Search NASA⌕ Search

SEARCH · Search NASA

Results for “Transcriptomics”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Perimatrix of middle ear cholesteatoma: A granulation tissue with a specific transcriptomic signature

Objectives/Hypothesis: To establish comprehensive transcriptomic profiles of cholesteatoma perimatrix tissue and granulation tissue from chronic otitis media (COM) that did not develop cholesteatoma, which can indicate molecular pathways involved in the cholesteatoma perimatrix pathology and invasiveness. Study Design: Retrospective Case Series. Methods: Transcriptome data were obtained from cholesteatoma perimatrix tissue and COM granulation tissue by an Illumina iScan microarray. Differentially expressed genes (DEGs) were subsequently analyzed using both bioinformatical functional annotation and network analysis. Expression of candidate genes (MMP9 and LCN2) was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on a larger group of samples. Results: Analysis of the transcriptome led to the identification of 169 differentially expressed genes between investigated tissues. Bioinformatic analysis suggested that most significant biological processes involving DEGs were previously described in cholesteatoma pathology. Network analysis identified ERBB2, TFAP2A, and TP63 as major hubs of the DEGs molecular network. Furthermore, it was observed that the cellular component most significantly enriched in DEGs was extracellular space containing 47 DEGs. Using qRT-PCR, it was confirmed that mRNA levels of the major extracellular hub (MMP9) are increased, whereas its interacting molecule (LCN2) mRNA levels were decreased in cholesteatoma perimatrix tissue compared to COM granulation tissue. Conclusions: The current study approach offers an overall look at molecular mechanisms that describe the cholesteatoma entity by focusing exclusively on the perimatrix processes in comparison to COM granulation tissue. The observed differences in gene expression between cholesteatoma perimatrix and COM granulation tissue could suggest novel markers potentially influenced by the perimatrix–matrix molecular interplay, which is not present in COM without cholesteatoma. Level of Evidence: NA. Laryngoscope, 130:E220–E227, 2020. © 2019 The American Laryngological, Rhinological and Otological Society, Inc.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Resveratrol induces dynamic changes to the microglia transcriptome, inhibiting inflammatory pathways and protecting against microglia-mediated photoreceptor apoptosis

Highlights: • Resveratrol blocks the pro-inflammatory response of microglia on the transcriptome level. • Resveratrol reduces microglial phagocytosis, migration, and radical secretion. • Resveratrol prevents microglia-induced photoreceptor apoptosis. Microglia activation is central to the pathophysiology of retinal degenerative disorders. Resveratrol, a naturally occurring non-flavonoid phenolic compound present in red wine has potent anti-inflammatory and immunomodulatory properties. However, molecular mechanisms by which resveratrol influences microglial inflammatory pathways and housekeeping functions remain unclear. Here, we first studied the immuno-modulatory effects of resveratrol on BV-2 microglial cells at the transcriptome level using DNA-microarrays and selected qRT-PCR analyses. We then analyzed resveratrol effects on microglia morphology, phagocytosis and migration and estimated their neurotoxicity on 661 W photoreceptors by quantification of caspase 3/7 levels. We found that resveratrol effectively blocked gene expression of a broad spectrum of lipopolysaccharide (LPS)-induced pro-inflammatory molecules, including cytokines and complement proteins. These transcriptomic changes were accompanied by potent inhibition of LPS-induced nitric oxide secretion and reduced microglia-mediated apoptosis of 661 W photoreceptor cultures. Our findings highlight novel targets involved in the anti-inflammatory and neuroprotective action of resveratrol against neuroinflammatory responses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Entire expressed peripheral blood transcriptome in pediatric severe malarial anemia

