Biology subjects

Kalsotra, A.

Publications and source records attributed to Kalsotra, A..

15 recordsLinked to original sources

bioRxiv2026

ExPert infers causal perturbations from cell states across biological and clinical contexts

Pooled single-cell CRISPR screens have driven substantial effort toward models predicting the response to a perturbation. We posed the inverse question: which perturbation produced an observed cell state? Here we developed ExPert (Extracting Perturbations), which learns one prototype per perturbation in a latent space shared by 12 cellular contexts, trained on 8,964 perturbations and 19.5 million cells, and ranks 19,895 candidate genes per query, over half absent from training. ExPert more than doubled held-out-context recall over the best existing method with 1,000-fold less compute, and transferred without retraining to drugs, protein degradation and combinations. In alcohol-associated liver disease it implicated mRNA export, unchanged in expression, and we experimentally confirmed nuclear poly(A) retention and isoform switching. In primary mitochondrial disease it recovered the affected complex from blood, localized the signal to monocytes, and flagged a misdiagnosed control. Perturbation atlases can thus be queried in reverse, from state to likely driver.

systems biology↗

bioRxiv2026

Inbred and outcrossed Drosophila embryos diverge in stress defense and developmental maintenance during heat stress

Early embryos must mount stress defenses without compromising the developmental programs that drive morphogenesis, but whether genetic background alters this balance remains unclear. We compared cellularizing Drosophila melanogaster embryos from inbred and outcrossed backgrounds under control conditions and after acute or chronic heat-stress. Both backgrounds developed successfully and showed modest transcriptomic differences at 18{degrees}C. Heat stress at 32{degrees}C, however, sharply separated their phenotypic and transcriptional responses. Inbred embryos showed poor larval hatching after acute and chronic heat, and this deficit persisted through adult eclosion. Their transcriptomes exhibited a greater stress-response burden, with enrichment of proteostasis, DNA repair, mitochondrial maintenance, RNA processing, and metabolic remodeling programs. Outcrossed embryos retained substantially greater developmental survival and higher abundance of transcripts associated with development, adhesion, epithelial organization, signaling, transport, sensory responses, and morphogenesis. Background-dependent divergence was greatest during acute heat. Under chronic heat, background-dependent differences were fewer, with most overlapping those seen during acute heat and a smaller set detected only under chronic heat. Transcript-origin analysis further showed that acute outcrossed-biased genes were predominantly maternally derived and showed no enrichment for purely zygotic transcripts, whereas chronic outcrossed-biased genes contained a larger zygotic component. In summary, the less viable inbred background mounted the broader molecular stress signature, whereas the more viable outcrossed background retained greater abundance of development-associated transcripts. These findings identify genetic background as a major determinant of how embryonic heat responses are distributed between molecular stress defense and developmental gene expression.

genetics↗

bioRxiv2025

Reducing Cofilin dosage makes embryos resilient to heat stress

In addition to regulating actin dynamics, Cofilin also senses and responds to physiological stress and can determine cell survival outcomes. Yet, the full picture of Cofilin's role in stress response is lacking. Here, we used imaging and RNA-seq methods to show that exposing early Drosophila melanogaster embryos to either acute or chronic heat stress (32{degrees}C) induces a Cofilin-mediated Actin Stress Response (ASR) and leads to upregulation of genes associated with both heat shock and Endoplasmic Reticulum (ER) unfolded protein responses. Reducing cofilin gene dosage (cofilin+/-) in heat-stressed embryos modulates all observed stress responses and partially rescues embryo survival. Unexpectedly we find that heat shock- and ER- stress response modulation arises because non-stressed cofilin+/- embryos already show upregulation of heat shock- and ER- stress response genes, prior to heat exposure. Our data support a model whereby cofilin heterozygosity activates specific stress responses associated with reduced protein homeostasis, thus priming embryos to be more resilient when they encounter subsequent heat stress. We conclude that Cofilin dosage serves as a determinant of stress outcomes, impacting both the actin cytoskeleton and inducible stress response pathways. In the embryo, we identify Cofilin as a novel link between inducible stress response and thermotolerance.

developmental biology↗

bioRxiv2024

Dysregulated RNA splicing induces regeneration failure in alcohol-associated liver disease

Individuals with progressive liver failure are at a high risk of mortality without liver transplantation. However, our understanding of derailed regenerative responses in failing livers is limited. Here, we performed comprehensive multi-omic profiling of healthy and diseased human livers using bulk and single-nucleus RNA-plus ATAC-seq. We report that hepatic immune milieu alterations in alcohol-associated liver disease (ALD) prevent hepatocytes from transitioning to a proliferative progenitor-like state, trapping them into an unproductive intermediate state. We discovered striking changes in RNA binding protein (RBP) expression, particularly ESRP, PTBP, and SR families, that cause misregulation of developmentally controlled RNA splicing in ALD. Our data pinpoint ESRP2 as a pivotal disease-sensitive RBP and support a causal role of its deficiency in ALD pathogenesis. Notably, splicing defects in ESRP2-targets Tcf4 and Slk, amongst others, directly alter their nuclear localization and activities, disrupting WNT and Hippo signaling pathways, which are critical for normal liver regeneration. We demonstrate that changes in stromal cell populations enrich failing ALD livers with TGF-{beta}, which suppresses ESRP2-driven epithelial splicing program and replaces functional parenchyma with quasi-progenitor-like cells lacking liver-specific functions. This unprecedented account of transcriptional and post-transcriptional dysregulation in ALD suggests that targeting misspliced RNAs could improve recovery and serve as biomarkers for poor ALD outcomes.

