ABSTRACT
For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.
Subject(s)
Cell Transdifferentiation , Hepatocytes , Liver Diseases , Liver , Humans , Biliary Tract/cytology , Biliary Tract/metabolism , Biliary Tract/pathology , Biopsy , Cell Plasticity , Chronic Disease , Disease Progression , Epithelial Cells/metabolism , Epithelial Cells/cytology , Epithelial Cells/pathology , Hepatocytes/metabolism , Hepatocytes/cytology , Hepatocytes/pathology , Insulin/metabolism , Liver/pathology , Liver/metabolism , Liver/cytology , Liver Diseases/pathology , Liver Diseases/metabolism , Liver Regeneration , Organoids/metabolism , Organoids/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA-Seq , Signal Transduction , Single-Cell Analysis , TOR Serine-Threonine Kinases/metabolismABSTRACT
Bulk sequencing experiments (single- and multi-omics) are essential for exploring wide-ranging biological questions. To facilitate interactive, exploratory tasks, coupled with the sharing of easily accessible information, we present bulkAnalyseR, a package integrating state-of-the-art approaches using an expression matrix as the starting point (pre-processing functions are available as part of the package). Static summary images are replaced with interactive panels illustrating quality-checking, differential expression analysis (with noise detection) and biological interpretation (enrichment analyses, identification of expression patterns, followed by inference and comparison of regulatory interactions). bulkAnalyseR can handle different modalities, facilitating robust integration and comparison of cis-, trans- and customised regulatory networks.
Subject(s)
MultiomicsABSTRACT
Germ line variants in the DDX41 gene have been linked to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) development. However, the risks associated with different variants remain unknown, as do the basis of their leukemogenic properties, impact on steady-state hematopoiesis, and links to other cancers. Here, we investigate the frequency and significance of DDX41 variants in 454 792 United Kingdom Biobank (UKB) participants and identify 452 unique nonsynonymous DNA variants in 3538 (1/129) individuals. Many were novel, and the prevalence of most varied markedly by ancestry. Among the 1059 individuals with germ line pathogenic variants (DDX41-GPV) 34 developed MDS/AML (odds ratio, 12.3 vs noncarriers). Of these, 7 of 218 had start-lost, 22 of 584 had truncating, and 5 of 257 had missense (odds ratios: 12.9, 15.1, and 7.5, respectively). Using multivariate logistic regression, we found significant associations of DDX41-GPV with MDS, AML, and family history of leukemia but not lymphoma, myeloproliferative neoplasms, or other cancers. We also report that DDX41-GPV carriers do not have an increased prevalence of clonal hematopoiesis (CH). In fact, CH was significantly more common before sporadic vs DDX41-mutant MDS/AML, revealing distinct evolutionary paths. Furthermore, somatic mutation rates did not differ between sporadic and DDX41-mutant AML genomes, ruling out genomic instability as a driver of the latter. Finally, we found that higher mean red cell volume (MCV) and somatic DDX41 mutations in blood DNA identify DDX41-GPV carriers at increased MDS/AML risk. Collectively, our findings give new insights into the prevalence and cognate risks associated with DDX41 variants, as well as the clonal evolution and early detection of DDX41-mutant MDS/AML.
Subject(s)
GATA2 Deficiency , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Humans , Prevalence , DEAD-box RNA Helicases/genetics , Myelodysplastic Syndromes/epidemiology , Myelodysplastic Syndromes/genetics , Leukemia, Myeloid, Acute/genetics , DNAABSTRACT
BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls. METHODS: Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature. RESULTS: We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1. CONCLUSIONS: Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.
ABSTRACT
The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.
