RESUMEN
Transcriptional downregulation caused by intronic triplet repeat expansions underlies diseases such as Friedreich's ataxia. This downregulation of gene expression is coupled with epigenetic changes, but the underlying mechanisms are unknown. Here, we show that an intronic GAA/TTC triplet expansion within the IIL1 gene of Arabidopsis thaliana results in accumulation of 24-nt short interfering RNAs (siRNAs) and repressive histone marks at the IIL1 locus, which in turn causes its transcriptional downregulation and an associated phenotype. Knocking down DICER LIKE-3 (DCL3), which produces 24-nt siRNAs, suppressed transcriptional downregulation of IIL1 and the triplet expansion-associated phenotype. Furthermore, knocking down additional components of the RNA-dependent DNA methylation (RdDM) pathway also suppressed both transcriptional downregulation of IIL1 and the repeat expansion-associated phenotype. Thus, our results show that triplet repeat expansions can lead to local siRNA biogenesis, which in turn downregulates transcription through an RdDM-dependent epigenetic modification.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Epigénesis Genética , Intrones , ARN de Planta/genética , ARN Interferente Pequeño/genética , Ribonucleasa III/genética , Transcripción Genética , Metilación de ADN , ADN Polimerasa beta/genética , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Oligonucleótidos Antisentido/genética , Fenotipo , Interferencia de ARN , Transgenes , Expansión de Repetición de TrinucleótidoRESUMEN
Insect-symbiont interactions are known to play key roles in host functions and fitness. The common insect endosymbiont Wolbachia can reduce the ability of several human pathogens, including arboviruses and the malaria parasite, to replicate in insect hosts. Wolbachia does not naturally infect Aedes aegypti, the primary vector of dengue virus, but transinfected Ae. aegypti have antidengue virus properties and are currently being trialled as a dengue biocontrol strategy. Here, we assess the impact of Wolbachia infection of Ae. aegypti on the microbiome of wild mosquito populations (adults and larvae) collected from release sites in Cairns, Australia, by profiling the 16S rRNA gene using next-generation sequencing. Our data indicate that Wolbachia reduces the relative abundance of a large proportion of bacterial taxa in Ae. aegypti adults, that is in accordance with the known pathogen-blocking effects of Wolbachia on a variety of bacteria and viruses. In adults, several of the most abundant bacterial genera were found to undergo significant shifts in relative abundance. However, the genera showing the greatest changes in relative abundance in Wolbachia-infected adults represented a low proportion of the total microbiome. In addition, there was little effect of Wolbachia infection on the relative abundance of bacterial taxa in larvae, or on species diversity (accounting for species richness and evenness together) detected in adults or larvae. These results offer insight into the effects of Wolbachia on the Ae. aegypti microbiome in a native setting, an important consideration for field releases of Wolbachia into the population.
Asunto(s)
Aedes/microbiología , Wolbachia/fisiología , Animales , Australia , Biodiversidad , Femenino , Genes Bacterianos , Secuenciación de Nucleótidos de Alto Rendimiento , Larva/microbiología , Microbiota/genética , Filogenia , ARN Ribosómico 16S/genética , Wolbachia/clasificaciónRESUMEN
Plants are highly sensitive to environmental changes and even small variations in ambient temperature have severe consequences on their growth and development. Temperature affects multiple aspects of plant development, but the processes and mechanisms underlying thermo-sensitive growth responses are mostly unknown. Here we exploit natural variation in Arabidopsis thaliana to identify and characterize novel components and processes mediating thermo-sensitive growth responses in plants. Phenotypic screening of wild accessions identified several strains displaying pleiotropic growth defects, at cellular and organism levels, specifically at high ambient temperatures. Positional cloning and characterization of the underlying gene revealed that ICARUS1 (ICA1), which encodes a protein of the tRNAHis guanylyl transferase (Thg1) superfamily, is required for plant growth at high temperatures. Transcriptome and gene marker analyses together with DNA content measurements show that ICA1 loss-of-function results in down regulation of cell cycle associated genes at high temperatures, which is linked with a block in G2/M transition and endoreduplication. In addition, plants with mutations in ICA1 show enhanced sensitivity to DNA damage. Characterization of additional strains that carry lesions in ICA1, but display normal growth, shows that alternative splicing is likely to alleviate the deleterious effects of some natural mutations. Furthermore, analyses of worldwide and regional collections of natural accessions indicate that ICA1 loss-of-function has arisen several times independently, and that these occur at high frequency in some local populations. Overall our results suggest that ICA1-mediated-modulation of fundamental processes such as tRNAHis maturation, modify plant growth responses to temperature changes in a quantitative and reversible manner, in natural populations.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proliferación Celular , Regulación de la Expresión Génica de las Plantas , Variación Genética , Nucleotidiltransferasas/genética , Alelos , Empalme Alternativo , Proteínas de Arabidopsis/metabolismo , Biología Computacional , Daño del ADN , Duplicación de Gen , Marcadores Genéticos , Calor , Datos de Secuencia Molecular , Mutación , Nucleotidiltransferasas/metabolismo , Hojas de la Planta/genética , TranscriptomaRESUMEN