molecular biology↗

bioRxiv2024

Chromatin-associated lncRNA-splicing factor condensates regulate hypoxia responsive RNA processing of genes pre-positioned near nuclear speckles

Hypoxia-induced alternative splicing (AS) regulates tumor progression and metastasis. Little is known about how such AS is controlled and whether higher-order genome and nuclear domain (ND) organizations dictate these processes. We observe that hypoxia-responsive alternatively spliced genes position near nuclear speckle (NS), the ND that enhances splicing efficiency. NS-resident MALAT1 long noncoding RNA, induced in response to hypoxia, regulates hypoxia-responsive AS. MALAT1 achieves this by organizing the SR-family of splicing factor, SRSF1, near NS and regulating the binding of SRSF1 to pre-mRNAs. Mechanistically, MALAT1 enhances the recruitment of SRSF1 to elongating RNA polymerase II (pol II) by promoting the formation of phase-separated condensates of SRSF1, which are preferentially recognized by pol II. During hypoxia, MALAT1 regulates spatially organized AS by establishing a threshold SRSF1 concentration near NSs, potentially by forming condensates, critical for pol II-mediated recruitment of SRSF1 to pre-mRNAs.

molecular biology↗

bioRxiv2024

ESRP2-microRNA-122 axis directs the postnatal onset of liver polyploidization and maturation

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.

developmental biology↗

bioRxiv2024

Cancer-associated snaR-A noncoding RNA interacts with core splicing machinery and disrupts processing of mRNA subpopulations

RNA polymerase III (Pol III) activity in cancer is linked to the production of small noncoding (nc)RNAs that are otherwise silent in most tissues. snaR-A (small NF90-associated RNA isoform A) - a hominid-specific ncRNA shown to enhance cell proliferation, migration, and invasion - is a cancer-emergent Pol III product that remains largely uncharacterized despite promoting growth phenotypes. Here, we applied a combination of genomic and biochemical approaches to study the biogenesis and subsequent protein interactions of snaR-A and to better understand its role as a putative driver of cancer progression. By profiling the chromatin landscapes across a multitude of primary tumor types, we show that predicted snaR-A upregulation is broadly linked with unfavorable outcomes among cancer patients. At the molecular level, we unexpectedly discover widespread interactions between snaR-A and mRNA splicing factors, including SF3B2 - a core component of the U2 small nuclear ribonucleoprotein (snRNP). We find that SF3B2 levels are sensitive to high snaR-A abundance and that depletion of snaR-A alone is sufficient to decrease intron retention levels across subpopulations of mRNA enriched for U2 snRNP occupancy. snaR-A sensitive genes are characterized by high GC content, close spatial proximity to nuclear bodies concentrated in pre-mRNA splicing factors, and functional enrichment for proteins involved in deacetylation and autophagy. We highlight examples of splicing misregulation and increased protein levels following snaR-A depletion for a wide-ranging set of factors, suggesting snaR-A-driven splicing defects may have far-reaching effects that re-shape the cellular proteome. These findings clarify the molecular activities and consequences of snaR-A in cancer, and altogether establish a novel mechanism through which Pol III overactivity may promote tumorigenesis.

molecular biology↗

bioRxiv2024

PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

molecular biology↗

bioRxiv2021

PTBP1/HNRNP I controls intestinal epithelial cell regeneration by maintaining stem cell survival and stemness

Properly controlled intestinal epithelial cell regeneration is not only vital for protection against insults from environmental hazards but also crucial for preventing intestinal cancer. Intestinal stem cells located in the crypt region provide the driving force for epithelial regeneration, and thus their survival and death must be precisely regulated. We show here that polypyrimidine tract binding protein 1 (PTBP1, also called heterogeneous nuclear ribonucleoprotein I, or HNRNP I), an RNA-binding protein that post-transcriptionally regulates gene expression, is critical for intestinal stem cell survival and stemness. Mechanistically, we show that PTBP1 inhibits the expression of PHLDA3, an AKT repressor, and thereby maintains AKT activity in the intestinal stem cell compartment to promote stem cell survival and proliferation. Furthermore, we show that PTBP1 inhibits the expression of PTBP2, a paralog of PTBP1 that is known to induce neuron differentiation, through repressing inclusion of alternative exon 10 to Ptbp2 transcript. Loss of PTBP1 results in a significant upregulation of PTBP2, which is accompanied by splicing changes in genes that are important for neuronal cell development. This finding suggests that PTBP1 prevents aberrant differentiation of intestinal stem cells into neuronal cells through inhibiting PTBP2. Our results thus reveal a novel mechanism whereby PTBP1 maintains intestinal stem cell survival and stemness through the control of gene function post-transcriptionally.