Subject(s)
Gene Expression Regulation, Leukemic , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Neoplasm Proteins/metabolism , Protein Biosynthesis , ZAP-70 Protein-Tyrosine Kinase/metabolism , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Male , Neoplasm Proteins/genetics , Tumor Cells, Cultured , ZAP-70 Protein-Tyrosine Kinase/geneticsABSTRACT
High-throughput sequencing enables an unprecedented resolution in transcript quantification, at the cost of magnifying the impact of technical noise. The consistent reduction of random background noise to capture functionally meaningful biological signals is still challenging. Intrinsic sequencing variability introducing low-level expression variations can obscure patterns in downstream analyses. We introduce noisyR, a comprehensive noise filter to assess the variation in signal distribution and achieve an optimal information-consistency across replicates and samples; this selection also facilitates meaningful pattern recognition outside the background-noise range. noisyR is applicable to count matrices and sequencing data; it outputs sample-specific signal/noise thresholds and filtered expression matrices. We exemplify the effects of minimizing technical noise on several datasets, across various sequencing assays: coding, non-coding RNAs and interactions, at bulk and single-cell level. An immediate consequence of filtering out noise is the convergence of predictions (differential-expression calls, enrichment analyses and inference of gene regulatory networks) across different approaches.
Subject(s)
Computational Biology/methods , Gene Expression Profiling/methods , Gene Regulatory Networks/genetics , RNA-Seq/methods , Single-Cell Analysis/methods , Algorithms , Animals , Arabidopsis/genetics , Computer Simulation , High-Throughput Nucleotide Sequencing/methods , Humans , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of ResultsABSTRACT
Respiratory coinfection of influenza with Staphylococcus aureus often causes severe disease; methicillin-resistant S. aureus (MRSA) coinfection is frequently fatal. Understanding disease pathogenesis may inform therapies. We aimed to identify host and pathogen transcriptomic (messenger RNA) signatures from the respiratory compartment of pediatric patients critically ill with influenza-S. aureus coinfection (ISAC), signatures that predict worse outcomes. Messenger RNA extracted from endotracheal aspirate samples was evaluated for S. aureus and host transcriptomic biosignatures. Influenza-MRSA outcomes were worse, but of 190 S. aureus virulence-associated genes, 6 were differentially expressed between MRSA-coinfected versus methicillin-susceptible S. aureus-coinfected patients, and none discriminated outcome. Host gene expression in patients with ISAC was compared with that in patients with influenza infection alone. Patients with poor clinical outcomes (death or prolonged multiorgan dysfunction) had relatively reduced expression of interferons and down-regulation of interferon γ-induced immune cell chemoattractants CXCL10 and CXCL11. In ISAC, airway host but not pathogen gene expression profiles predicted worse clinical outcomes.
Subject(s)
Coinfection , Influenza, Human , Methicillin-Resistant Staphylococcus aureus , Pneumonia, Staphylococcal , Staphylococcal Infections , Chemotactic Factors , Child , Coinfection/pathology , Humans , Influenza, Human/complications , Influenza, Human/genetics , Interferon-gamma , Methicillin , Methicillin-Resistant Staphylococcus aureus/genetics , Pneumonia, Staphylococcal/genetics , Pneumonia, Staphylococcal/pathology , RNA, Messenger , Staphylococcal Infections/complications , Staphylococcal Infections/genetics , Staphylococcus aureus/genetics , TranscriptomeABSTRACT
MicroRNAs (miRNAs) are short, non-coding RNAs that modulate the translation-rate of messenger RNAs (mRNAs) by directing the RNA-induced silencing complex to sequence-specific targets. In plants, this typically results in cleavage and subsequent degradation of the mRNA. Degradome sequencing is a high-throughput technique developed to capture cleaved mRNA fragments and thus can be used to support miRNA target prediction. The current criteria used for miRNA target prediction were inferred on a limited number of experimentally validated A. thaliana interactions and were adapted to fit these specific interactions; thus, these fixed criteria may not be optimal across all datasets (organisms, tissues or treatments). We present a new tool, PAREameters, for inferring targeting criteria from small RNA and degradome sequencing datasets. We evaluate its performance using a more extensive set of experimentally validated interactions in multiple A. thaliana datasets. We also perform comprehensive analyses to highlight and quantify the differences between subsets of miRNA-mRNA interactions in model and non-model organisms. Our results show increased sensitivity in A. thaliana when using the PAREameters inferred criteria and that using data-driven criteria enables the identification of additional interactions that further our understanding of the RNA silencing pathway in both model and non-model organisms.
