Biology subjects

Bora, P.

Publications and source records attributed to Bora, P..

10 recordsLinked to original sources

bioRxiv2026

Probing RNA-protein interactions in the early mouse embryo

The union of two germ cells to form a zygote, and subsequent early embryo development, are marked by radical remodeling of virtually every major class of biomolecules as the specialized germline states give way to the rapid and active growth that marks early development. In recent years, advances in ultra-low input genome-wide methods have enabled systematic analyses of mRNA abundance, and of chromatin organization, throughout early development in a variety of model systems. Here, we extend these efforts to the study of RNA binding protein (RBP) function in early mouse embryos, adapting REMORA 1 - based on fusing an RNA-editing enzyme to an RBP of interest - for use in early embryos. We benchmark our approach for several well-studied RBPs, successfully recovering expected features of their RNA cargos, and assayed the RNA cargos for 17 RBPs of interest for early gene regulation. Analysis of changes in mRNA metabolism following knockdowns of the RBPs surveyed here allowed us to identify direct regulatory functions for a subset of RBPs in the early mammalian embryo, including an unanticipated role for the RNA export adaptor Alyref in control of 3 polyadenylation sites. Together, our data provide a proof of concept resource for systematically exploring RBP functions in mammalian embryogenesis.

molecular biology↗

bioRxiv2026

Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

cell biology↗

bioRxiv2025

Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells

Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) - the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2-deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

cell biology↗

bioRxiv2025

Optimizing PDIA1 Inhibition as a Strategy to Inhibit NLRP3 Inflammasome Activation and Activity

The NLRP3 inflammasome is a protein complex that promotes pro-inflammatory signaling as part of the innate immune response. Hyperactivation of the NLRP3 inflammasome has been implicated in many inflammatory and neurodegenerative diseases, leading to significant effort in developing strategies to limit its activation to intervene in these disorders. We previously showed that pharmacologic inhibition of the endoplasmic reticulum (ER)-localized protein disulfide isomerase PDIA1 suppresses NLRP3 activation and activity, identifying PDIA1 as a potential therapeutic target to mitigate hyperactive NLRP3 activity. Herein, we screen PDIA1 inhibitors to identify highly-potent compounds, including P1 and PACMA31, that pharmacologically target PDIA1 and block NLRP3 inflammasome assembly and activity. While sustained treatment with these PDIA1 inhibitors reduces THP1 viability, we show that acute treatment with these compounds is sufficient to both fully modify PDIA1 and inhibit NLRP3 inflammasome activity independent of any overt cellular toxicity. These results establish a treatment paradigm that can be exploited to develop highly-selective PDIA1 inhibitors to mitigate hyperactive NLRP3 inflammasome activity implicated in etiologically-diverse diseases.

biochemistry↗

bioRxiv2024

The Glycolytic Metabolite Methylglyoxal Covalently Inactivates the NLRP3 Inflammasome.

The NLRP3 inflammasome promotes inflammation in disease, yet the full repertoire of mechanisms regulating its activity are not well delineated. Among established regulatory mechanisms, covalent modification of NLRP3 has emerged as a common route for pharmacological inactivation of this protein. Here, we show that inhibition of the glycolytic enzyme PGK1 results in the accumulation of methylglyoxal, a reactive metabolite whose increased levels decrease NLRP3 assembly and inflammatory signaling in cells. We find that methylglyoxal inactivates NLRP3 via a non-enzymatic, covalent crosslinking-based mechanism, promoting inter- and intra-protein MICA posttranslational linkages within NLRP3. This work establishes NLRP3 as capable of sensing a host of electrophilic chemicals, both exogenous small molecules and endogenous reactive metabolites, and suggests a mechanism by which glycolytic flux can moderate the activation status of a central inflammatory signaling pathway.

cell biology↗

bioRxiv2023

Spatial positioning of preimplantation mouse embryo blastomeres is regulated by mTORC1 and 7mG-cap dependent translation at the 8- to 16-cell transition.

Preimplantation stages of mouse embryo development involve temporal and spatial specification and segregation of three late blastocyst cell lineages; trophectoderm (TE), primitive endoderm (PrE) and epiblast (EPI). Spatial separation of the outer TE lineage from the two inner cell mass (ICM) lineages (PrE and EPI) starts with the 8- to 16-cell transition and concludes following transit through the 16- to 32-cell stages. This results in an early blastocyst ICM derived from descendants of primary founding inner cells and a secondarily contributed population, of which subsequent relative EPI versus PrE potencies are subject to debate. Here, we report generation of primary but not the secondary ICM populations is highly dependent on temporally discreet activation of the mammalian target of Rapamycin (mTOR - specifically mTORC1) during M-phase entry at the 8-cell stage. This role is mediated via regulation of the 7-methylguanosine-(7mG) cap binding initiation complex (EIF4F), linked to translation of a subset of key mRNAs containing 5 UTR terminal oligopyrimidine (TOP-) or TOP-like sequence motifs; as knockdown of identified TOP-like motif containing transcripts also impairs generation of 16-cell stage primary ICM founders. However, mTOR inhibition induced ICM cell number deficits at the early blastocyst stage can be compensated by the late blastocyst stage, in the absence of inhibition. This compensation is likely initiated at the 32-cell stage when supernumerary outer cells in mTOR-inhibited embryos exhibit molecular characteristics of inner cells. Collectively, the data identify a novel mechanism specifically governing initial spatial segregation of blastomeres in the mouse embryo, that is distinct from those directing subsequent inner cell formation and contributes to germane segregation of late blastocyst lineages.

developmental biology↗

bioRxiv2021

DDX21 is a p38-MAPK sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification.