Abstract This study on severe malarial anemia (SMA: Hb < 6.0 g/dL), a leading global cause of childhood morbidity and mortality, compares the entire expressed whole blood host transcriptome between Kenyan children (3-48 mos.) with non-SMA (Hb ≥ 6.0 g/dL, n = 39) and SMA ( n = 18). Differential expression analyses reveal 1403 up-regulated and 279 down-regulated transcripts in SMA, signifying impairments in host inflammasome activation, cell death, and innate immune and cellular stress responses. Immune cell profiling shows decreased memory responses, antigen presentation, and immediate pathogen clearance, suggesting an immature/improperly regulated immune response in SMA. Module repertoire analysis of blood-specific gene signatures identifies up-regulation of erythroid genes, enhanced neutrophil activation, and impaired inflammatory responses in SMA. Enrichment analyses converge on disruptions in cellular homeostasis and regulatory pathways for the ubiquitin-proteasome system, autophagy, and heme metabolism. Pathway analyses highlight activation in response to hypoxic conditions [Hypoxia Inducible Factor (HIF)−1 target and Reactive Oxygen Species (ROS) signaling] as a central theme in SMA. These signaling pathways are also top-ranking in protein abundance measures and a Ugandan SMA cohort with available transcriptomic data. Targeted RNA-Seq validation shows strong concordance with our entire expressed transcriptome data. These findings identify key molecular themes in SMA pathogenesis, offering potential targets for new malaria therapies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Analyses of transcriptomes and the first complete genome of Leucocalocybe mongolica provide new insights into phylogenetic relationships and conservation

In this study, we report a de novo assembly of the first high-quality genome for a wild mushroom species Leucocalocybe mongolica (LM). We performed high-throughput transcriptome sequencing to analyze the genetic basis for the life history of LM. Our results show that the genome size of LM is 46.0 Mb, including 26 contigs with a contig N50 size of 3.6 Mb. In total, we predicted 11,599 protein-coding genes, of which 65.7% (7630) could be aligned with high confidence to annotated homologous genes in other species. We performed phylogenetic analyses using genes form 3269 single-copy gene families and showed support for distinguishing LM from the genus Tricholoma (L.) P.Kumm., in which it is sometimes circumscribed. We believe that one reason for limited wild occurrences of LM may be the loss of key metabolic genes, especially carbohydrate-active enzymes (CAZymes), based on comparisons with other closely related species. The results of our transcriptome analyses between vegetative (mycelia) and reproductive (fruiting bodies) organs indicated that changes in gene expression among some key CAZyme genes may help to determine the switch from asexual to sexual reproduction. Taken together, our genomic and transcriptome data for LM comprise a valuable resource for both understanding the evolutionary and life history of this species.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

High-throughput Single-Cell Proteomics and Transcriptomics from the Same Cells with a Nanoliter-Scale Spin-Transfer Approach

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

multi 'omics↗
DOE OSTI2020

Decoding co-/post-transcriptional complexities of plant transcriptomes and epitranscriptome using next-generation sequencing technologies

Next-generation sequencing (NGS) technologies - Illumina RNA-seq, Pacific Biosciences isoform sequencing (PacBio Iso-seq), and Oxford Nanopore direct RNA sequencing (DRS) - have revealed the complexity of plant transcriptomes and their regulation at the co-/post-transcriptional level. Global analysis of mature mRNAs, transcripts from nuclear run-on assays, and nascent chromatin-bound mRNAs using short as well as full-length and single-molecule DRS reads have uncovered potential roles of different forms of RNA polymerase II during the transcription process, and the extent of co-transcriptional pre-mRNA splicing and polyadenylation. These tools have also allowed mapping of transcriptome-wide start sites in cap-containing RNAs, poly(A) site choice, poly(A) tail length, and RNA base modifications. The emerging theme from recent studies is that reprogramming of gene expression in response to developmental cues and stresses at the co-/post-transcriptional level likely plays a crucial role in eliciting appropriate responses for optimal growth and plant survival under adverse conditions. Although the mechanisms by which developmental cues and different stresses regulate co-/post-transcriptional splicing are largely unknown, a few recent studies indicate that the external cues target spliceosomal and splicing regulatory proteins to modulate alternative splicing. In this review, we provide an overview of recent discoveries on the dynamics and complexities of plant transcriptomes, mechanistic insights into splicing regulation, and discuss critical gaps in co-/post-transcriptional research that need to be addressed using diverse genomic and biochemical approaches.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Spatial profiling of the interplay between cell type- and vision-dependent transcriptomic programs in the visual cortex

