Nuclear Transcriptome Research Articles

Effects of oxycodone on placental lineages: Evidence from the transcriptome profile of mouse trophoblast giant cells

Pregnant women are often prescribed or abuse opioid drugs. The placenta is likely the key to understanding how opioids cause adverse pregnancy outcomes. Maternal oxycodone (OXY) exposure of pregnant mice leads to disturbances in the layer of invasive parietal trophoblast giant cells (pTGC) that forms the interface between the placenta and uterus. These cells are analogous to extravillous trophoblasts of the human placenta. They are crucial to coordinating the metabolic needs of the conceptus with those of the mother and are primary participants in the placenta-brain axis. Their large nuclear size, however, has precluded both single-cell (sc) and single-nucleus (sn) RNA-seq analyses beyond embryonic day (E) 8.5. Here, we compared the transcriptomes of placentas from pregnant mice exposed to OXY with unexposed controls at E12.5, with particular emphasis on the pTGC. The nonfluidic Parse snRNA-seq approach permitted characterization of the nuclear transcriptomes of all the major placental cell lineages and their presumed progenitors at E12.5. OXY exposure had a negligible effect on components of the placental labyrinth, including the two syncytial cell layers, but caused transcriptomic changes consistent with metabolic stress throughout the spongiotrophoblast. Most notably, there was loss of the majority of pTGC, whose normal gene expression is consistent with elevated energy demand relating to biosynthesis of multiple secretory products, especially hormones, and endoduplication of DNA. This unusual sensitivity of pTGC presumably puts the pregnancy and future health of the offspring at particular risk to OXY exposure.

Single-nucleus RNA sequencing reveals cell types, genes, and regulatory factors influencing melanogenesis in the breast muscle of Xuefeng black-bone chicken

The black-bone chicken, known for its high melanin content, holds significant economic value due to this unique trait. Particularly notable is the prominent melanin deposition observed in its breast muscle. However, the molecular mechanisms governing melanin synthesis and deposition in the breast muscle of black-bone chickens remain largely unknown. This study employed a single-nucleus transcriptome assay to identify genes associated with melanin deposition in the breast muscle of black-bone chickens, which are presumed to influence pigmentation levels. A comprehensive analysis of the nuclear transcriptome was conducted on the breast muscle of Xuefeng black-bone chickens, encompassing 18 distinct cell types, including melanocytes. Our findings revealed that STIMATE, LRRC7, ENSGALG00000049990, and GLDC play pivotal regulatory roles in melanin deposition within the breast muscle. Further exploration into the molecular mechanisms unveiled transcription factors and protein interactions suggesting that RARB, KLF15, and PRDM4 may be crucial regulators of melanin accumulation in the breast muscle. Additionally, HPGDS, GSTO1, and CYP1B1 may modulate melanin production and deposition in the breast muscle by influencing melanocyte metabolism. Our findings also suggest that melanocyte function in the breast muscle may be intertwined with intercellular signaling pathways such as PTPRK-WNT5A, NOTCH1-JAG1, IGF1R-IGF1, IDE-GCG, and ROR2-WNT5A. Leveraging advanced snRNA-seq technology, we generated a comprehensive single-cell nuclear transcriptome atlas of the breast muscle of Xuefeng black-bone chickens. This facilitated the identification of candidate genes, regulatory factors, and cellular signals potentially influencing melanin deposition and melanocyte function. Overall, our study provides crucial insights into the molecular basis of melanin deposition in chicken breast muscle, laying the groundwork for future breeding programs aimed at enhancing black-bone chicken cultivation.

Single-cell and single-nucleus RNA-sequencing from paired normal-adenocarcinoma lung samples provide both common and discordant biological insights.

Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be context-dependent. Herein, a head-to-head comparison was performed in matched normal-adenocarcinoma human lung samples to assess biological insights derived from scRNA-seq versus snRNA-seq and better understand the cellular transition that occurs from normal to tumoral tissue. Here, the transcriptome of 160,621 cells/nuclei was obtained. In non-tumor lung, cell type proportions varied widely between scRNA-seq and snRNA-seq with a predominance of immune cells in the former (81.5%) and epithelial cells (69.9%) in the later. Similar results were observed in adenocarcinomas, in addition to an overall increase in cell type heterogeneity and a greater prevalence of copy number variants in cells of epithelial origin, which suggests malignant assignment. The cell type transition that occurs from normal lung tissue to adenocarcinoma was not always concordant whether cells or nuclei were examined. As expected, large differential expression of the whole-cell and nuclear transcriptome was observed, but cell-type specific changes of paired normal and tumor lung samples revealed a set of common genes in the cells and nuclei involved in cancer-related pathways. In addition, we showed that the ligand-receptor interactome landscape of lung adenocarcinoma was largely different whether cells or nuclei were evaluated. Immune cell depletion in fresh specimens partly mitigated the difference in cell type composition observed between cells and nuclei. However, the extra manipulations affected cell viability and amplified the transcriptional signatures associated with stress responses. In conclusion, research applications focussing on mapping the immune landscape of lung adenocarcinoma benefit from scRNA-seq in fresh samples, whereas snRNA-seq of frozen samples provide a low-cost alternative to profile more epithelial and cancer cells, and yield cell type proportions that more closely match tissue content.

Identification of core genes of craniopharyngioma angiogenesis based on single-cell nuclear transcriptome sequencing.

This study aimed to explore the core genes of craniopharyngioma angiogenesis for targeted vascular therapy based on single-cell nuclear transcriptome sequencing. For single-cell nuclear transcriptome sequencing, we collected six samples from the tumor center and adjacent hypothalamic tumor tissues from three patients with craniopharyngioma, as well as four normal brain tissues based on Gene Expression Omnibus. We screened genes with differential up-regulation between vascular endothelial cells of craniopharyngioma and those of normal brain tissues, performed GO and KEGG analysis, constructed the protein-protein interaction network, and selected key genes verified using immunofluorescence. After data cleaning and quality control, 623 craniopharyngioma endothelial cells and 439 healthy brain endothelial cells were obtained. Compared with normal brain endothelial cells, craniopharyngioma endothelial cells were screened for 394 differentially up-expressed genes (DEGs). GO and KEGG results showed that DEGs probably modulated endothelial cells, adherens junction, focal adhesion, migration, actin cytoskeleton, and invasion via the PI3K-AKT, Rap1, Ras, Wnt, and Hippo pathways. The core genes screened were CTNNB1, PTK2, ITGB1, STAT3, FYN, HIF1A, VCL, SMAD3, PECAM1, FOS, and CDH5. This study obtained possible anti-angiogenic genes in craniopharyngioma. Our results shed novel insights into molecular mechanisms and craniopharyngioma treatment.

Unraveling the mechanisms of NK cell dysfunction in aging and Alzheimer's disease: insights from GWAS and single-cell transcriptomics.

Aging is an important factor in the development of Alzheimer's disease (AD). The senescent cells can be recognized and removed by NK cells. However, NK cell function is gradually inactivated with age. Therefore, this study used senescence as an entry point to investigate how NK cells affect AD. The study validated the correlation between cognition and aging through a prospective cohort of the National Health and Nutrition Examination Survey database. A cellular trajectory analysis of the aging population was performed using single-cell nuclear transcriptome sequencing data from patients with AD and different ages. The genome-wide association study (GWAS) cohort of AD patients was used as the outcome event, and the expression quantitative trait locus was used as an instrumental variable. Causal associations between genes and AD were analyzed by bidirectional Mendelian randomization (MR) and co-localization. Finally, clinical cohorts were constructed to validate the expression of key genes. A correlation between cognition and aging was demonstrated using 2,171 older adults over 60 years of age. Gene regulation analysis revealed that most of the highly active transcription factors were concentrated in the NK cell subpopulation of AD. NK cell trajectories were constructed for different age populations. MR and co-localization analyses revealed that CHD6 may be one of the factors influencing AD. We explored different levels of AD and aging from population cohorts, single-cell data, and GWAS cohorts and found that there may be some correlations of NK cells between aging and AD. It also provides some basis for potential causation.

