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Functional Atlas of Primary miRNA Maturation by the Microprocessor.
Primary microRNAs (miRNAs) are the precursors of miRNAs that modulate the expression of most mRNAs in humans. They fold up into a hairpin structure that is cleaved at its base by an enzyme complex known as the Microprocessor (Drosha/DGCR8). While many of the molecular details are known, a complete understanding of what features distinguish primary miRNA from hairpin structures in other transcripts is still lacking. We develop a massively parallel functional assay termed Dro-seq (Drosha sequencing) that enables testing of hundreds of known primary miRNA substrates and thousands of single-nucleotide variants. We find an additional feature of primary miRNAs, called Shannon entropy, describing the structural ensemble important for processing. In a deep mutagenesis experiment, we observe particular apical loop U bases, likely recognized by DGCR8, are important for efficient processing. These findings build on existing knowledge about primary miRNA maturation by the Microprocessor and further explore the substrate RNA sequence-structure relationship.
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A spatiotemporal atlas of orchiectomy-induced androgen deprivation-mediated modulation of cellular composition and gene expression in the mouse prostate.
Androgen deprivation therapy (ADT) remains a cornerstone in the treatment of prostate cancer (PCa), yet most tumors eventually develop resistance. Murine models are widely used to study PCa progression and ADT response, but a detailed understanding of the prostate's biological response to androgen deprivation in these models is lacking. Here, we present a comprehensive spatiotemporal analysis of cellular and transcriptional dynamics in the mouse prostate following orchiectomy (ORX)-induced androgen deprivation. We observed progressive involution across all prostate lobes (dorsal, ventral, lateral, and anterior) and distinct lobe-specific temporal gene expression changes post-ORX. Immune cell infiltration markedly increased over time, highlighting a shift in the prostate's cellular landscape. Single-cell RNA sequencing uncovered a previously undescribed fibroblast subtype-termed ORX-induced fibroblast (OIF)-characterized by high expression of Wnt2, Rorb, and Wif1, with distinct spatial localization. Pathway analysis revealed upregulation of amide and peptide binding functions, alongside suppression of peptidase and endopeptidase activity. Furthermore, dynamic changes in ligand-receptor interactions across lobes underscored the evolving intercellular communication in the post-ORX prostate. By integrating spatial transcriptomics with single-cell profiling, our study generates a high-resolution atlas of the murine prostate's response to androgen deprivation. These findings provide a foundational resource for interpreting ADT responses in preclinical models of PCa.
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Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics.
To understand the brain, molecular details need to be overlaid onto neural wiring diagrams so that synaptic mode, neuromodulation and critical signaling operations can be considered. Single-cell transcriptomics provide a unique opportunity to collect this information. Here we present an initial analysis of thousands of individual cells from Drosophila midbrain, that were acquired using Drop-Seq. A number of approaches permitted the assignment of transcriptional profiles to several major brain regions and cell-types. Expression of biosynthetic enzymes and reuptake mechanisms allows all the neurons to be typed according to the neurotransmitter or neuromodulator that they produce and presumably release. Some neuropeptides are preferentially co-expressed in neurons using a particular fast-acting transmitter, or monoamine. Neuromodulatory and neurotransmitter receptor subunit expression illustrates the potential of these molecules in generating complexity in neural circuit function. This cell atlas dataset provides an important resource to link molecular operations to brain regions and complex neural processes.
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A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.
Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.
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Single-Cell RNA Sequencing Delineates the Atlas and Cell Interactions of the Testicular Cells in Chinese Soft-Shelled Turtle (Pelodiscus sinensis).