How early sensory experience during “critical periods” of postnatal life affects the organization of the mammalian neocortex at the resolution of neuronal cell types is poorly understood. We previously reported that the functional and molecular profiles of layer 2/3 (L2/3) cell types in the primary visual cortex (V1) are vision-dependent [S. Chenget al.,Cell185, 311–327.e24 (2022)]. Here, we characterize the spatial organization of L2/3 cell types with and without visual experience. Spatial transcriptomic profiling based on 500 genes recapitulates the zonation of L2/3 cell types along the pial–ventricular axis in V1. By applying multitasking theory, we suggest that the spatial zonation of L2/3 cell types is linked to the continuous nature of their gene expression profiles, which can be represented as a 2D manifold bounded by three archetypal cell types. By comparing normally reared and dark reared L2/3 cells, we show that visual deprivation-induced transcriptomic changes comprise two independent gene programs. The first, induced specifically in the visual cortex, includes immediate-early genes and genes associated with metabolic processes. It manifests as a change in cell state that is orthogonal to cell-type-specific gene expression programs. By contrast, the second program impacts L2/3 cell-type identity, regulating a subset of cell-type-specific genes and shifting the distribution of cells within the L2/3 cell-type manifold. Through an integrated analysis of spatial transcriptomics with single-nucleus RNA-seq data, we describe how vision patterns cortical L2/3 cell types during the critical period.

Science & Technology - Other Topics↗
DOE OSTI2023

Transcriptome and DNA methylome divergence of inflorescence development between 2 ecotypes in Panicum hallii

The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation. Hall's panicgrass (Panicum hallii, P. hallii) is a wild perennial grass that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the 2 major ecotypes in P. hallii, the upland ecotype (P. hallii var hallii, HAL2 genotype) with compact inflorescence and large seed and the lowland ecotype (P. hallii var filipes, FIL2 genotype) with an open inflorescence and small seed. Here we conducted a comparative analysis of the transcriptome and DNA methylome, an epigenetic mark that influences gene expression regulation, across different stages of inflorescence development using genomic references for each ecotype. Global transcriptome analysis of differentially expressed genes (DEGs) and co-expression modules underlying the inflorescence divergence revealed the potential role of cytokinin signaling in heterochronic changes. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of P. hallii inflorescence. We found that a large proportion of differentially methylated regions (DMRs) were located in the flanking regulatory regions of genes. Intriguingly, we observed a substantial bias of CHH hypermethylation in the promoters of FIL2 genes. The integration of DEGs, DMRs, and $K_a$/$K_s$ ratio results characterized the evolutionary features of DMR-associated DEGs that contribute to the divergence of the P. hallii inflorescence. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a genomic resource for perennial grass biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Transcriptome and secretome profiling of sensory neurons reveals sex differences in pathways relevant to insulin sensing and insulin secretion

Sensory neurons in the dorsal root ganglia (DRG) convey somatosensory and metabolic cues to the central nervous system and release substances from stimulated terminal endings in peripheral organs. Sex-biased variations driven by the sex chromosome complement (XX and XY) have been implicated in the sensory–islet crosstalk. However, the molecular underpinnings of these male–female differences are not known. Here, we aim to characterize the molecular repertoire and the secretome profile of the lower thoracic spinal sensory neurons and to identify molecules with sex-biased insulin sensing- and/or insulin secretion-modulating activity that are encoded independently of circulating gonadal sex hormones. We used transcriptomics and proteomics to uncover differentially expressed genes and secreted molecules in lower thoracic T5-12 DRG sensory neurons derived from sexually immature 3-week-old male and female C57BL/6J mice. Comparative transcriptome and proteome analyses revealed differential gene expression and protein secretion in DRG neurons in males and females. Furthermore, the transcriptome analysis identified, among others, higher insulin signaling/sensing capabilities in female DRG neurons; secretome screening uncovered several sex-specific candidate molecules with potential regulatory functions in pancreatic β cells. Together, these data suggest a putative role of sensory interoception of insulin in the DRG–islet crosstalk with implications in sensory feedback loops in the regulation of β-cell activity in a sex-biased manner. Finally, we provide a valuable resource of molecular and secretory targets that can be leveraged for understanding insulin interoception and insulin secretion and inform the development of novel studies/approaches to fathom the role of the sensory–islet axis in the regulation of energy balance in males and females.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Nutri‐cereal tissue‐specific transcriptome atlas during development: Functional integration of gene expression to identify mineral uptake pathways in little millet ( Panicum sumatrense )