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell-fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (Ddx21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear Ddx21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation; a localisation dependent on active p38-MAPK. Efficient siRNA mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute extra significance to emerging importance of lineage specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.

developmental biology↗

bioRxiv2020

p38-MAPK mediated rRNA processing and translation regulation enables PrE differentiation during mouse blastocyst maturation.

Backgroundp38-MAPKs are stress-activated kinases necessary for placental development and nutrient and oxygen transfer during murine post-implantation development. In preimplantation development, p38-MAPK activity is required for blastocyst formation. Additionally, we have previously reported its role in regulating specification of inner cell mass (ICM) towards primitive endoderm (PrE), although a comprehensive mechanistic understanding is currently limited. Adopting live embryo imaging, proteomic and transcriptomic approaches, we report experimental data that directly address this deficit. ResultsChemical inhibition of p38-MAPK activity during blastocyst maturation causes impaired blastocyst cavity expansion, most evident between the third and tenth hours post inhibition onset. We identify an overlapping minimal early blastocyst maturation window of p38-MAPKi inhibition (p38-MAPKi) sensitivity, that is sufficient to impair PrE cell fate by the late blastocyst (E4.5) stage. Comparative proteomic analyses reveal substantial downregulation of ribosomal proteins, the mRNA transcripts of which are also significantly upregulated. Ontological analysis of the differentially expressed transcriptome during this developmental period reveals "translation" related gene transcripts as being most significantly, yet transiently, affected by p38-MAPKi. Moreover, combined assays consistently report concomitant reductions in de novo translation that are associated with accumulation of unprocessed rRNA precursors. Using a phosphoproteomic approach, {+/-} p38-MAPKi, we identified Mybpp1a, an rRNA transcription and processing regulator gene, as a potential p38-MAPK effector. We report that siRNA mediated clonal knockdown of Mybpp1a is associated with significantly diminished PrE contribution. Lastly, we show that defective PrE specification caused by p38-MAPKi (but not MEK/ERK signalling inhibition) can be partially rescued by activating the archetypal mTOR mediated translation regulatory pathway. ConclusionsActivated p38-MAPK controls blastocyst maturation in an early and distinctly transient developmental window by regulating gene functionalities related to translation, that creates a permissive environment for appropriate specification of ICM cell fate.

developmental biology↗

bioRxiv2019

Wwc2 is a novel mitotic/meiotic cell-cycle regulator and cell fate related gene, during preimplantation mouse embryo development and oogenesis

Formation of a mature and hatching mouse blastocyst marks the end of the preimplantation development, whereby regulated cell cleavages culminate in the formation of three distinct lineages. We report dysregulated expression of Wwc2, an ill-characterised paralog of the Hippo-signalling activator Kibra/Wwc1, is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities, typified by imbalanced daughter cell chromatin segregation. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the fertilisation competent stage of meiosis II metaphase arrest, characterised by spindle defects and failed Aurora-A kinase (AURKA) activation. Such cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal dysregulation implicates Wwc2 in maintaining the pluripotent late blastocyst stage epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell-fate.

developmental biology↗

bioRxiv2019

p38-mitogen activated kinases mediate a developmental regulatory response to amino acid depletion and associated oxidative stress in mouse blastocyst embryos

Maternal starvation coincident with preimplantation development has profound consequences for placental-foetal development, with various identified pathologies persisting/manifest in adulthood; the Developmental Origin of Health and Disease (DOHaD) hypothesis/model. Despite evidence describing DOHaD-related incidence, supporting mechanistic and molecular data relating to preimplantation embryos themselves are comparatively meagre. We recently identified the classically recognised stress-related p38-mitogen activated kinases (p38-MAPK) as regulating formation of the extraembryonic primitive endoderm (PrE) lineage within mouse blastocyst inner cell mass (ICM). Thus, we wanted to assay if PrE differentiation is sensitive to amino acid availability, in a manner regulated by p38-MAPK. Although blastocysts appropriately mature, without developmental/morphological or cell fate defects, irrespective of amino acid supplementation status, we found the extent of p38-MAPK inhibition induced phenotypes was more severe in the absence of amino acid supplementation. Specifically, both PrE and epiblast (EPI) ICM progenitor populations remained unspecified and there were fewer cells and smaller blastocyst cavities. Such phenotypes could be ameliorated, to resemble those observed in groups supplemented with amino acids, by addition of the anti-oxidant NAC (N-acetyl-cysteine), although PrE differentiation deficits remained. Therefore, p38-MAPK performs a hitherto unrecognised homeostatic early developmental regulatory role (in addition to direct specification of PrE), by buffering blastocyst cell number and ICM cell lineage specification (relating to EPI) in response to amino acid availability, partly by counteracting induced oxidative stress; with clear implications for the DOHaD model.

developmental biology↗