Biology subjects

Pancher, M.

Publications and source records attributed to Pancher, M..

3 recordsLinked to original sources

bioRxiv2025

Polysomal profiling coupled to allele-specific proteomics reveals an EIF4H tranSNP allele possessing higher mRNA translation potential.

To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116-derived cell lines, computing allelic fractions in total and polysome-associated RNA from RNA-seq data. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants associated with allele-specific mRNA translation, of which 16 are nonsynonymous. To validate instances of allele-specific translation, a proof-of-concept proteomics approach was developed to quantify the relative expression of pairs of endogenous proteins resulting from the decoding of alleles containing the nonsynonymous heterozygous variants. In particular, the G>A, R183H missense SNV rs1554710467 in the EIF4H gene was investigated. The alternative peptide containing H183 was significantly more abundant than the corresponding one containing R183, consistent with the overrepresentation of the alternative allele in polysomal RNA in HCT116 cells. A dual-fluorescence ribosome-stalling assay confirmed the enhanced translation potential of the variant allele. The two EIF4H allelic proteins exhibited similar stability and subcellular localization. These findings support the classification of rs1554710467 as a gain-of-function allele. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels, exploiting heterozygous coding variants. Overall, our approach extends the toolbox available to investigate allele-specific differences in mRNA translation potential, a relatively underexplored layer of gene expression control that could underlie inter-individual differences in disease-relevant phenotypes.

genetics↗

bioRxiv2025

A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

Relatively few studies have examined the link between SNPs and mRNA translation, despite the established importance of translational regulation in shaping cell phenotypes. We developed a pipeline analyzing the allelic imbalance in total and polysome-bound mRNAs from paired RNA-seq data of HCT116 cells and identified 40 candidate tranSNPs, i.e. SNPs associated with allele-specific translation. Among them, the SNP rs1053639 (T/A) on DNA damage-inducible transcript 4 (DDIT4) 3UTR was identified, with the reference T allele showing a higher polysome association. rs1053639 TT clones generated by genome editing exhibited significantly higher DDIT4 protein levels than AA ones. The difference in DDIT4 proteins was even greater when cells were treated with Thapsigargin or Nutlin, two perturbations that induce DDIT4 transcription. The RNA-binding protein RBMX influenced these allele-dependent differences in DDIT4 protein expression, as shown by RNA-EMSA, RIP, and smiFISH assays. RBMX depletion reduced DDIT4 protein in TT clones to the AA levels. Functionally, TT clones more effectively repressed mTORC1 under ER stress, while AA clones outcompeted TT clones in vitro or when injected in zebrafish embryos. RBMX depletion increased the fitness of TT cells in co-culture experiments. The rs1053639 AA genotype, under a recessive model, correlates with poor prognosis in TCGA cancer data. Key points- Translatome analysis in HCT116 cells revealed allele-specific mRNA translation for 40 SNPs - rs1053639 (T/A) in DDIT4 3UTR showed allelic differences in mRNA localization & protein expression - AA cells showed weaker mTOR inhibition & higher proliferation; AA individuals had poorer prognosis

molecular biology↗

bioRxiv2024

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The intrinsic biocompatibility, stability in biofluids, and heterogeneous molecular cargo of EVs promise advancements in targeted therapy applications. However, predicting cell-targeting spectrum and cargo delivery are fundamental challenges for exploiting EVs or hybrid formulations. In this work, we combined cell-based and biochemical approaches to understand if secreted EVs show predictable EV-cell interactions and consequent cargo delivery. We exploited the tetraspanin CD81 to encode full-length recombinant proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. These fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs when transiently over-expressed in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used in EV-cell interaction assays. In time-frames where the global GFP spot distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 manipulation in isogenic cells remarkably affected the tropism of heterologous EVs. In this line, secreted doxorubicin-EVs, which showed improved efficacy compared to the free drug, had a reduced cell-killing activity on SK-BR-3 with a knocked-out HER2 receptor. Interestingly, the fusion protein-corresponding transcripts also present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, as detected by ddPCR in tissue biopsies, improving our sensitivity in detecting bioavailable cargoes. These data show multiple mechanisms underlying EV-cell interactions and prioritize the profiling of surfaceomes for better comprehension of cell engagement and design new generations of EV-based nanovehicles.

molecular biology↗