molecular biology↗

bioRxiv2021

Splicing Factor SRSF1 Deficiency in the Liver Triggers NASH-like Pathology via R-Loop Induced DNA Damage and Cell Death

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA-DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver failure.

cell biology↗

bioRxiv2020

Single-cell analyses of the corneal epithelium: Unique cell types and gene expression profiles

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal epithelial cells from stage 49-51 Xenopus larvae. We identified five main clusters with distinct molecular signatures, which represent apical, basal and keratocyte cell types as well as two discrete proliferative cell types in the bi-layered epithelium. Our data reveal several novel genes expressed in corneal cells and spatiotemporal changes in gene expression during corneal differentiation. Through gene regulatory network analysis, we identified key developmental gene regulons, which guide these different cell states. Our study offers a detailed atlas of single-cell transcriptomes in the frog corneal epithelium. In future, this work will be useful to elucidate the function of novel genes in corneal homeostasis, wound healing and cornea regeneration, which includes lens regeneration in Xenopus. SUMMARY STATEMENTThis study identifies cell types and transcriptional heterogeneity in the corneal epithelium that regulate its differentiation, and facilitates the search for corneal stem cell markers.

developmental biology↗

bioRxiv2020

Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver

The adult liver has exceptional ability to regenerate, but how it sustains normal metabolic activities during regeneration remains unclear. Here, we use partial hepatectomy (PHx) in tandem with single-cell transcriptomics to track cellular transitions and heterogeneities of ~22,000 liver cells through the initiation, progression, and termination phases of mouse liver regeneration. Our results reveal that following PHx, a subset of hepatocytes transiently reactivates an early-postnatal-like gene expression program to proliferate, while a distinct population of metabolically hyperactive cells appears to compensate for any temporary deficits in liver function. Importantly, through combined analysis of gene regulatory networks and cell-cell interaction maps, we find that regenerating hepatocytes redeploy key developmental gene regulons, which are guided by extensive ligand-receptor mediated signaling events between hepatocytes and non-parenchymal cells. Altogether, our study offers a detailed blueprint of the intercellular crosstalk and cellular reprogramming that balances the metabolic and proliferation requirements of a regenerating liver.

genomics↗

bioRxiv2020

SimiC: A Single Cell Gene Regulatory Network Inference method with Similarity Constraints

Single-cell RNA-Sequencing has made it possible to infer high-resolution gene regulatory networks (GRNs), providing deep biological insights by revealing regulatory interactions at single-cell resolution. However, current single-cell GRN analysis methods produce only a single GRN per input dataset, potentially missing relationships between cells from different phenotypes. To address this issue, we present SimiC, a single-cell GRN inference method that produces a GRN per phenotype while imposing a similarity constraint that forces a smooth transition between GRNs, allowing for a direct comparison between different states, treatments, or conditions. We show that jointly inferring GRNs can uncover variation in regulatory relationships across phenotypes that would have otherwise been missed. Moreover, SimiC can recapitulate complex regulatory dynamics across a range of systems, both model and non-model alike. Taken together, we establish a new approach to quantitating regulatory architectures between the GRNs of distinct cellular phenotypes, with far-reaching implications for systems biology.

bioinformatics↗

bioRxiv2019

Overexpression of a non-muscle RBFOX2 isoform triggers cardiac conduction defects in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by a CTG trinucleotide repeat expansion in the 3' untranslated region of DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients and are the second leading cause of DM1-related deaths. Despite these figures, the mechanisms underlying cardiac-based DM1 phenotypes are unknown. Herein, we report that upregulation of a non-muscle splice isoform of RNA binding protein RBFOX2 in DM1 heart tissue--due to altered splicing factor and microRNA activities--induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX2 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac-conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from both DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX2-driven splicing defects in the voltage-gated sodium and potassium channels, which can alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX2 isoform in DM1 cardiac pathogenesis.

genetics↗

bioRxiv2019

Constitutive Androstane Receptor contributes towards increased drug clearance in cholestasis

Understanding the alterations in drug metabolism in different liver diseases is crucial for appropriate therapeutic intervention. We performed high-throughput RNA sequencing on various liver injury models, including cholestasis, diet-induced steatosis, and regeneration. Comparative liver transcriptome analysis revealed overlapping and distinct gene profiles among different liver diseases. Particularly, cholestatic livers displayed robust induction of drug metabolizing genes. This upregulation is not a generic hepatic stress response, as it was suppressed or unchanged in other models of liver diseases. Consistently, drug metabolic gene profiles were induced in a subset of biliary atresia patients, but not in individuals with hepatitis B or C viral infection, and alcoholic hepatitis. Further analysis revealed this induction was specific to genes regulated by nuclear receptor CAR (Constitutive Androstane Receptor). To test this, we challenged cholestatic mice with a paralytic agent, zoxazolamine. Compared to controls, these mice displayed significantly reduced paralysis time, reflecting increased drug metabolism, and this effect was lost upon inhibition of CAR. Thus, CAR activation can alter therapeutic efficacy of certain drugs in a subset of cholestatic individuals.

physiology↗