Cellular and molecular mechanisms of highly active mesenchymal stem cells in the treatment of senescence of rhesus monkey ovary

BackgroundRecent studies have shown that umbilical cord mesenchymal stem cells have an anti-aging effect in ovaries, but the cellular and molecular mechanisms of HA-MSC ovarian anti-aging remain to be studied. Therefore, we conducted a 10X Genomics single-nucleus transcriptome sequencing experiment on the ovaries of macaque monkeys after HA-MSC treatment.MethodsThe results of cell subgroup classification were visualized by 10X Genomics single nuclear transcriptome sequencing. The aging model of hGCs was established, and the migration ability of the cells was determined after coculture of HA-MSCs and aging hGCs. The genes screened by single nuclear transcriptional sequencing were verified in vitro by qPCR.ResultsCompared with the aging model group, the number of cell receptor pairs in each subgroup of the HA-MSC-treated group increased overall. Treatment with 200 μmol/L H2O2 for 48 h was used as the optimum condition for the induction of hGC senescence. After coculture of noncontact HA-MSCs with senescent hGCs, it was found that HA-MSCs can reverse the cell structure, proliferation ability, senescence condition, expression level of senescence-related genes, and expression level of key genes regulating the senescence pathway in normal hGCs.ConclusionsHA-MSC therapy can improve the tissue structure and secretion function of the ovary through multiple cellular and molecular mechanisms to resist ovarian aging. In vitro validation experiments further supported the results of single-cell sequencing, which provides evidence supporting a new option for stem cell treatment of ovarian senescence.

A High-Efficiency Capture-Based NGS Approach for Comprehensive Analysis of Mitochondrial Transcriptome.

The transcription of the mitochondrial genome is pivotal for maintenance of mitochondrial functions, and the deregulated mitochondrial transcriptome contributes to various pathological changes. Despite substantial progress having been achieved in uncovering the transcriptional complexity of the nuclear transcriptome, many unknowns and controversies remain for the mitochondrial transcriptome, partially owing to the lack of a highly efficient mitochondrial RNA (mtRNA) sequencing and analysis approach. Here, we first comprehensively evaluated the influence of essential experimental protocols, including strand-specific library construction, two RNA enrichment strategies, and optimal rRNA depletion, on accurately profiling mitochondrial transcriptome in whole-transcriptome sequencing (WTS) data. Based on these insights, we developed a highly efficient approach specifically suitable for targeted sequencing of whole mitochondrial transcriptome, termed capture-based mtRNA seq (CAP), in which strand-specific library construction and optimal rRNA depletion were applied. Compared with WTS, CAP has a great decrease of required data volume without affecting the sensitivity and accuracy of detection. In addition, CAP also characterized the unannotated mt-tRNA transcripts whose expression levels are below the detection limits of conventional WTS. As a proof-of-concept characterization of mtRNAs, the transcription initiation sites and mtRNA cleavage ratio were accurately identified in CAP data. Moreover, CAP had very reliable performance in plasma and single-cell samples, highlighting its wide application. Altogether, the present study has established a highly efficient pipeline for targeted sequencing of mtRNAs, which may pave the way toward functional annotation of mtRNAs and mtRNA-based diagnostic and therapeutic strategies in various diseases.

