Biology subjects

Toposiviric, I.

Publications and source records attributed to Toposiviric, I..

5 recordsLinked to original sources

bioRxiv2026

RAPPL-Seq Enables Rapid, High-Sensitivity Ribosome Profiling of Scarce Samples

Ribosome profiling is a powerful method for transcriptome-wide monitoring of translation activity in vivo, but its use is limited by lengthy protocols, reliance on specialized equipment and inefficient recovery of ribosome-protected fragments (RPFs). To overcome these challenges, we developed RAPPL-Seq, a magnetic bead-based affinity capture method that enables fast and efficient RPF isolation. RAPPL-Seq achieves high recovery efficiency, short processing time, and robust performance with limited input material. It is broadly applicable across multiple sample types, including cell lines, primary tissues, and human biospecimens. RAPPL-Seq can also be paired with translation inhibitors to characterize inhibitor- and tissue-specific mRNA translation. RAPPL-Seq efficiently captured translatomes of glioblastoma (GBM) and adjacent non-tumor brain tissues from patient biopsies. Together, these findings establish RAPPL-Seq as a straightforward, easily accessible, and highly sensitive approach that extends translatome studies to scarce experimental and clinical samples, opening new avenues for mechanistic insight and therapeutic discovery.

molecular biology↗

bioRxiv2025

A platform for the systematic interrogation of genetic interactions in human cells: 26 gene by genome-wide double knockout screens as a proof of concept

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term "gene by genome-wide" knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca).

genetics↗

bioRxiv2020

The MNK1/2-eIF4E axis contributes to phenotype switching, melanoma progression, and resistance to immunotherapy.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Melanoma plasticity exhibited as phenotype switching contributes to immunotherapy resistance, however the mechanisms are not completely understood and thus therapeutically unexploited. Here, using a transgenic melanoma mouse model, we demonstrated a critical role of the MNK1/2-eIF4E axis in melanoma plasticity and resistance to immunotherapy. We showed that phospho-eIF4E deficient murine melanomas express high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified that phospho-eIF4E controls the translation of NGFR, a critical effector of phenotype switching. In patients with melanoma, the expression of MKNK1, the kinase for eIF4E, positively correlated with markers of immune exhaustion. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors and increased CD8+ T cell infiltrates. Blocking phospho-eIF4E, using MNK1/2 inhibitors, offers a new strategy to inhibit melanoma plasticity and improve the survival response to anti-PD-1 immunotherapy.

cancer biology↗

bioRxiv2019

The mTORC1/S6K/PDCD4/eIF4A axis determines outcome of mitosis.

mTOR is a serine/threonine kinase which acts a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in mitotic arrested cells across a variety of cancer and normal cell lines. Prevention of raptor phosphorylation during mitosis resulted in reactivation of mTORC1 in a rapamycin-sensitive manner. Importantly, expression of a non-phosphorylatable raptor mutant caused a dramatic reduction in cytotoxicity of the spindle poison Taxol. This effect was mediated via degradation of Programmed Cell Death Protein 4 (PDCD4), a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A was able to enhance the effects of taxol and restore sensitivity in Taxol resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in death vs. slippage decision during prolonged mitotic arrest and may be exploited to gain a clinical benefit in treating cancers resistant to anti-mitotic drugs.

molecular biology↗

bioRxiv2018

Lysosome expansion by selective translation of lysosomal transcripts during phagocyte activation

The mechanisms that govern organelle adaptation and remodeling remain poorly defined. The endo-lysosomal system degrades cargo from various routes including endocytosis, phagocytosis and autophagy. For phagocytes, endosomes and lysosomes (endo-lysosomes) are kingpin organelles since they are essential to kill pathogens and process and present antigens. During phagocyte activation, endo-lysosomes undergo a morphological transformation, going from a collection of dozens of globular structures to a tubular network in a process that requires the phosphatidylinositol-3-kinase-AKT-mTOR signalling pathway. Here, we show that the endo-lysosomal system undergoes an expansion in volume and holding capacity during phagocyte activation within 2 h of LPS stimulation. Endo-lysosomal expansion was paralleled by an increase in lysosomal protein levels, but this was unexpectedly largely independent of TFEB and TFE3 transcription factors, known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of acute organelle expansion via mTORC1-dependent increase in translation, which appears to be mediated by both, S6Ks and 4E-BPs. Moreover, we show that stimulation of RAW cells with LPS alters translation of a subset but not all of mRNAs encoding endo-lysosomal proteins, thereby suggesting that endo-lysosome expansion is accompanied by functional remodelling. Importantly, mTORC1-dependent increase in translation activity was necessary for efficient and rapid antigen presentation by dendritic cells. Collectively, we identified a previously unknown and functionally relevant mechanism for endo-lysosome expansion that relies on mTORC1-dependent translation to stimulate endo-lysosome biogenesis in response to an infection signal.

cell biology↗