Spermatogenesis is a complicated process of sexual reproduction, involving cell proliferation and differentiation, and depends on intricate interactions between testicular somatic cells and germ cells. For further investigations on male germ cells' development and differentiation in Chinese soft-shelled turtle (Pelodiscus sinensis), it is crucial to define testicular cell types and their molecular regulators. Here, the 10 × Genomics single-cell RNA sequencing was adopted to study the transcriptomic profiles of single cells during the spermatogenesis of adult Chinese soft-shelled turtle. In total, 7317 individual cell transcriptomes were collected for analysis, and 11 cell types were identified with known differentially expressed genes, including Leydig cells, Sertoli cells, spermatogonia, spermatocytes, and spermatids. Likewise, the top 10 marker genes and top-enriched gene pathways were analyzed in each cell type. Intriguingly, ligand-receptor analysis showed that the strongest interaction between Leydig cells and germ cells was using CellChat. Moreover, a primary developmental trajectory of male germ cells was constructed from spermatogonia to spermatids, as well as some important cell-specific regulators were identified for labeling the germ cells at different stages, including PCNA and Stra8, validated by immunostaining fluorescence. In addition, the transcriptomic profiles of male germ cells at different stages were comparatively analyzed among fruit fly, fish, mammals, and Chinese soft-shelled turtle, and the conserved and divergent regulators of male germ cells were summarized across species. In conclusion, this study provided novel insights into the testicular cells' atlas in turtles, and the findings would facilitate the development of techniques for manipulating germ cells, such as isolating cells and defining stages of differentiation.
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Identification of an anoikis-related gene signature and characterization of immune infiltration in skin cutaneous melanoma.
Anoikis is considered strongly associated with a biological procession of tumors. Herein, we utilized anoikis-related genes (ARGs) to predict the prognosis and immunotherapeutic efficacy for skin cutaneous melanoma (SKCM). RNA-seq data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. After dividing patients into novel subtypes based on the expression of prognostic ARGs, K-M survival was conducted to compare the survival status. Subsequently, differentially expressed ARGs were identified and the predictive model was established. The predictive effects were validated using the areas under the curve about the receiver operating characteristic. Moreover, tumor mutation burden, the enriched functional pathway, immune cells and functions, and the immunotherapeutic response were also analyzed and compared. The distribution of model genes at cell level was visualized by the single-cell seq with tumor immune single-cell hub database. Patients of The Cancer Genome Atlas-SKCM cohort were divided into 2 clusters, the cluster 1 performed a better prognosis. Cluster 2 was more enriched in metabolism-related pathways whereas cluster 1 was more associated with immune pathways. A predictive risk model was established with 6 ARGs, showing the areas under the curves of 1-year, 3-year, and 5-year ROC were 0.715, 0,720, and 0.731, respectively. Moreover, risk score was negatively associated with tumor mutation burden and immune-related pathways enrichment. In addition, patients with high-risk scores performed immunosuppressive status but the decreasing scores enhanced immune cell infiltration, immune function activation, and immunotherapeutic response. In this study, we established a novel signature in predicting prognosis and immunotherapy. It can be considered reliable to formulate the complex treatment for SKCM patients.
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Single-cell multiomics gene regulatory landscape reveals impaired spermatogonial stem cells and macrophage-driven inflammaging during testicular aging.
Testicular aging is a key driver of declining male reproductive health, but a comprehensive understanding of its underlying epigenetic drivers is lacking. To address this, we construct a multiomics aging atlas by integrating single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), and spatial transcriptomics of young and aged mouse testes. Our analysis reveals that altered chromatin accessibility accompanies transcriptional dysregulation and identifies spermatogonial stem cells (SSCs) as the most epigenetically vulnerable population. We further pinpoint ZFX as a consistently downregulated transcription factor in early germ cells, linking its decline to compromised SSC maintenance. In the somatic niche, we define disrupted regulatory networks that underpin impaired blood-testis barrier integrity in Sertoli cells and steroidogenic dysfunction in Leydig cells. Notably, we uncover an aberrant expansion of CD206+H2-Eb1- macrophages that propagates local inflammation via the secreted factor CXCL13, which we demonstrate is sufficient to disrupt the SSC niche. Cross-species analysis identifies conserved aging-associated regulons, including BRCA1 and E2F1. This work provides resources for understanding testicular senescence and nominates ZFX and CXCL13 as potential therapeutic targets for mitigating age-related reproductive decline.
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Identification of DNA damage and repair gene-related markers in pancreatic ductal adenocarcinoma by single-cell and bulk RNA sequencing.
