Biology subjects

Banerjee, U.

Publications and source records attributed to Banerjee, U..

10 recordsLinked to original sources

bioRxiv2025

CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

The transcription-associated cyclin dependent kinases (CDKs) 12 and 13 play important roles in the expression of RNAs involved in DNA damage repair and ribosomal proteins, yet their roles if any, in the regulation of ribosomal DNA expression are unclear. Here we show that these CDKs play critical roles in the processing and degradation of noncoding RNAs in the nucleolus and that inhibition of their function leads to the upregulation of unprocessed ribosomal RNA (rRNA) transcripts in the intergenic spacer (IGS) regions between ribosomal subunits. These upregulated RNAs are aberrantly polyadenylated by the TRAMP complex polymerase TENT4, and are represented as polyadenylated rings surrounding the nucleolus. The accumulation of these noncoding RNAs is associated with increased RNA polymerase II (Pol II)-mediated transcription coupled with failure of degradation by the MTREX (SKIV2L2) RNA helicase, a member of the RNA exosome complex whose expression is prematurely terminated by CDK12/13 inhibition. Altogether, these findings provide new insights into Pol II-mediated transcriptional regulation by CDK12/13 within the nucleolus.

cancer biology↗

bioRxiv2024

Distance between miRNA responsive elements selectively enhances target specificity with reduced cellular toxicity by miRNA-mediated synergism

Despite the emergence of miRNAs as promising therapeutic tools in cancer management, most clinical trials have not been successful due to their cytotoxic effects. Here, we have investigated the factors regulating the effect of miRNA-mimic pairs in enhancing target gene specificity while reducing cellular toxicity. Synergistic reductions by the miRNA-mimic pairs were observed for the 3-UTRs of common target genes with miRNA-responsive elements (MREs) preferentially located at a distance of 200-800bp. Deletion of either of the miRNA seed sequences resulted in a loss of synergism. Furthermore, we performed small RNA-sequencing to identify significantly downregulated miRNAs in Oral cancer. Consequently, we transfected let-7c-5p and miR-125b-5p miRNA mimics either alone or in combination at half-dose concentrations and determined the expression levels of their common and unique target genes in oral cancer cells. Significant reductions in target gene expression were observed for genes containing MREs for both miRNA mimics within the preferential distance. Proteomic data revealed that the let-7c-5p and miR-125b-5p miRNA-mimic pairs synergistically reduced the expression of their common target genes, hexokinase 2 (HK2) and branched-chain amino acid transaminase 1 (BCAT1), in cancer cells. However, unique target genes did not exhibit any significant alterations in their protein levels. The effect of HK2 and BCAT1 downregulation was also reflected in the metabolomic profiling of cancer cells, specifically affecting glycolysis and the BCAA degradation pathways. As a result of the metabolic impairment caused by the synergistic effect of miRNA mimic pairs, oral cancer cells showed a significant reduction in proliferation, migration, and spheroid formation compared to cells treated with either single miRNA. The synergistic effect of these miRNA mimics was lost in normal keratinocytes, possibly due to low expression levels of the target oncogenes, suggesting a cancer cell-specific effect of this mechanism.

cancer biology↗

bioRxiv2024

The MYCN 5' UTR as a therapeutic target in neuroblastoma

Tumor cell amplification of the MYCN transcription factor is seen in half of patients with high-risk neuroblastoma, where it functions as an oncogenic driver associated with metastatic disease and poor survival. Yet, direct targeting of MYCN has been met with little success, prompting efforts to inhibit its expression at multiple levels. MYCN-amplified neuroblastoma cells have an increased requirement for protein synthesis to meet the overwhelming transcriptional burden imposed by oncogenic MYCN. Here, we take advantage of this vulnerability to interrogate the therapeutic potential of inhibiting the activity of the eukaryotic translation initiation factor 4A1 (eIF4A1), an RNA-helicase responsible for resolving structural barriers such as polypurine preponderance within 5' untranslated regions (UTRs). We observed that eIF4A1 is a key regulator of transcript-specific mRNA recruitment in MYCN-overexpressing neuroblastomas and MYCN-associated transcripts rank highly in polypurine-rich 5' UTR sequences, the majority of which have critical roles in cell proliferation. Using CMLD012824, a novel synthetic amidino-rocaglate (ADR) derivative, we demonstrate selectively increased eIF4A1 affinity for polypurine-rich 5' UTRs, including the MYCN mRNA, leading to translation inhibition and cytotoxicity in human neuroblastoma cell lines and animal models. Through ribosome profiling and PAR-CLIP analysis, we show that ADR-mediated clamping of eIF4A1 onto mRNA spans the full lengths of target transcripts, whereas translational inhibition is mediated selectively through 5' UTR binding. Both cap-dependent and cap-independent translation of MYCN are disrupted, pointing to the ability of CMLD012824 to disrupt non-canonical translation initiation. Our studies provide insights into the functional role of eIF4A1 in meeting the increased protein synthesis demands of MYCN-amplified neuroblastoma and suggest that its disruption may be therapeutically beneficial in this disease.

