Biology subjects

Stoilov, P.

Publications and source records attributed to Stoilov, P..

9 recordsLinked to original sources

bioRxiv2026

Erythropoietin Mediates Glycerophospholipid Remodeling During Human Early Erythropoiesis

The unique biconcave morphology and deformability of mature red blood cells (RBCs) require a specific membrane lipid composition. Disruption of this special lipid profile is seen in multiple types of anemia and bone marrow (BM) failure diseases, yet molecular mechanisms regulating lipid metabolism during normal erythroid differentiation remain poorly defined. Here, we identify a previously undescribed role for erythropoietin (Epo) in contributing to the appropriate lipid composition of differentiating human erythroid cells. Using single-cell transcriptomic profiling of ex vivo cultures of human BM-derived hematopoietic stem and progenitor cells cultured with or without Epo, we delineated transcriptional dynamics across differentiation stages, identifying the Epo-dependent transition of burst-forming unit erythroid (BFU-E) to colony-forming unit erythroid (CFU-E) progenitors. Comparative analysis revealed the activation of canonical erythroid programs involving heme biosynthesis, globin expression, and iron regulation, and additionally, uncovered a transient upregulation of lipid metabolic pathways during the BFU-E to CFU-E transition. Complementary untargeted lipidomics demonstrated Epo-dependent alterations in specific glycerophospholipid (GPL) species, consistent with differential expression of GPL biosynthesis genes in the single cell dataset. Intracellular flow cytometry further confirmed the requirement of Epo for maintaining enzymes critical for phosphatidylcholine and phosphatidylethanolamine synthesis in erythroid cells. Together, these multiomic findings reveal a new role for Epo in modulating lipid metabolism during early erythropoiesis and provide mechanistic insight into how membrane lipid composition is dynamically regulated to support normal red cell development.

cell biology↗

bioRxiv2025

A single Musashi gene allele is sufficient to maintain mouse photoreceptor cells

In vertebrates, two genes, Musashi1 (Msi1) and Musashi2 (Msi2), encode for highly similar Musashi protein paralogs. The Musashi proteins are known to bind to 3-UTRs and control translation. In photoreceptor cells, the Musashi proteins promote the inclusion of photoreceptor-specific alternative exons by binding to the proximal downstream of their introns. While the Musashi proteins are expressed in various cell types, their role in regulating splicing appears to be confined to photoreceptor cells, where the two proteins have exceptionally high expression levels. To test if the photoreceptor-specific role of MSI1 and MSI2 in splicing is due to their expression levels in photoreceptor cells, we generated combined Msi1 and Msi2 knockouts that progressively reduced the number of Musashi alleles in photoreceptor cells. We analyzed the splicing of photoreceptor-specific exons in the Cc2d2a, Cep290, Prom1, and Ttc8 genes and the function of photoreceptor cells in the knockouts. We found that a single allele from either Msi1 or Msi2 is sufficient to maintain photoreceptor function and support high inclusion levels of the photoreceptor-specific exons.

genetics↗

bioRxiv2025

Molecular Mechanisms Limiting the Therapeutic Window of AAV Gene Therapy in Mouse Models of Blue Cone Monochromacy

Blue cone monochromacy (BCM) is an X-linked retinal disorder caused by mutations in the OPN1LW/OPN1MW gene locus, resulting in impaired cone function and structural degeneration. We conducted a comparative analysis of AAV-mediated gene therapy in Opn1lw/Opn1mw double knockout (DKO) and Opn1mwC198R/Opn1sw-/- (C198R) BCM mouse models and evaluated the therapeutic window, efficacy, and longevity. Our results demonstrate that the AAV8-Y733F capsid achieved superior cone rescue compared to AAV5. While both DKO and C198R models showed similar therapeutic windows and rescue longevity, treatment efficacy decreased markedly in older mutant mice. Structural analysis revealed that aged cones in both models displayed degenerative changes, including mislocalized mitochondria and compromised connecting cilia. At the molecular level, we observed reduced AAV-mediated transgene expression in DKO and C198R older cones, which may result from decreased transduction efficiency, decreased circular episome stability, genome-wide transcription/translation downregulation, targeted mRNA/protein degradation, or overall cone degeneration. Notably, the cone-specific promoters for Pde6c and Cngb3 maintained robust activity in degenerating cones. These findings suggest that combining an efficient AAV serotype with an optimized cone promoter could be a viable approach to extend the therapeutic window and enhance treatment longevity for BCM patients.

molecular biology↗

bioRxiv2024

Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals

Viruses continue to pose a significant health burden to the human population, and recent history has shown a concerning surge in viral threats. Treatment options for viral infections are limited, and viruses have proven adept at evolving resistance to many existing therapies, highlighting a significant vulnerability in our defenses. In response to this challenge, we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Many viruses depend on the host cells RNA splicing machinery, and small alterations to this host process results in catastrophic changes in viral protein production, ultimately inhibiting virus replication. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191-mediated reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 534219-resistant variants of coronavirus or influenza. As with HIV-1, 5342191s inhibition of coronaviruses and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for the replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential of greater resilience in combating viral infections.

microbiology↗

bioRxiv2024

Erythropoietin-dependent Acquisition of CD71hiCD105hi Phenotype within CD235a- Early Erythroid Progenitors