Subject(s)
Arabidopsis/genetics , Computational Biology/methods , Gene Expression Regulation, Plant , MicroRNAs/genetics , RNA, Messenger/genetics , RNA, Plant/genetics , Software , Arabidopsis/metabolism , Base Sequence , Datasets as Topic , Flowers/genetics , Flowers/metabolism , High-Throughput Nucleotide Sequencing , MicroRNAs/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , RNA Cleavage , RNA, Messenger/metabolism , RNA, Plant/metabolism , Sensitivity and Specificity , Sequence Analysis, RNA , TranscriptomeABSTRACT
The queen-worker caste system of eusocial insects represents a prime example of developmental polyphenism (environmentally-induced phenotypic polymorphism) and is intrinsic to the evolution of advanced eusociality. However, the comparative molecular basis of larval caste determination and subsequent differentiation in the eusocial Hymenoptera remains poorly known. To address this issue within bees, we profiled caste-associated gene expression in female larvae of the intermediately eusocial bumblebee Bombus terrestris. In B. terrestris, female larvae experience a queen-dependent period during which their caste fate as adults is determined followed by a nutrition-sensitive period also potentially affecting caste fate but for which the evidence is weaker. We used mRNA-seq and qRT-PCR validation to isolate genes differentially expressed between each caste pathway in larvae at developmental stages before and after each of these periods. We show that differences in gene expression between caste pathways are small in totipotent larvae, then peak after the queen-dependent period. Relatively few novel (i.e., taxonomically-restricted) genes were differentially expressed between castes, though novel genes were significantly enriched in late-instar larvae in the worker pathway. We compared sets of caste-associated genes in B. terrestris with those reported from the advanced eusocial honeybee, Apis mellifera, and found significant but relatively low levels of overlap of gene lists between the two species. These results suggest both the existence of low numbers of shared toolkit genes and substantial divergence in caste-associated genes between Bombus and the advanced eusocial Apis since their last common eusocial ancestor.
Subject(s)
Bees , Behavior, Animal , Gene Expression Profiling , Animals , Bees/genetics , Female , Gene Expression , Larva/geneticsABSTRACT
Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this.
Subject(s)
Computational Biology , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , RNA, Small Untranslated , Sequence Analysis, RNA , Software , Computational Biology/methods , Computational Biology/standards , High-Throughput Nucleotide Sequencing/methods , High-Throughput Nucleotide Sequencing/standards , Reproducibility of Results , Sequence Analysis, RNA/methods , Sequence Analysis, RNA/standards , WorkflowABSTRACT
Socio-sexual environments have profound effects on fitness. Local sex ratios can alter the threat of sexual competition, to which males respond via plasticity in reproductive behaviors and ejaculate composition. In Drosophila melanogaster, males detect the presence of conspecific, same-sex mating rivals prior to mating using multiple, redundant sensory cues. Males that respond to rivals gain significant fitness benefits by altering mating duration and ejaculate composition. Here we investigated the underlying genome-wide changes involved. We used RNA-seq to analyze male transcriptomic responses 2, 26, and 50 h after exposure to rivals, a time period that was previously identified as encompassing the major facets of male responses to rivals. The results showed a strong early activation of multiple sensory genes in the head-thorax (HT), prior to the expression of any phenotypic differences. This gene expression response was reduced by 26 h, at the time of maximum phenotypic change, and shut off by 50 h. In the abdomen (A), fewer genes changed in expression and gene expression responses appeared to increase over time. The results also suggested that different sets of functionally equivalent genes might be activated in different replicates. This could represent a mechanism by which robustness is conferred upon highly plastic traits. Overall, our study reveals that mRNA-seq can identify subtle genomic signatures characteristic of flexible behavioral phenotypes.
