameshifting are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions, analysis of genetic variation and quantitative genome-wide data on translation for characterisation of regulatory modulators of translation.
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AIMS/HYPOTHESIS: Correctly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)-MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes. METHODS: We extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped HNF1A variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers' phenotype and treatment response to sulfonylurea. RESULTS: In total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing, revealing 19 with HNF1A variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic. CONCLUSIONS/INTERPRETATION: Based on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY.
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Diabetes Mellitus Tipo 2 , Humanos , Niño , Proyectos Piloto , Diabetes Mellitus Tipo 2/metabolismo , Fenotipo , Autoanticuerpos/genética , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Noruega/epidemiología , Compuestos de Sulfonilurea , MutaciónRESUMEN
RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
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Nanoporos , Transcriptoma , Animales , Ratones , ADN Complementario/genética , Pez Cebra/genética , Pez Cebra/metabolismo , Poli A/genética , Poli A/metabolismo , Perfilación de la Expresión Génica , ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.
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Nanoporos , Conformación de Ácido Nucleico , ARN , Análisis de Secuencia de ARN , Riboswitch , ARN/genética , ARN/química , Análisis de Secuencia de ARN/métodos , TranscriptomaRESUMEN
Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.
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Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Cultivadas/fisiología , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Edición Génica/métodos , Reparación del ADN/genética , Reparación del ADN/fisiología , HumanosRESUMEN
BACKGROUND: With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. RESULTS: Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5'UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5' UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. CONCLUSION: In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment. AVAILABILITY: http://bioconductor.org/packages/ORFik .
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Biosíntesis de Proteínas , Ribosomas , Regiones no Traducidas 5' , Secuenciación de Nucleótidos de Alto Rendimiento , Sistemas de Lectura Abierta/genética , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.
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The poly(A) tail is a homopolymeric stretch of adenosine at the 3'-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr-an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing-from library preparation to downstream data analysis with tailfindr.
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Secuenciación de Nanoporos/métodos , Poli A/análisis , ARN/análisis , Análisis de Secuencia de ARN/métodos , Animales , Estudios de Factibilidad , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Poli A/genética , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , ARN/química , ARN/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/análisis , ARN Mensajero/genética , Transcriptoma , Pez Cebra/genéticaRESUMEN
The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on-target editing) with minimal editing at unintended locations (off-target editing). Although laboratory validation should ideally be used to detect off-target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA-DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user-friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock-out, sequence knock-in, and RNA knock-down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock-out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock-in Alternate Protocol 2: Design of gRNAs for knock-in using non-homologous end joining Basic Protocol 4: Design of gRNAs for knock-in using Cas9 nickases.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Sistemas CRISPR-Cas , Genoma , ARN Guía de Kinetoplastida/genéticaRESUMEN
Advances in genomics and molecular biology have revealed an abundance of small open reading frames (sORFs) across all types of transcripts. While these sORFs are often assumed to be non-functional, many have been implicated in physiological functions and a significant number of sORFs have been described in human diseases. Thus, sORFs may represent a hidden repository of functional elements that could serve as therapeutic targets. Unlike protein-coding genes, it is not necessarily the encoded peptide of an sORF that enacts its function, sometimes simply the act of translating an sORF might have a regulatory role. Indeed, the most studied sORFs are located in the 5'UTRs of coding transcripts and can have a regulatory impact on the translation of the downstream protein-coding sequence. However, sORFs have also been abundantly identified in non-coding RNAs including lncRNAs, circular RNAs and ribosomal RNAs suggesting that sORFs may be diverse in function. Of the many different experimental methods used to discover sORFs, the most commonly used are ribosome profiling and mass spectrometry. These can confirm interactions between transcripts and ribosomes and the production of a peptide, respectively. Extensions to ribosome profiling, which also capture scanning ribosomes, have further made it possible to see how sORFs impact the translation initiation of mRNAs. While high-throughput techniques have made the identification of sORFs less difficult, defining their function, if any, is typically more challenging. Together, the abundance and potential function of many of these sORFs argues for the necessity of including sORFs in gene annotations and systematically characterizing these to understand their potential functional roles. In this review, we will focus on the high-throughput methods used in the detection and characterization of sORFs and discuss techniques for validation and functional characterization.
