Biology subjects

Alunno, L.

Publications and source records attributed to Alunno, L..

3 recordsLinked to original sources

bioRxiv2026

Allele-resolved monosome and polysome sequencing identifies functional cis-acting variants affecting mRNA translation efficiency

To prioritize germline genetic variants affecting mRNA fate at the post-transcriptional and translational levels, we leveraged sucrose-gradient-based isolation of 80S monosomes and polysomes, followed by mRNA retrieval and paired-end sequencing. Total cytoplasmic RNA was also sequenced for comparison. Experiments were performed in the non-transformed cell line RPE-1, cultured under basal conditions or upon p53 activation by Nutlin. Differential gene expression analysis confirmed a canonical p53 response. Heterozygous SNPs and SNVs were identified from the RNA-seq data, and allelic fractions (AF) were calculated for total, monosomal, and polysomal mRNAs. Variants showing reproducible AF differences across fractions beyond experimental variability were defined as tranSNPs. Among nearly 7000 heterozygous variants analyzable in polysomal or total RNA and over 5000 in monosomal mRNA, 1155 displayed a significant imbalance. Reporter assays performed in both RPE-1 and HCT116 cells validated allelic or haplotype effects for 17 selected variants in UTRs and coding regions, confirming differences in 15 cases, with evidence of cell line-specific responses. Proteomic analysis further supported allelic imbalance for selected missense variants. Overall, tranSNPs were identified in a non-transformed cell line at frequencies comparable to those in cancer cells, thereby extending their implications in human physiology. Further, monosome profiling enabled improved detection sensitivity of tranSNPs without positional bias, suggesting that 80S profiling improves detection of allele-specific translational regulation in RPE-1 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/723424v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f10281org.highwire.dtl.DTLVardef@db91ccorg.highwire.dtl.DTLVardef@140ea91org.highwire.dtl.DTLVardef@e80c99_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

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↗