Transcriptomic profiling of nuclei from paraformaldehyde-fixed and formalin-fixed paraffin-embedded brain tissues

BackgroundParaformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens. Single-cell gene expression workflows have recently been developed for PFA-fixed and FFPE tissue specimens. However, for tissues where intact cells are hard to recover, including tissues containing highly interconnected neurons, single-nuclear transcriptomics is beneficial. Moreover, since RNA is very unstable, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens like FFPE samples are largely anecdotal. ResultsWe evaluated the effects of polyformaldehyde (PFA) fixation and paraffin-embedding on transcriptional profiles of the mouse hippocampus obtained by RNA sequencing (RNA-seq). The transcriptomic signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were comparable to those of cryopreserved samples. However, more differentially expressed genes were obtained for brains after PFA fixation for more than 3 days than in fresh PFA-fixed samples, especially genes involved in spliceosome and synaptic-related pathways. Importantly, the real cell states were destroyed, with oligodendrocyte precursor cells depleted in the 1day fixed hippocampus. After fixation for 3 days, the proportions of neuronal cells and oligodendrocytes decreased and microglia increased; however, relative frequencies remained constant for longer fixation durations. The storage time of FFPE samples had a negligible effect on the cell composition. SignificanceThis represents the first work to investigate the effects of fixation and storage time of brains on its nuclear transcriptome signatures in detail. The fixation time had more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1–3 days. These findings are expected to guide sample preparation for single-nucleus RNA-seq of FFPE samples, particularly in transcriptomic studies focused on brain diseases.

Mitochondrially mediated RNA interference, a retrograde signaling system affecting nuclear gene expression.

Several functional classes of short noncoding RNAs are involved in manifold regulatory processes in eukaryotes, including, among the best characterized, miRNAs. One of the most intriguing regulatory networks in the eukaryotic cell is the mito-nuclear crosstalk: recently, miRNA-like elements of mitochondrial origin, called smithRNAs, were detected in a bivalve species, Ruditapes philippinarum. These RNA molecules originate in the organelle but were shown in vivo to regulate nuclear genes. Since miRNA genes evolve easily de novo with respect to protein-coding genes, in the present work we estimate the probability with which a newly arisen smithRNA finds a suitable target in the nuclear transcriptome. Simulations with transcriptomes of 12 bivalve species suggest that this probability is high and not species specific: one in a hundred million (1 × 10-8) if five mismatches between the smithRNA and the 3' mRNA are allowed, yet many more are allowed in animals. We propose that novel smithRNAs may easily evolve as exaptation of the pre-existing mitochondrial RNAs. In turn, the ability of evolving novel smithRNAs may have played a pivotal role in mito-nuclear interactions during animal evolution, including the intriguing possibility of acting as speciation trigger.

Response of the organellar and nuclear (post)transcriptomes of Arabidopsis to drought.

Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used RNA-sequencing after ribosomal RNA depletion to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0. Concerning the changes detected in the organellar transcriptomes, chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Conversely, alternative splicing (AS) affected nearly 1,500 genes (9% of expressed nuclear genes). Of these, 42% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Moreover, we identified 927 isoform switching events. We provide a table of the most interesting candidates, and as proof of principle, increased drought tolerance of the carbonic anhydrase ca1 and ca2 mutants is shown. In addition, altering the relative contributions of the spliced isoforms could increase drought resistance. For example, our data suggest that the accumulation of a nonfunctional FLM (FLOWERING LOCUS M) isoform and not the ratio of FLM-ß and -δ isoforms may be responsible for the phenotype of early flowering under long-day drought conditions. In sum, our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought.

Multi-stage nuclear transcriptomic insights of morphogenesis and biparental role changes in Lentinula edodes.