BACKGROUND: The DNA damage response (DDR) has a major impact on the development and progression of pancreatic ductal adenocarcinoma (PDAC). Investigating biomarkers linked to the DDR may facilitate prognostic assessment and prediction of immunological characteristics for patients with PDAC. METHODS: The single-cell RNA sequencing (scRNA-seq) dataset GSE212966 was obtained from the GEO database, whereas the bulk RNA-seq data were sourced from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Least absolute shrinkage and selection operator (LASSO) and univariate Cox regression analyses were used to select genes to construct a prognostic risk model. Finally, the correlations of the model score with drug sensitivity, immunological checkpoints, and immune infiltration were assessed. RESULTS: We used 16 DDR marker genes to construct a predictive model. Furthermore, we established that the model had strong performance in both the training and validation cohorts. For PDAC, the model risk score served as an independent predictor of prognosis. There were notable differences in the proportions of the immune cells in the tumor microenvironment and drug sensitivity between the high and low risk score groups. The study confirmed that the risk score model is useful for predicting the immunotherapy response. Our experiments verified that knockdown of LY6D inhibits cell proliferation, promotes apoptosis and DNA damage. CONCLUSION: Our creative integration of bulk RNA sequencing and scRNA-seq data allowed us to construct a DDR-related prognostic model. Our model can be used to predict the immunological features, treatment response and prognosis of PDAC with a relatively high degree of accuracy.
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A molecularly defined and spatially resolved cell atlas of the whole mouse brain.
In mammalian brains, tens of millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells in the brain have so far hindered our understanding of the molecular and cellular basis of its functions. Recent advances in spatially resolved single-cell transcriptomics have allowed systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1-3. However, these approaches have only been applied to a few brain regions1-11 and a comprehensive cell atlas of the whole brain is still missing. Here, we imaged a panel of >1,100 genes in ~8 million cells across the entire adult mouse brain using multiplexed error-robust fluorescence in situ hybridization (MERFISH)12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating MERFISH and single-cell RNA-sequencing (scRNA-seq) data. Using this approach, we generated a comprehensive cell atlas of >5,000 transcriptionally distinct cell clusters, belonging to ~300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of the MERFISH images to the common coordinate framework (CCF) of the mouse brain further allowed systematic quantifications of the cell composition and organization in individual brain regions defined in the CCF. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes in the gene-expression profiles of cells. Finally, this high-resolution spatial map of cells, with a transcriptome-wide expression profile associated with each cell, allowed us to infer cell-type-specific interactions between several hundred pairs of molecularly defined cell types and predict potential molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a valuable resource for future functional investigations of neural circuits and their dysfunction in diseases.
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Single-Cell Atlas of the Chinese Tongue Sole (Cynoglossus semilaevis) Ovary Reveals Transcriptional Programs of Oogenesis in Fish.
Oogenesis is a highly orchestrated process that depends on regulation by autocrine/paracrine hormones and growth factors. However, many details of the molecular mechanisms that regulate fish oogenesis remain elusive. Here, we performed a single-cell RNA sequencing (scRNA-seq) analysis of the molecular signatures of distinct ovarian cell categories in adult Chinese tongue sole (Cynoglossus semilaevis). We characterized the successive stepwise development of three germ cell subtypes. Notably, we identified the cellular composition of fish follicle walls, including four granulosa cell types and one theca cell type, and we proposed important transcription factors (TFs) showing high activity in the regulation of cell identity. Moreover, we found that the extensive niche-germline bidirectional communications regulate fish oogenesis, whereas ovulation in fish is accompanied by the coordination of simultaneous and tightly sequential processes across different granulosa cells. Additionally, a systems biology analysis of the homologous genes shared by Chinese tongue sole and macaques revealed remarkably conserved biological processes in germ cells and granulosa cells across vertebrates. Our results provide key insights into the cell-type-specific mechanisms underlying fish oogenesis at a single-cell resolution, which offers important clues for exploring fish breeding mechanisms and the evolution of vertebrate reproductive systems.