SUMMARY Little millet (Panicum sumatrenseRoth ex Roem. & Schult.) is an essential minor millet of southeast Asia and Africa's temperate and subtropical regions. The plant is stress‐tolerant, has a short life cycle, and has a mineral‐rich nutritional profile associated with unique health benefits. We report the developmental gene expression atlas of little millet (genotype JK‐8) from ten tissues representing different stages of its life cycle, starting from seed germination and vegetative growth to panicle maturation. The developmental transcriptome atlas led to the identification of 342 827 transcripts. The BUSCO analysis and comparison with the transcriptomes of related species confirm that this study presents high‐quality, in‐depth coverage of the little millet transcriptome. In addition, the eFP browser generated here has a user‐friendly interface, allowing interactive visualizations of tissue‐specific gene expression. Using these data, we identified transcripts, the orthologs of which inArabidopsisand rice are involved in nutrient acquisition, transport, and response pathways. The comparative analysis of the expression levels of these transcripts holds great potential for enhancing the mineral content in crops, particularly zinc and iron, to address the issue of “hidden hunger” and to attain nutritional security, making it a valuable asset for translational research.

Plant Sciences↗
DOE OSTI2025

Homoeolog expression divergence contributes to time of day changes in transcriptomic and glucosinolate responses to prolonged water limitation in Brassica napus

SUMMARY Water availability is a major determinant of crop production, and rising temperatures from climate change are leading to more extreme droughts. To combat the effects of climate change on crop yields, we need to develop varieties that are more tolerant to water‐limited conditions. We aimed to determine how diverse crop types (winter/spring oilseed, tuberous, and leafy) of the allopolyploid Brassica napus , a species that contains the economically important rapeseed oilseed crop, respond to prolonged water limitation. We exposed plants to an 80% reduction in water and assessed growth and color on a high‐throughput phenotyping system over 4 weeks and ended the experiment with tissue collection for a time course transcriptomic study. We found an overall reduction in growth across cultivars but to varying degrees. Diel transcriptome analyses revealed significant accession‐specific changes in time‐of‐day regulation of photosynthesis, carbohydrate metabolism, and sulfur metabolism. Interestingly, there was extensive variation in which homoeologs from the two parental subgenomes responded to water limitation across crop types that could be due to differences in regulatory regions in these allopolyploid lines. Follow‐up experiments on select cultivars confirmed that plants maintained photosynthetic health during the prolonged water limitation while slowing growth. In two cultivars examined, we found significant time of day changes in levels of glucosinolates, sulfur‐ and nitrogen ‐rich specialized metabolites, consistent with the diel transcriptomic responses. These results suggest that these lines are adjusting their sulfur and nitrogen stores under water‐limited conditions through distinct time of day regulation.

Ricono, Angela [University of Minnesota St. Paul M↗
DOE OSTI2026

A tissue‐resolved, network‐based transcriptomic framework for abiotic stress responses in sorghum

Developing climate‐resilient crops requires a detailed understanding of stress‐induced gene expression dynamics, as maladaptive responses can compromise their productivity and survival. Sorghum, a globally important cereal with exceptional tolerance to multiple abiotic stresses, provides a powerful system for investigating these dynamics. However, how stress type, tissue specificity, and temporal progression jointly shape transcriptomic responses in crops remains poorly understood. Here, we present a comparative, time‐resolved transcriptomic atlas of sorghum responses to drought, heat, and salinity stress across shoot and root tissues. Integrative analyses revealed that tissue specificity is the dominant determinant of abiotic stress‐induced gene reprogramming across all three stresses. Building on these global comparisons, we focused on heat stress, as it elicited the most coherent and pronounced transcriptional and regulatory responses, enabling deeper network‐level interrogation. Co‐expression network analysis identified tissue‐specific modules enriched for phytohormone‐responsive genes, while gene regulatory network (GRN) mapping and cistrome analyses uncovered transcription factors (TFs) controlling key hub genes within these modules. Together, this study provides a foundational transcriptomic and network‐based resource for dissecting the regulatory architecture of abiotic stress responses in sorghum and offers prioritized candidates for future functional validation and engineering of climate‐resilient crops.