Based on six offspring with different mitochondrial (M) and parental nuclear (N) genotypes, the multi-stage morphological characteristics and nuclear transcriptomes of Lentinula edodes were compared to investigate morphogenesis mechanisms during cultivation, the key reason for cultivar resistance to genotype changes, and regulation related to biparental role changes. Six offspring had specific transcriptomic data and morphological characteristics that were mainly regulated by the two parental nuclei, followed by the cytoplasm, at different growth stages. Importing a wild N genotype easily leads to failure or instability of fruiting; however, importing wild M genotypes may improve cultivars. Major facilitator superfamily (MFS) transporter genes encoding specific metabolites in spawns may play crucial roles in fruiting body formation. Pellets from submerged cultivation and spawns from sawdust substrate cultivation showed different carbon metabolic pathways, especially in secondary metabolism, degradation of lignin, cellulose and hemicellulose, and plasma membrane transport (mainly MFS). When the stage of small young pileus (SYP) was formed on the surface of the bag, the spawns inside were mainly involved in nutrient accumulation. Just broken pileus (JBP) showed a different expression of plasma membrane transporter genes related to intracellular material transport compared to SYP and showed different ribosomal proteins and cytochrome P450 functioning in protein biosynthesis and metabolism than near spreading pileus (NSP). Biparental roles mainly regulate offspring metabolism, growth, and morphogenesis by differentially expressing specific genes during different vegetative growth stages. Additionally, some genes encoding glycine-rich RNA-binding proteins, F-box, and folliculin-interacting protein repeat-containing proteins may be related to multi-stage morphogenesis. KEY POINTS: • Replacement of nuclear genotype is not suitable for cultivar breeding of L. edodes. • Some genes show a biparental role-divergent expression at mycelial growth stage. • Transcriptomic changes of some sawdust substrate cultivation stages have been elucidated.

Regulation of alternative splicing by retrograde and light signals converges to control chloroplast proteins.

Retrograde signals sent by chloroplasts control transcription in the nucleus. These signals antagonistically converge with light signals to coordinate the expression of genes involved in chloroplast functioning and seedling development. Although significant advances have been made in understanding the molecular interplay between light and retrograde signals at the transcriptional level, little is known about their interconnection at the post-transcriptional level. By using different publicly available datasets, this study addresses the influence of retrograde signaling on alternative splicing and defines the molecular and biological functions of this regulation. These analyses revealed that alternative splicing mimics transcriptional responses triggered by retrograde signals at different levels. First, both molecular processes similarly depend on the chloroplast-localized pentatricopeptide-repeat protein GUN1 to modulate the nuclear transcriptome. Secondly, as described for transcriptional regulation, alternative splicing coupled with the nonsense-mediated decay pathway effectively downregulates expression of chloroplast proteins in response to retrograde signals. Finally, light signals were found to antagonistically control retrograde signaling-regulated splicing isoforms, which consequently generates opposite splicing outcomes that likely contribute to the opposite roles these signals play in controlling chloroplast functioning and seedling development.

Single-Cell Joint Profiling of Open Chromatin and Transcriptome by Paired-Seq.

Simultaneous detection of chromatin accessibility and transcription from the same cells promises to greatly facilitate the dissection of cell-type-specific gene regulatory programs in complex tissues. Paired-seq enables joint analysis of open chromatin and nuclear transcriptome from up to a million cells in parallel. It achieves ultra-high-throughput single-cell multiomics with the use of a combinatorial barcoding strategy involving sequential ligation of multiplexed DNA barcodes to chromatin DNA fragments and reverse transcription products, followed by high-throughput DNA sequencing of the resulting DNA libraries and deconvolution of single-cell multiomic maps based on cell-specific barcodes.

LAFITE Reveals the Complexity of Transcript Isoforms in Subcellular Fractions.

Characterization of the subcellular distribution of RNA is essential for understanding the molecular basis of biological processes. Here, the subcellular nanopore direct RNA-sequencing (DRS) of four lung cancer cell lines (A549, H1975, H358, and HCC4006) is performed, coupled with a computational pipeline, Low-abundance Aware Full-length Isoform clusTEr (LAFITE), to comprehensively analyze the full-length cytoplasmic and nuclear transcriptome. Using additional DRS and orthogonal data sets, it is shown that LAFITE outperforms current methods for detecting full-length transcripts, particularly for low-abundance isoforms that are usually overlooked due to poor read coverage. Experimental validation of six novel isoforms exclusively identified by LAFITE further confirms the reliability of this pipeline. By applying LAFITE to subcellular DRS data, the complexity of the nuclear transcriptome is revealed in terms of isoform diversity, 3'-UTR usage, m6A modification patterns, and intron retention. Overall, LAFITE provides enhanced full-length isoform identification and enables a high-resolution view of the RNA landscape at the isoform level.