Subject(s)
Competitive Behavior/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Genomics/methods , Sexual Behavior, Animal/physiology , Animals , Drosophila melanogaster/physiology , Gene Expression Profiling/methods , Gene Expression Regulation , Gene Regulatory Networks , Male , Sequence Analysis, RNA/methodsABSTRACT
Motivation: RNA interference, a highly conserved regulatory mechanism, is mediated via small RNAs (sRNA). Recent technical advances enabled the analysis of larger, complex datasets and the investigation of microRNAs and the less known small interfering RNAs. However, the size and intricacy of current data requires a comprehensive set of tools, able to discriminate the patterns from the low-level, noise-like, variation; numerous and varied suggestions from the community represent an invaluable source of ideas for future tools, the ability of the community to contribute to this software is essential. Results: We present a new version of the UEA sRNA Workbench, reconfigured to allow an easy insertion of new tools/workflows. In its released form, it comprises of a suite of tools in a user-friendly environment, with enhanced capabilities for a comprehensive processing of sRNA-seq data e.g. tools for an accurate prediction of sRNA loci (CoLIde) and miRNA loci (miRCat2), as well as workflows to guide the users through common steps such as quality checking of the input data, normalization of abundances or detection of differential expression represent the first step in sRNA-seq analyses. Availability and implementation: The UEA sRNA Workbench is available at: http://srna-workbench.cmp.uea.ac.uk. The source code is available at: https://github.com/sRNAworkbenchuea/UEA_sRNA_Workbench. Supplementary information: Supplementary data are available at Bioinformatics online.
Subject(s)
MicroRNAs/genetics , RNA, Small Interfering/genetics , Sequence Analysis, RNA/methods , Software , RNA Interference , WorkflowABSTRACT
Highly precise, yet flexible and responsive coordination of expression across groups of genes underpins the integrity of many vital functions. However, our understanding of gene regulatory networks (GRNs) is often hampered by the lack of experimentally tractable systems, by significant computational challenges derived from the large number of genes involved or from difficulties in the accurate identification and characterization of gene interactions. Here we used a tractable experimental system in which to study GRNs: the genes encoding the seminal fluid proteins that are transferred along with sperm (the 'transferome') in Drosophila melanogaster fruit flies. The products of transferome genes are core determinants of reproductive success and, to date, only transcription factors have been implicated in the modulation of their expression. Hence, as yet, we know nothing about the post-transcriptional mechanisms underlying the tight, responsive and precise regulation of this important gene set. We investigated this omission in the current study. We first used bioinformatics to identify potential regulatory motifs that linked the transferome genes in a putative interaction network. This predicted the presence of putative microRNA (miRNA) 'hubs'. We then tested this prediction, that post-transcriptional regulation is important for the control of transferome genes, by knocking down miRNA expression in adult males. This abolished the ability of males to respond adaptively to the threat of sexual competition, indicating a regulatory role for miRNAs in the regulation of transferome function. Further bioinformatics analysis then identified candidate miRNAs as putative regulatory hubs and evidence for variation in the strength of miRNA regulation across the transferome gene set. The results revealed regulatory mechanisms that can underpin robust, precise and flexible regulation of multiple fitness-related genes. They also help to explain how males can adaptively modulate ejaculate composition.