Animals experience near constant infection with microorganisms. A significant proportion of these microbiota reside in the alimentary tract. There is a growing appreciation for the roles gut microbiota play in host biology. The gut microbiota of insects, for example, have been shown to help the host overcome pathogen infection either through direct competition or indirectly by stimulating host immunity. These defenses may also be supplemented by coinfecting maternally inherited microbes such as Wolbachia. The presence of Wolbachia in a host can delay and/or reduce death caused by RNA viruses. Whether the gut microbiota of the host interacts with Wolbachia, or vice versa, the precise role of Wolbachia in antiviral protection is not known. In this study, we used 16S rDNA sequencing to characterise changes in gut microbiota composition in Drosophila melanogaster associated with Wolbachia infection and antibiotic treatment. We subsequently tested whether changes in gut composition via antibiotic treatment altered Wolbachia-mediated antiviral properties. We found that both antibiotics and Wolbachia significantly reduced the biodiversity of the gut microbiota without changing the total microbial load. We also showed that changing the gut microbiota composition with antibiotic treatment enhanced Wolbachia density but did not confer greater antiviral protection against Drosophila C virus to the host. We concluded there are significant interactions between Wolbachia and gut microbiota, but changing gut microbiota composition is not likely to be a means through which Wolbachia conveys antiviral protection to its host.
Asunto(s)
Drosophila melanogaster/microbiología , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Virosis/microbiología , Wolbachia , Animales , Antibacterianos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Virus de Insectos , Virus ARNRESUMEN
BACKGROUND: Next generation sequencing for oncology patient management is now routine in clinical pathology laboratories. Although wet lab, sequencing and pipeline tasks are largely automated, the analysis of variants for clinical reporting remains largely a manual task. The increasing volume of sequencing data and the limited availability of genetic experts to analyse and report on variants in the data is a key scalability limit for molecular diagnostics. METHOD: To determine the impact and size of the issue, we examined the longitudinally compiled genetic variants from 48,036 cancer patients over a six year period in a large cancer hospital from ten targeted cancer panel tests in germline, solid tumour and haematology contexts using hybridization capture and amplicon assays. This testing generated 24,168,398 sequenced variants of which 23,255 (8214 unique) were clinically reported. RESULTS: Of the reported variants, 17,240 (74.1%) were identified in more than one assay which allowed curated variant data to be reused in later reports. The remainder, 6015 (25.9%) were not subsequently seen in later assays and did not provide any reuse benefit. The number of new variants requiring curation has significantly increased over time from 1.72 to 3.73 variants per sample (292 curated variants per month). Analysis of the 23,255 variants reported, showed 28.6% (n = 2356) were not present in common public variant resources and therefore required de novo curation. These in-house only variants were enriched for indels, tumour suppressor genes and from solid tumour assays. CONCLUSION: This analysis highlights the significant percentage of variants not present within common public variant resources and the level of non-recurrent variants that consequently require greater curation effort. Many of these variants are unique to a single patient and unlikely to appear in other patients reflecting the personalised nature of cancer genomics. This study depicts the real-world situation for pathology laboratories faced with curating increasing numbers of low-recurrence variants while needing to expedite the process of manual variant curation. In the absence of suitably accurate automated methods, new approaches are needed to scale oncology diagnostics for future genetic testing volumes.
Asunto(s)
Neoplasias , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patología , Patología Molecular , Medicina de Precisión/métodosRESUMEN
BACKGROUND: The increasing affordability of DNA sequencing has allowed it to be widely deployed in pathology laboratories. However, this has exposed many issues with the analysis and reporting of variants for clinical diagnostic use. Implementing a high-throughput sequencing (NGS) clinical reporting system requires a diverse combination of capabilities, statistical methods to identify variants, global variant databases, a validated bioinformatics pipeline, an auditable laboratory workflow, reproducible clinical assays and quality control monitoring throughout. These capabilities must be packaged in software that integrates the disparate components into a useable system. RESULTS: To meet these needs, we developed a web-based application, PathOS, which takes variant data from a patient sample through to a clinical report. PathOS has been used operationally in the Peter MacCallum Cancer Centre for two years for the analysis, curation and reporting of genetic tests for cancer patients, as well as the curation of large-scale research studies. PathOS has also been deployed in cloud environments allowing multiple institutions to use separate, secure and customisable instances of the system. Increasingly, the bottleneck of variant curation is limiting the adoption of clinical sequencing for molecular diagnostics. PathOS is focused on providing clinical variant curators and pathology laboratories with a decision support system needed for personalised medicine. While the genesis of PathOS has been within cancer molecular diagnostics, the system is applicable to NGS clinical reporting generally. CONCLUSIONS: The widespread availability of genomic sequencers has highlighted the limited availability of software to support clinical decision-making in molecular pathology. PathOS is a system that has been developed and refined in a hospital laboratory context to meet the needs of clinical diagnostics. The software is available as a set of Docker images and source code at https://github.com/PapenfussLab/PathOS .