abiotic stress↗
DOE OSTI2023

A transcriptomic atlas of acute stress response to low pH in multiple Issatchenkia orientalis strains

Because of its natural stress tolerance to low pH, Issatchenkia orientalis (a.k.a. Pichia kudriavzevii) is a promising non-model yeast for bio-based production of organic acids. Yet, this organism is relatively unstudied, and specific mechanisms of its tolerance to low pH are poorly understood, limiting commercial use. In this study, we selected 12 I. orientalis strains with varying acid stress tolerance (six tolerant and six susceptible) and profiled their transcriptomes in different pH conditions to study potential mechanisms of pH tolerance in this species. We identified hundreds of genes whose expression response is shared by tolerant strains but not by susceptible strains, or vice versa, as well as genes whose responses are reversed between tolerant and susceptible strains. We mapped regulatory mechanisms of transcriptomic responses via motif analysis as well as differential network reconstruction, identifying several transcription factors, including Stb5, Mac1, and Rtg1/Rtg3, some of which are known for their roles in acid response in Saccharomyces cerevisiae. Functional genomics analysis of short-listed genes and transcription factors suggested significant roles for energy metabolism and translation-related processes, as well as the cell wall integrity pathway and RTG-dependent retrograde signaling pathway. Finally, we conducted additional experiments for two organic acids, 3-hydroxypropionate and citramalate, to eliminate acid-specific effects and found potential roles for glycolysis and trehalose biosynthesis specifically for response to low pH. In summary, our approach of comparative transcriptomics and phenotypic contrasting, along with a multi-pronged bioinformatics analysis, suggests specific mechanisms of tolerance to low pH in I. orientalis that merit further validation through experimental perturbation and engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of 56 Fe, 16 O, and 28 Si irradiation on the induction of murine hepatocellular carcinoma

One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC. We performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n 56 Fe (0.2 Gy), 1 GeV/n 16 O (0.2 Gy), and 350 MeV/n 28 Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes (e.g., Acute Phase Response Signaling, B Cell Receptor Signaling, IL-3 Signaling), and RNA Transcription/Expression. A large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Linking crop traits to transcriptome differences in a progeny population of tetraploid potato

Background: Potato is the third most consumed crop in the world. Breeding for traits such as yield, product quality and pathogen resistance are main priorities. Identifying molecular signatures of these and other important traits is important in future breeding efforts. In this study, a progeny population from a cross between a breeding line, SW93–1015, and a cultivar, Désirée, was studied by trait analysis and RNA-seq in order to develop understanding of segregating traits at the molecular level and identify transcripts with expressional correlation to these traits. Transcript markers with predictive value for field performance applicable under controlled environments would be of great value for plant breeding.Results: A total of 34 progeny lines from SW93–1015 and Désirée were phenotyped for 17 different traits in a field in Nordic climate conditions and controlled climate settings. A master transcriptome was constructed with all 34 progeny lines and the parents through a de novo assembly of RNA-seq reads. Gene expression data obtained in a controlled environment from the 34 lines was correlated to traits by different similarity indices, including Pearson and Spearman, as well as DUO, which calculates the co-occurrence between high and low values for gene expression and trait. Our study linked transcripts to traits such as yield, growth rate, high laying tubers, late and tuber blight, tuber greening and early flowering. We found several transcripts associated to late blight resistance and transcripts encoding receptors were associated to Dickeya solani susceptibility. Transcript levels of a UBX-domain protein was negatively associated to yield and a GLABRA2 expression modulator was negatively associated to growth rate.Conclusion: In our study, we identify 100’s of transcripts, putatively linked based on expression with 17 traits of potato, representing both well-known and novel associations. This approach can be used to link the transcriptome to traits. We explore the possibility of associating the level of transcript expression from controlled, optimal environments to traits in a progeny population with different methods introducing the application of DUO for the first time on transcriptome data. We verify the expression pattern for five of the putative transcript markers in another progeny population.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