Subject(s)
Drosophila melanogaster/genetics , Insect Proteins/metabolism , Semen/metabolism , Animals , Drosophila melanogaster/metabolism , Gene Expression Regulation , Gene Knockdown Techniques/methods , Gene Regulatory Networks , Genetic Fitness , Insect Proteins/genetics , Male , MicroRNAs , Sexual Behavior, Animal , Transcription FactorsABSTRACT
MOTIVATION: MicroRNAs are a class of â¼21-22 nt small RNAs which are excised from a stable hairpin-like secondary structure. They have important gene regulatory functions and are involved in many pathways including developmental timing, organogenesis and development in eukaryotes. There are several computational tools for miRNA detection from next-generation sequencing datasets. However, many of these tools suffer from high false positive and false negative rates. Here we present a novel miRNA prediction algorithm, miRCat2. miRCat2 incorporates a new entropy-based approach to detect miRNA loci, which is designed to cope with the high sequencing depth of current next-generation sequencing datasets. It has a user-friendly interface and produces graphical representations of the hairpin structure and plots depicting the alignment of sequences on the secondary structure. RESULTS: We test miRCat2 on a number of animal and plant datasets and present a comparative analysis with miRCat, miRDeep2, miRPlant and miReap. We also use mutants in the miRNA biogenesis pathway to evaluate the predictions of these tools. Results indicate that miRCat2 has an improved accuracy compared with other methods tested. Moreover, miRCat2 predicts several new miRNAs that are differentially expressed in wild-type versus mutants in the miRNA biogenesis pathway. AVAILABILITY AND IMPLEMENTATION: miRCat2 is part of the UEA small RNA Workbench and is freely available from http://srna-workbench.cmp.uea.ac.uk/. CONTACT: v.moulton@uea.ac.uk or s.moxon@uea.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Computational Biology/methods , Genetic Loci , High-Throughput Nucleotide Sequencing/methods , MicroRNAs/genetics , Software , Algorithms , Animals , Entropy , Plants/genetics , Plants/metabolism , Sequence Analysis, DNA/methods , Sequence Analysis, RNA/methodsABSTRACT
Plants substantially alter their developmental programme upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15°C, 21°C, and 27°C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive microRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature-dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/metabolism , CCAAT-Binding Factor/physiology , Gene Expression Regulation, Plant , Mixed Function Oxygenases/physiology , RNA, Plant/metabolism , RNA, Small Untranslated/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/metabolism , Blotting, Northern , CCAAT-Binding Factor/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , MicroRNAs/physiology , Mixed Function Oxygenases/metabolism , RNA, Plant/genetics , RNA, Plant/physiology , RNA, Small Untranslated/genetics , RNA, Small Untranslated/physiology , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA , TemperatureABSTRACT
BACKGROUND: Mycoviruses are viruses that naturally infect and replicate in fungi. Aspergillus fumigatus, an opportunistic pathogen causing fungal lung diseases in humans and animals, was recently shown to harbour several different types of mycoviruses. A well-characterised defence against virus infection is RNA silencing. The A. fumigatus genome encodes essential components of the RNA silencing machinery, including Dicer, Argonaute and RNA-dependent RNA polymerase (RdRP) homologues. Active silencing of double-stranded (ds)RNA and the generation of small RNAs (sRNAs) has been shown for several mycoviruses and it is anticipated that a similar mechanism will be activated in A. fumigatus isolates infected with mycoviruses. RESULTS: To investigate the existence and nature of A. fumigatus sRNAs, sRNA-seq libraries of virus-free and virus-infected isolates were created using Scriptminer adapters and compared. Three dsRNA viruses were investigated: Aspergillus fumigatus partitivirus-1 (AfuPV-1, PV), Aspergillus fumigatus chrysovirus (AfuCV, CV) and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1, NK) which were selected because they induce phenotypic changes such as coloration and sectoring. The dsRNAs of all three viruses, which included two conventionally encapsidated ones PV and CV and one unencapsidated example NK, were silenced and yielded characteristic vsiRNAs together with co-incidental silencing of host fungal genes which shared sequence homology with the viral genomes. CONCLUSIONS: Virus-derived sRNAs were detected and characterised in the presence of virus infection. Differentially expressed A. fumigatus microRNA-like (miRNA-like) sRNAs and small interfering RNAs (siRNAs) were detected and validated. Host sRNA loci which were differentially expressed as a result of virus infection were also identified. To our knowledge, this is the first study reporting the sRNA profiles of A. fumigatus isolates.
Subject(s)
Aspergillus fumigatus/genetics , Aspergillus fumigatus/virology , Fungal Viruses/physiology , RNA Viruses/physiology , RNA, Small Untranslated/genetics , Sequence Analysis, RNA , Genetic Loci/genetics , High-Throughput Nucleotide SequencingABSTRACT
A new satellite RNA (satRNA) of grapevine fanleaf virus (GFLV) was identified by high-throughput sequencing of high-definition (HD) adapter libraries from grapevine plants of the cultivar Panse precoce (PPE) affected by enation disease. The complete nucleotide sequence was obtained by automatic sequencing using primers designed based on next-generation sequencing (NGS) data. The full-length sequence, named satGFLV-PPE, consisted of 1119 nucleotides with a single open reading frame from position 15 to 1034. This satRNA showed maximum nucleotide sequence identity of 87 % to satArMV-86 and satGFLV-R6. Symptomatic grapevines were surveyed for the presence of the satRNA, and no correlation was found between detection of the satRNA and enation symptom expression.
