Rahul Sinha

Current single-cell RNA atlases largely capture polyadenylated transcripts while missing critical regulatory layers from noncoding RNA. To address this, we develop a generalizable framework that adapts total RNA profiling for use in standard droplet-based platforms and captures a broad complement of coding and noncoding RNAs using a unified pipeline. Applying this approach to the developing human brain, we generate a dataset mapping diverse RNA biotypes across all neuronal and non-neuronal lineages, revealing biotype-specific expression programs with cell-type and temporal specificity. Tracking microRNA dynamics in Cajal-Retzius neurons, transient and early-born neurons in the cortex, we show the enrichment and target anticorrelation of MIR137, associated with schizophrenia and intellectual disability, suggesting tight regulatory control. We apply TotalX to human peripheral blood mononuclear cells and identify transcriptional modules combining coding and noncoding RNAs and tRNA dynamics. In addition, we analyze dengue-infected hepatocytes and capture non-adenylated viral transcripts that distinguish infection states. This expanded coverage helps with understanding cellular identity and gene regulation at the atlas scale.

Hematopoietic stem cells (HSCs) are multipotent self-renewing cells that give rise to all types of blood cells. Past research has identified that long-term hematopoietic stem cells in young mice and humans produce a balanced output of lymphoid and myeloid cells, while in old age, they are largely replaced by myeloid-biased HSCs (My-HSC). It has not yet been determined whether this transition results from epigenetic changes in a single population of HSC or if two or more subsets of HSCs exist that gain or lose dominance with age via processes of selection. Whether epigenetic change or competition, several characteristics of each may exist to ensure that the appropriate subset is placed in niches that support them. HSC can be mobilized into the blood and home selectively to target tissues via expression of “homing receptors,” but these molecules do not determine their intraorgan migration to appropriate niches. Chemokines are the class of molecules that determine intraorgan migration of cells. Here, we show that the chemokine receptor CCR5 is mainly expressed on My-HSCs, and therefore, the frequency of CCR5 ⁺ HSCs increases with age. Aged HSCs negative for CCR5 expression generate lower frequency of myeloid cells than lymphoid cells upon transplantation into recipients. Additionally, disruption of the CCL5–CCR5 signaling axis changes frequency of lymphoid populations in peripheral blood of aged mice, supporting research that shows the depletion of My-HSCs can result in the rejuvenation of adaptive immunity.

Non-coding RNAs represent a widespread and diverse layer of post-transcriptional regulation across cell types and states, yet much of their diversity remains uncharted at single-cell resolution. This gap stems from the limitations of widely used single-cell RNA-sequencing protocols, which focus on polyadenylated transcripts and miss many short or non-polyadenylated RNAs. Here, we adapted single-cell RNA-sequencing on the 10x Genomics platform to capture a broad complement of coding and non-coding RNAs—including miRNAs, tRNAs, lncRNAs, histone RNAs, and non-adenylated viral transcripts. This approach enabled the discovery of rich, dynamic non-coding RNA programs across immune cells, virally infected hepatocytes, and the developing human brain. In dengue virus-infected hepatocytes, we detect non-adenylated viral transcripts and distinguish active from transcriptionally quiescent infected states, each with distinct host regulatory signatures. In brain tissue, we identify biotype-specific, cell-type–restricted non-coding RNAs, including miRNAs whose expression anticorrelates with predicted targets, consistent with post-transcriptional regulatory relationships. We show that MIR137, one of the strongest GWAS loci associated with schizophrenia and intellectual disability, is expressed specifically in Cajal-Retzius cells, an early-born but transient population that guides subsequent cortical neuron migration. These findings demonstrate the importance of non-coding RNAs in defining cell identity and state, and show how expanded transcriptome coverage can reveal additional layers of gene control—now accessible through practical and scalable single-cell profiling.

Mouse lemurs are the smallest and fastest reproducing primates, as well as one of the most abundant, and they are emerging as a model organism for primate biology, behaviour, health and conservation. Although much has been learnt about their ecology and phylogeny in Madagascar and their physiology, little is known about their cellular and molecular biology. Here we used droplet-based and plate-based single-cell RNA sequencing to create Tabula Microcebus, a transcriptomic atlas of 226,000 cells from 27 mouse lemur organs opportunistically obtained from four donors clinically and histologically characterized. Using computational cell clustering, integration and expert cell annotation, we define and biologically organize more than 750 lemur molecular cell types and their full gene expression profiles. This includes cognates of most classical human cell types, including stem and progenitor cells, and differentiating cells along the developmental trajectories of spermatogenesis, haematopoiesis and other adult tissues. We also describe dozens of previously unidentified or sparsely characterized cell types. We globally compare expression profiles to define the molecular relationships of cell types across the body, and explore primate cell and gene expression evolution by comparing lemur transcriptomes to those of human, mouse and macaque. This reveals cell-type-specific patterns of primate specialization and many cell types and genes for which the mouse lemur provides a better human model than mouse¹. The atlas provides a cellular and molecular foundation for studying this model primate and establishes a general approach for characterizing other emerging model organisms.

Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3 , CTLA4 , and PDCD1 ), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3 ), and loss of Treg-mediated suppression.

Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

Supplementary Figure 1. IDH2 is mutated in preleukemic hematopoietic stem cells in relapse causing clones in AML. Supplementary Figure 2. Single-cell RNA sequencing and combined genotyping reveal distinct transcriptomic profiles of IDH1-mutant pHSCs in AML Supplementary Figure 3. Modeling human pHSCs using CRISPR/Cas9 with HDR show reduced proliferation and blocked differentiation caused by IDH1 mutations Supplementary Figure 4. IDH1 mutations reduce 5-hydroxymethylcytosine in gene bodies Supplementary Figure 5. IDH1 mutations drives transcriptomic alterations Supplementary Figure 6. IDH1-mutant pHSCs are sensitive to inhibition of oxidative phosphorylation

Supplementary Table 1. Clinical characteristics of the 17 IDH-mutant AML patients assessed. Supplementary Table 2. Summary of antibodies and flow cytometry reagents.

Supplementary Figure 1. IDH2 is mutated in preleukemic hematopoietic stem cells in relapse causing clones in AML. Supplementary Figure 2. Single-cell RNA sequencing and combined genotyping reveal distinct transcriptomic profiles of IDH1-mutant pHSCs in AML Supplementary Figure 3. Modeling human pHSCs using CRISPR/Cas9 with HDR show reduced proliferation and blocked differentiation caused by IDH1 mutations Supplementary Figure 4. IDH1 mutations reduce 5-hydroxymethylcytosine in gene bodies Supplementary Figure 5. IDH1 mutations drives transcriptomic alterations Supplementary Figure 6. IDH1-mutant pHSCs are sensitive to inhibition of oxidative phosphorylation

Rare preleukemic hematopoietic stem cells (pHSC) harboring only the initiating mutations can be detected at the time of acute myeloid leukemia (AML) diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSC). We confirm that IDH1-driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in the complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wild-type HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent the development and relapse of leukemia.Significance:A high burden of pHSCs is associated with worse overall survival in AML. Using single-cell sequencing, metabolic assessment, and gene-edited human models, we find human pHSCs with IDH1 mutations to be metabolically vulnerable and sensitive to eradication by complex I inhibition.See related commentary by Steensma, p. 83.This article is featured in Selected Articles from This Issue, p. 80