The development of committed erythroid progenitors and their continued maturation into mature erythrocytes requires the cytokine erythropoietin (Epo). Here, we describe the immunophenotypic identification of a unique Epo-dependent colony-forming unit-erythroid (CFU-E) cell subtype that forms during early erythropoiesis (EE). This previously undescribed CFU-E subtype, termed late-CFU-E (lateC), lacks surface expression of the characteristic erythroid marker CD235a (glycophorin A) but has high levels of CD71 and CD105. LateCs could be prospectively detected in human bone marrow (BM) cells and, upon isolation and reculture, exhibited the potential to form CFU-E colonies in medium containing only Epo (no other cytokines) and continued differentiation along the erythroid trajectory. Analysis of ex vivo cultures of BM CD34+ cells showed that acquisition of the CD7hiCD105hi phenotype in lateCs is gradual and occurs through the formation of four EE cell subtypes. Of these, two are CD34+ burst-forming unit-erythroid (BFU-E) cells, distinguishable as CD7loCD105lo early BFU-E and CD7hiCD105lo late BFU-E, and two are CD34- CFU-Es, also distinguishable as CD71loCD105lo early CFU-E and CD7hiCD105lo mid-CFU-E. The transition of these EE populations is accompanied by a rise in CD36 expression, such that all lateCs are CD36+. Single cell RNA-sequencing analysis confirmed Epo-dependent formation of a CFU-E cluster that exhibits high coexpression of CD71, CD105, and CD36 transcripts. Gene set enrichment analysis revealed the involvement of genes specific to fatty acid and cholesterol metabolism in lateC formation. Overall, in addition to identifying a key Epo-dependent EE cell stage, this study provides a framework for investigation into mechanisms underlying other erythropoiesis-stimulating agents.

developmental biology↗

bioRxiv2022

The Musashi proteins are post-transcriptional activators of protein expression and alternative exon splicing in vertebrate photoreceptors

The Musashi proteins, MSI1 and MSI2, are conserved RNA binding proteins with a role in the maintenance and renewal of stem cells. Contrasting with this role, retina and terminally differentiated photoreceptor cells express high levels of MSI1 and MSI2, indicating that the two proteins have a role unrelated to maintaining undifferentiated cell state. Here we show that the Musashi proteins are essential in mature photoreceptors. Combined knockout of Msi1 and Msi2 lead to loss of the retina response to light and progressive photoreceptor cell death. The two proteins are fully redundant as individual deletion of Msi1 or Msi2 did not produce a phenotype. To define the molecular functions underlying the requirement for Musashi in photoreceptors, we delineated their RNA targets and analyzed the effect of the combined Msi1/Msi2 knockout on transcript levels, pre-mRNA splicing, and protein expressions. We show distinct nuclear and cytoplasmic functions for the Musashi proteins in photoreceptor cells. In the nucleus, Musashi binding to the downstream proximal intron promotes splicing of alternative exons. Surprisingly, four conserved photoreceptor-specific alternative exons in genes critical for vision proved to be dispensable, leaving open questions about the selective pressures that lead to the conservation of these exons and the contribution of alternative splicing to the phenotype of the Musashi knockout. In the photoreceptor cell cytoplasm MSI1 and MSI2 act as activators of protein expression. The combined knockout of Msi1 and Msi2 reduced the levels of multiple proteins including proteins required for vision and photoreceptor survival.

neuroscience↗

bioRxiv2021

The Global Protein-RNA Interaction map of Epithelial Splicing Regulatory Protein 1 defines a post-transcriptional program that is essential for epithelial cell function

The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3 and 5 untranslated regions (UTRs) of genes involved in epithelial cell function suggesting that its post-transcriptional functions extend beyond splicing regulation.

molecular biology↗

bioRxiv2020

A critical role for MSI1 and MSI2 in photoreceptor morphogenesis

Musashi family of RNA-binding proteins are known for their role in stem-cell renewal and are negative regulators of cell differentiation. Interestingly, in the retina, Musashi proteins, MSI1 and MSI2 are differentially expressed throughout the cycle of retinal development including robust expression in the adult retinal tissue. To study the role of Musashi proteins in the retina, we generated a pan-retinal and rod photoreceptor neuron specific conditional knockout mouse lacking MSI1 and MSI2. Independent of sex, photoreceptor neurons with simultaneous deletion of Msi1 and Msi2 were unable to respond to light and displayed severely disrupted OS morphology and ciliary defects. The retina lacking Musashi exhibited neuronal degeneration with complete loss of photoreceptors by six months. In concordance with our earlier studies that proposed a role for Musashi in regulating alternative splicing, the loss of Musashi prevented the use of photoreceptor-specific exons in transcripts critical for OS morphogenesis, ciliogenesis and synaptic transmission. Overall, we demonstrate a critical role for Musashi in the morphogenesis of terminally differentiated photoreceptor neurons. This role is in stark contrast with the canonical function of these two proteins in maintenance and renewal of stem cells.

developmental biology↗

bioRxiv2018

Uncovering the signaling landscape controlling breast cancer cell migration identifies novel metastasis driver genes

Metastasis is the major cause of death in cancer patients and migration of cancer cells from the primary tumor to distant sites is the prerequisite of metastasis formation. Here we applied an imaging-based RNAi phenotypic cell migration screen using two highly migratory basal breast cancer cell lines (Hs578T and MDA-MB-231) to provide a repository for signaling determinants that functionally drive cancer cell migration. We screened ~4,200 individual target genes covering most cell signaling components and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, of which 43 genes were common denominators of cell migration. Interaction networks of candidate migratory modulators were in common with networks of different clinical breast cancer prognostic and metastasis classifiers. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF were amplified in human primary breast tumors and the expression was associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF caused primarily down-regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B was essential for triple negative breast cancer cell migration and critical for breast cancer metastasis formation in vivo, making PRPF4B a candidate for further drug development. Our systematic phenotypic screen provides an important repository of candidate metastasis drug targets.

cancer biology↗