cancer biology↗

bioRxiv2023

Wnt signaling couples G2 phase control with differentiation during hematopoiesis

During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, we show that a Wnt6/EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in the G2 phase by a {beta}-catenin-independent Wnt6 pathway that restricts Cdc25 nuclear entry and promotes cell growth. Canonical {beta}-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine-kinases EGFR and Abl, which promote nuclear entry of {beta}-catenin and facilitate exit from G2. This strategy combines transcription-dependent and - independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

developmental biology↗

bioRxiv2023

Epithelioids: Self-sustaining 3D epithelial cultures to study long-term processes

Studying long-term biological processes such as the colonization of aging epithelia by somatic mutant clones has been slowed by the lack of suitable culture systems. Here we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least a year, recapitulating the 3D structure, cell dynamics, transcriptome, and genomic stability of the esophagus. Live imaging over 5 months showed epithelioids replicate in vivo cell dynamics. Epithelioids enable the study of cell competition and mutant selection in 3D epithelia, and how anti-cancer treatments modulate the competition between transformed and wild type cells. Epithelioids are a novel method with a wide range of applications in epithelial tissues, particularly the study of long term processes, that cannot be accessed using other culture models.

cell biology↗

bioRxiv2021

Injury-induced inflammatory signaling and hematopoiesis in Drosophila

Inflammatory response in Drosophila to sterile (axenic) injury in embryos and adults has received some attention in recent years, and most concentrate on the events at the injury site. Here we focus on the effect sterile injury has on the hematopoietic organ, the lymph gland, and the circulating blood cells in the larva, the developmental stage at which major events of hematopoiesis are evident. In mammals, injury activates Toll-like receptor (TLR)/NF{kappa}B signaling in macrophages, which then express and secrete secondary, pro-inflammatory cytokines. In Drosophila larvae, distal puncture injury of the body wall epidermis causes a rapid activation of Toll and Jun kinase (JNK) signaling throughout the hematopoietic system and the differentiation of a unique blood cell type, the lamellocyte. Furthermore, we find that Toll and JNK signaling are coupled in their activation. Secondary to this Toll/JNK response, a cytokine, Upd3, is induced as a Toll pathway transcriptional target, which then promotes JAK/STAT signaling within the blood cells. Toll and JAK/STAT signaling are required for the emergence of the injury-induced lamellocytes. This is akin to the derivation of specialized macrophages in mammalian systems. Upstream, at the injury site, a Duox- and peroxide-dependent signal causes the activation of the proteases Grass and SPE needed for the activation of the Toll-ligand Spz, but microbial sensors or the proteases most closely associated with them during septic injury are not involved in the axenic inflammatory response.

genetics↗

bioRxiv2021

Paths and Pathways that Generate Cell-Type Heterogeneity and Developmental Progression in Hematopoiesis

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single cell RNA-Seq and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, while inhibiting canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.

developmental biology↗

bioRxiv2020

Intermediate Progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants

Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated due to lack of appropriate genetic tools. Here we report a split-GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs comprise a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. Additionally, through their dynamic control of the Notch ligand, Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. The split-GAL4 strategy is amenable for adoption in mammalian systems and would be invaluable in assigning trajectories that stem and progenitor populations follow as they develop into mature blood cells.

developmental biology↗

bioRxiv2020

Metabolic Plasticity drives Development during Mammalian Embryogenesis

Preimplantation mouse embryos interact minimally with their environment, and development is largely driven by metabolic processes. During the earliest cleavage stages, metabolism is rigid, with maternal deposits enforcing a redox state that facilitates zygotic genome activation. As maternal control falls, metabolic shuttles are activated, increasing glycolysis and equilibrating the TCA cycle. The resulting flexibility of nutrient utilization and metabolic plasticity facilitates unidirectional developmental progression such that later stage embryos proceed to form blastocysts without any exogenously added nutrients. We explore the mechanisms that govern this choreographed sequence that balances the deposition, degradation, synthesis and function of metabolic enzymes with redox control, bioenergetics and biosynthesis. Cancer cells follow a distinct metabolic strategy from that of the preimplantation embryo. However, important shared features emerge under reductive stress. We conclude that metabolic plasticity drives normal development while stress conditions mimic hallmark events in Cancer Metabolism.

developmental biology↗

bioRxiv2019

Glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis

The mouse embryo undergoes compaction at the 8-cell stage and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. We report here, that this first cell fate specification event is controlled by glucose metabolism. Glucose does not fuel mitochondrial ATP (energy) generation and glycolysis is dispensable for blastocyst formation. Glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1, and the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of AP-2{gamma}. YAP1, TEAD4 and AP-2{gamma} physically interact to form a nuclear complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, but this cascade of events constituting \"Developmental Metabolism\" specifically controls the fate of TE cells.

developmental biology↗