Gene loss, genome rearrangement, and accelerated substitution rates in plastid genome of Hypericum ascyron (Hypericaceae)

BackgroundComparative genomic analysis exhibits dynamic evolution of plastid genome (plastome) in the clusioid clade of Malpighiales, which comprise five families, including multiple inversions and gene losses. Little is known about the plastome evolution in Hypericaceae, a large family in the clade. Only the plastome of one species, Cratoxylum cochinchinense, has been published.ResultsWe generated a complete plastome sequence for Hypericum ascyron, providing the first complete plastome from the tribe Hypericeae (Hypericaceae). The H. ascyron plastome exhibits dynamic changes in gene and intron content, structure, and sequence divergence compared to the C. cochinchinense plastome from the tribe Cratoxyleae (Hypericaceae). Transcriptome data determined the evolutionary fate of the missing plastid genes infA, rps7, rps16, rpl23, and rpl32 in H. ascyron. Putative functional transfers of infA, rps7, and rpl32 were detected to the nucleus, whereas rps16 and rpl23 were substituted by nuclear-encoded homologs. The plastid rpl32 was integrated into the nuclear-encoded SODcp gene. Our findings suggested that the transferred rpl32 had undergone subfunctionalization by duplication rather than alternative splicing. The H. ascyron plastome rearrangements involved seven inversions, at least three inverted repeat (IR) boundary shifts, which generated gene relocations and duplications. Accelerated substitution rates of plastid genes were observed in the H. ascyron plastome compared with that of C. cochinchinense plastid genes. The higher substitution rates in the accD and clpP were correlated with structural change, including a large insertion of amino acids and losses of two introns, respectively. In addition, we found evidence of positive selection of the clpP, matK, and rps3 genes in the three branches related to H. ascyron. In particular, the matK gene was repeatedly under selection within the family Hypericaceae. Selective pressure in the H. ascyron matK gene was associated with the loss of trnK-UUU and relocation into the IR region.ConclusionsThe Hypericum ascyron plastome sequence provides valuable information for improving the understanding of plastome evolution among the clusioid of the Malpighiales. Evidence for intracellular gene transfer from the plastid to the nucleus was detected in the nuclear transcriptome, providing insight into the evolutionary fate of plastid genes in Hypericaceae.

Characterization of transcript enrichment and detection bias in single-nucleus RNA-seq for mapping of distinct human adipocyte lineages.

Single-cell RNA sequencing (scRNA-seq) enables molecular characterization of complex biological tissues at high resolution. The requirement of single-cell extraction, however, makes it challenging for profiling tissues such as adipose tissue, for which collection of intact single adipocytes is complicated by their fragile nature. For such tissues, single-nucleus extraction is often much more efficient and therefore single-nucleus RNA sequencing (snRNA-seq) presents an alternative to scRNA-seq. However, nuclear transcripts represent only a fraction of the transcriptome in a single cell, with snRNA-seq marked with inherent transcript enrichment and detection biases. Therefore, snRNA-seq may be inadequate for mapping important transcriptional signatures in adipose tissue. In this study, we compare the transcriptomic landscape of single nuclei isolated from preadipocytes and mature adipocytes across human white and brown adipocyte lineages, with whole-cell transcriptome. We show that snRNA-seq is capable of identifying the broad cell types present in scRNA-seq at all states of adipogenesis. However, we also explore how and why the nuclear transcriptome is biased and limited, as well as how it can be advantageous. We robustly characterize the enrichment of nuclear-localized transcripts and adipogenic regulatory lncRNAs in snRNA-seq, while also providing a detailed understanding for the preferential detection of long genes upon using this technique. To remove such technical detection biases, we propose a normalization strategy for a more accurate comparison of nuclear and cellular data. Finally, we show successful integration of scRNA-seq and snRNA-seq data sets with existing bioinformatic tools. Overall, our results illustrate the applicability of snRNA-seq for the characterization of cellular diversity in the adipose tissue.