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BACKGROUND: The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes. RESULTS: In this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters. CONCLUSIONS: Methylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.
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Metilación de ADN/genética , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/genética , Epigénesis Genética/genética , Femenino , Humanos , Posmenopausia , Valores de ReferenciaRESUMEN
Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5' UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.
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Iniciación de la Cadena Peptídica Traduccional/genética , Humanos , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
OBJECTIVE: Neonatal diabetes has been shown to be associated with high neuropsychiatric morbidity in a genotype-phenotype-dependent manner. However, the specific impact of different mutations on intellectual functioning is still insufficiently characterized. Specifically, only a small number of subjects with developmental delay have been comprehensively assessed, creating a knowledge gap about patients carrying the heaviest burden. RESEARCH DESIGN AND METHODS: We assessed the intellectual functioning and mental health of the complete Norwegian population with KATP channel neonatal diabetes. Eight sulfonylurea-treated children (five with the p.V59M genotype [KCNJ11]) were assessed using age-matched control subjects with type 1 diabetes. The investigations included a physical and motor developmental examination, cerebral MRI, psychometrical examination, and questionnaires assessing intellectual capabilities and psychiatric morbidity. RESULTS: A strong genotype-phenotype correlation was found, revealing the p.V59M genotype as highly associated with substantial intellectual disability, with no significant correlation with the time of sulfonylurea initiation. Consistent with previous studies, other genotypes were associated with minor cognitive impairment. Cerebral MRI verified normal brain anatomy in all but one child. CONCLUSIONS: We here presented a comprehensive assessment of intellectual functioning in the largest cohort of p.V59M subjects to date. The level of intellectual disability revealed not only changes the interpretation of other psychological measures but downplays a strong protective effect of sulfonylurea. Within the scope of this study, we could not find evidence supporting an early treatment start to be beneficial, although a weaker effect cannot be ruled out.
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Diabetes Mellitus Tipo 1/congénito , Diabetes Mellitus Tipo 1/genética , Discapacidad Intelectual/genética , Canales de Potasio de Rectificación Interna/genética , Adolescente , Sustitución de Aminoácidos , Estudios de Casos y Controles , Niño , Preescolar , Codón sin Sentido , Estudios de Cohortes , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/epidemiología , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Recién Nacido , Enfermedades del Recién Nacido/genética , Enfermedades del Recién Nacido/psicología , Discapacidad Intelectual/epidemiología , Masculino , Metionina/genética , Mutación Missense , Noruega/epidemiología , Receptores de Sulfonilureas/genética , Valina/genéticaRESUMEN
In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remain poorly understood. Here, we generated chromatin accessibility maps with ATAC-seq from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hypertranscribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.
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Ensamble y Desensamble de Cromatina , Regulación del Desarrollo de la Expresión Génica , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOX/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Cromatina/genética , Embrión no Mamífero/metabolismo , Impresión Genómica , Pez CebraRESUMEN
BACKGROUND: In phylogenetically diverse organisms, the 5' ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic. RESULTS: We quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, consistent with a role of the SL sequence in nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continued to filter-feed, but arrested growth until favorable conditions returned. Upon release from unfavorable conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development. CONCLUSION: Our results are consistent with a proposed role of trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under growth-limiting conditions.
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Regulación de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Trans-Empalme , Transcripción Genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Femenino , Mamíferos/genética , Motivos de Nucleótidos , Oocitos/metabolismo , ARN Mensajero/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Urocordados/genéticaRESUMEN
Polyadenylation at the 3'-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3'-ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.