transcription start and end sites↗
DOE OSTI2020

Assessing the Ability of Developmentally Precocious Estrogen Signaling to Recapitulate Ovarian Transcriptomes and Follicle Dynamics in Alligators from a Contaminated Lake

Concern has grown in recent decades over anthropogenic contaminants that interfere with the functioning of endocrine hormones. However, mechanisms connecting developmental processes to pathologies associated with endocrine-disrupting chemical (EDC) exposure are poorly understood in naturally exposed populations. We sought to a) characterize divergence in ovarian transcriptomic and follicular profiles between alligators originating from a historically EDC-contaminated site, Lake Apopka, and a reference site; b) test the ability of developmentally precocious estrogen exposure to recapitulate site-associated patterns of divergence; and c) test whether treatment with exogenous follicle-stimulating hormone (FSH) is capable of rescuing phenotypes associated with contaminant exposure and/or embryonic estrogen treatment. Alligators eggs were collected from a contaminated site and a reference site, and a subset of eggs from the reference site were treated with estradiol (E2) during embryonic development prior to gonadal differentiation. After hatching, alligators were raised under controlled laboratory settings for 5 months. Juveniles from both sites were divided and treated with exogenous FSH. Histological analyses and RNA-sequencing were conducted to characterize divergence in ovarian follicle dynamics and transcriptomes between sites, between reference and E2-treated animals, and between FSH-treated and nontreated animals. We observed broad site-of-origin divergence in ovarian transcriptomes and reductions in ovarian follicle density between juvenile alligators from Lake Apopka and the reference site. Treating embryos from the reference site with E2 overwhelmingly recapitulated transcriptional and histological alterations observed in Lake Apopka juveniles. Ovarian phenotypes observed in Lake Apopka alligators or resulting from estrogen treatment were only partially rescued by treatment with exogenous FSH. Recapitulation of ovarian abnormalities by precocious E2 revealed a relatively simple mechanism underlying contaminant-induced pathologies in a historical example of environmental endocrine disruption. Findings reported here support a model where the developmental timing of estrogen signaling has the potential to permanently alter ovarian organization and function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative transcriptome analysis of rainbow trout gonadal cells (RTG-2) infected with U and J genogroup infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is the causative pathogen of infectious hematopoietic necrosis, outbreaks of which are responsible for significant losses in rainbow trout aquaculture. Strains of IHNV isolated worldwide have been classified into five major genogroups, J, E, L, M, and U. To date, comparative transcriptomic analysis has only been conducted individually for the J and M genogroups. In this study, we compared the transcriptome profiles in U genogroup and J genogroup IHNV-infected RTG-2 cells with mock-infected RTG-2 cells. The RNA-seq results revealed 17,064 new genes, of which 7,390 genes were functionally annotated. Differentially expressed gene (DEG) analysis between U and J IHNV-infected cells revealed 2,238 DEGs, including 1,011 downregulated genes and 1,227 upregulated genes. Among the 2,238 DEGs, 345 new genes were discovered. The DEGs related to immune responses, cellular signal transduction, and viral diseases were further analyzed. RT-qPCR validation confirmed that the changes in expression of the immune response-related genes trpm2 , sting , itgb7 , ripk2 , and irf1 , cellular signal transduction-related genes irl , cacnb2 , bmp2l , gadd45α , and plk2 , and viral disease-related genes mlf1, mtor, armc5 , pik3r1 , and c-myc were consistent with the results of transcriptome analysis. Taken together, our findings provide a comprehensive transcriptional analysis of the differential virulence of the U and J genogroups of IHNV, and shed new light on the pathogenic mechanisms of IHNV strains.

Zhao, Jing-Zhuang↗