A survey of RNA editing at single-cell resolution links interneurons to schizophrenia and autism.

Conversion of adenosine to inosine in RNA by ADAR enzymes, termed “RNA editing,” is essential for healthy brain development. Editing is dysregulated in neuropsychiatric diseases, but has not yet been investigated at scale at the level of individual neurons. We quantified RNA editing sites in nuclear transcriptomes of 3055 neurons from six cortical regions of a neurotypical female donor, and found 41,930 sites present in at least ten nuclei. Most sites were located within Alu repeats in introns or 3′ UTRs, and approximately 80% were cataloged in public RNA editing databases. We identified 9285 putative novel editing sites, 29% of which were also detectable in unrelated donors. Intersection with results from bulk RNA-seq studies provided cell-type and spatial context for 1730 sites that are differentially edited in schizophrenic brain donors, and 910 such sites in autistic donors. Autism-related genes were also enriched with editing sites predicted to modify RNA structure. Inhibitory neurons showed higher overall transcriptome editing than excitatory neurons, and the highest editing rates were observed in the frontal cortex. We used generalized linear models to identify differentially edited sites and genes between cell types. Twenty nine genes were preferentially edited in excitatory neurons, and 43 genes were edited more heavily in inhibitory neurons, including RBFOX1, its target genes, and genes in the autism-associated Prader–Willi locus (15q11). The abundance of SNORD115/116 genes from locus 15q11 was positively associated with editing activity across the transcriptome. We contend that insufficient editing of autism-related genes in inhibitory neurons may contribute to the specific perturbation of those cells in autism.

Single-nucleus RNA-seq2 reveals functional crosstalk between liver zonation and ploidy

Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity, we have developed a single-nucleus RNA-seq2 method tailored for the comprehensive analysis of the nuclear transcriptome from frozen tissues, allowing the dissection of all cell types present in the liver, regardless of cell size or cellular fragility. We use this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that ploidy states are associated with different metabolic potential, and gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobule. Our work reveals a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Polymorphism in the Chloroplast ATP Synthase Beta-Subunit Is Associated with a Maternally Inherited Enhanced Cold Recovery in Cucumber.

Cucumber (Cucumis sativus L.) is a warm-season crop that is sensitive to chilling temperatures and a maternally inherited cold tolerance exists in the heirloom cultivar ‘Chipper’ (CH). Because the organelles of cucumber show differential transmission (maternal for chloroplast and paternal for mitochondrion), this cold tolerance is hypothesized to be chloroplast-associated. The goal of this research was to characterize the cold tolerant phenotype from CH and determine its genetic basis. Doubled haploid (DH) lines were produced from CH and cold susceptible cucumbers, reciprocal hybrids with identical nuclear genotypes were produced, and plants were subjected to cold treatments under lights at 4 °C for 5.5 h. Hybrid plants with CH as the maternal parent had significantly higher fresh and dry weights 14 days after cold treatment compared to the reciprocal hybrid, revealing an enhanced cold recovery phenotype maternally conferred by CH. Results from analyses of the nuclear transcriptome and reactive oxygen species (ROS) between reciprocal hybrids were consistent with the cold recovery phenotype. Sequencing of the chloroplast genome and transcriptome of the DH parents and reciprocal hybrids, respectively, revealed one maternally transmitted non-synonymous single nucleotide polymorphism (SNP) in the chloroplast F1FO-ATP synthase (CF1FO-ATPase) beta-subunit gene (atpB) of CH which confers an amino acid change from threonine to arginine. Protein modeling revealed that this change is located at the interface of the alpha- and beta-subunits in the CF1FO-ATPase complex. Polymorphisms in the CF1FO-ATPase complex have been associated with stress tolerances in other plants, and selection for or creation of polymorphic beta-subunit proteins by chloroplast transformation or gene editing could condition improved recovery from cold stress in plants.