Asunto(s)
Servicios de Laboratorio Clínico , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Programas Informáticos , Humanos , Neoplasias/diagnóstico , Medicina de Precisión , Análisis de Secuencia de ADN/métodosRESUMEN
Increasing global temperatures have an impact on flowering, and the underlying mechanisms are just beginning to be unravelled(1,2). Elevated temperatures can induce flowering, and different mechanisms that involve either activation or de-repression of FLOWERING LOCUS T (FT) by transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or the FLOWERING LOCUS M (FLM)-SHORT VEGETATIVE PHASE (SVP) complex, respectively, have been suggested to be involved(3-6). Thermosensitivity in flowering has been mapped to FLM(5), which encodes a floral repressor(7,8). FLM undergoes alternative splicing(8) and it has been suggested that temperature-dependent alternative splicing leads to differential accumulation of the FLM-ß and FLM-δ transcripts, encoding proteins with antagonistic effects, and that their ratio determines floral transition(4). Here we show that high temperatures downregulate FLM expression by alternative splicing coupled with nonsense-mediated mRNA decay (AS-NMD). We identify thermosensitive splice sites in FLM and show that the primary effect of temperature is explained by an increase in NMD target transcripts. We also show that flm is epistatic to pif4, which suggests that most of the PIF4 effects are FLM dependent. Our findings suggest a model in which the loss of the floral repressor FLM occurs through mRNA degradation in response to elevated temperatures, signifying a role for AS-NMD in conferring environmental responses in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Flores/crecimiento & desarrollo , Proteínas de Dominio MADS/genética , Degradación de ARNm Mediada por Codón sin Sentido , ARN de Planta/genética , Empalme Alternativo , Proteínas de Arabidopsis/metabolismo , Regulación hacia Abajo , Flores/genética , Flores/metabolismo , Calor , Proteínas de Dominio MADS/metabolismo , ARN de Planta/metabolismoRESUMEN
Triplet repeat expansions underlie several human genetic diseases such as Huntington's disease and Friedreich's ataxia. Although such mutations are primarily known from humans, a triplet expansion associated genetic defect has also been reported at the IIL1 locus in the Bur-0 accession of the model plant Arabidopsis thaliana. The IIL1 triplet expansion is an example of cryptic genetic variation as its phenotypic effects are seen only under genetic or environmental perturbation, with high temperatures resulting in a growth defect. Here we demonstrate that the IIL1 triplet expansion associated growth defect is not a general stress response and is specific to particular environmental perturbations. We also confirm and map genetic modifiers that suppress the effect of IIL1 triplet repeat expansion. By collecting and analyzing accessions from the island of Ireland, we recover the repeat expansion in wild populations suggesting that the repeat expansion has persisted at least 60 years in Ireland. Through genome-wide genotyping, we show that the repeat expansion is present in diverse Irish populations. Our findings indicate that even deleterious alleles can persist in populations if their effect is conditional. Our study demonstrates that analysis of groups of wild populations is a powerful tool for understanding the dynamics of cryptic genetic variation.
RESUMEN
Most strains of the widespread endosymbiotic bacterium Wolbachia pipientis are benign or behave as reproductive parasites. The pathogenic strain wMelPop is a striking exception, however: it overreplicates in its insect hosts and causes severe life shortening. The mechanism of this pathogenesis is currently unknown. We have sequenced the genomes of three variants of wMelPop and of the closely related nonpathogenic strain wMelCS. We show that the genomes of wMelCS and wMelPop appear to be identical in the nonrepeat regions of the genome and differ detectably only by the triplication of a 19-kb region that is unlikely to be associated with life shortening, demonstrating that dramatic differences in the host phenotype caused by this endosymbiont may be the result of only minor genetic changes. We also compare the genomes of the original wMelPop strain from Drosophila melanogaster and two sequential derivatives, wMelPop-CLA and wMelPop-PGYP. To develop wMelPop as a novel biocontrol agent, it was first transinfected into and passaged in mosquito cell lines for approximately 3.5 years, generating wMelPop-CLA. This cell line-passaged strain was then transinfected into Aedes aegypti mosquitoes, creating wMelPop-PGYP, which was sequenced after 4 years in the insect host. We observe a rapid burst of genomic changes during cell line passaging, but no further mutations were detected after transinfection into mosquitoes, indicating either that host preadaptation had occurred in cell lines, that cell lines are a more selectively permissive environment than animal hosts, or both. Our results provide valuable data on the rates of genomic and phenotypic change in Wolbachia associated with host shifts over short time scales.