Subject(s)
Nepovirus/genetics , RNA, Satellite/genetics , Vitis/virology , Base Sequence , High-Throughput Nucleotide Sequencing , Molecular Sequence Data , Phylogeny , Plant Diseases/virologyABSTRACT
BACKGROUND: The 7B-1 tomato line (Solanum lycopersicum cv. Rutgers) is a photoperiod-sensitive male-sterile mutant, with potential application in hybrid seed production. Small RNAs (sRNAs) in tomato have been mainly characterized in fruit development and ripening, but none have been studied with respect to flower development and regulation of male-sterility. Using sRNA sequencing, we identified miRNAs that are potentially involved in anther development and regulation of male-sterility in 7B-1 mutant. RESULTS: Two sRNA libraries from 7B-1 and wild type (WT) anthers were sequenced and thirty two families of known miRNAs and 23 new miRNAs were identified in both libraries. MiR390, miR166, miR159 were up-regulated and miR530, miR167, miR164, miR396, miR168, miR393, miR8006 and two new miRNAs, miR#W and miR#M were down-regulated in 7B-1 anthers. Ta-siRNAs were not differentially expressed and likely not associated with 7B-1 male-sterility. miRNA targets with potential roles in anther development were validated using 5'-RACE. QPCR analysis showed differential expression of miRNA/target pairs of interest in anthers and stem of 7B-1, suggesting that they may regulate different biological processes in these tissues. Expression level of most miRNA/target pairs showed negative correlation, except for few. In situ hybridization showed predominant expression of miR159, GAMYBL1, PMEI and cystatin in tapetum, tetrads and microspores. CONCLUSION: Overall, we identified miRNAs with potential roles in anther development and regulation of male-sterility in 7B-1. A number of new miRNAs were also identified from tomato for the first time. Our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops.
Subject(s)
MicroRNAs/genetics , Solanum lycopersicum/genetics , Flowers/genetics , Flowers/physiology , Gene Expression Profiling , Solanum lycopersicum/physiology , Plant Infertility/genetics , Plant Infertility/physiologyABSTRACT
BACKGROUND: Marine phytoplankton are responsible for 50% of the CO2 that is fixed annually worldwide and contribute massively to other biogeochemical cycles in the oceans. Diatoms and coccolithophores play a significant role as the base of the marine food web and they sequester carbon due to their ability to form blooms and to biomineralise. To discover the presence and regulation of short non-coding RNAs (sRNAs) in these two important phytoplankton groups, we sequenced short RNA transcriptomes of two diatom species (Thalassiosira pseudonana, Fragilariopsis cylindrus) and validated them by Northern blots along with the coccolithophore Emiliania huxleyi. RESULTS: Despite an exhaustive search, we did not find canonical miRNAs in diatoms. The most prominent classes of sRNAs in diatoms were repeat-associated sRNAs and tRNA-derived sRNAs. The latter were also present in E. huxleyi. tRNA-derived sRNAs in diatoms were induced under important environmental stress conditions (iron and silicate limitation, oxidative stress, alkaline pH), and they were very abundant especially in the polar diatom F. cylindrus (20.7% of all sRNAs) even under optimal growth conditions. CONCLUSIONS: This study provides first experimental evidence for the existence of short non-coding RNAs in marine microalgae. Our data suggest that canonical miRNAs are absent from diatoms. However, the group of tRNA-derived sRNAs seems to be very prominent in diatoms and coccolithophores and maybe used for acclimation to environmental conditions.
Subject(s)
Diatoms/genetics , Microalgae/genetics , RNA, Small Untranslated/genetics , Diatoms/physiology , Interspersed Repetitive Sequences , Microalgae/physiology , Oxidative Stress , RNA, Small Untranslated/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Sequence Analysis, RNA , Up-RegulationABSTRACT
Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.