Mining common genetic Variants impacting on Allele-Specific Translation and cancer risk

The mRNA translation regulation is a critical factor in protein expression and cellular homeostasis, with deep implications for cancer biology. The work described in this thesis identifies, validates, and investigates allele-specific differences in translation efficiency mediated by tranSNPs: genetic variants that influence mRNA translation. By leveraging RNA-seq data from total and polysomal RNA, a pipeline was developed to identify tranSNPs and validate their functional consequences. Two case studies, rs1053639 in the DDIT4’s 3’UTR and rs1554710467 in eIF4H’s coding sequence, highlight the effectiveness of this approach in uncovering cis regulators of allele-specific translation. The first study focuses on rs1053639 (T>A), a single-nucleotide polymorphism located in the 3'UTR of the DNA damage-inducible transcript 4 (DDIT4) gene, a key regulator of mTORC1 signaling and a p53 target. Polysomal profiling and Sanger sequencing revealed a higher abundance of the reference T allele in polysome-associated versus total RNA fractions of TA heterozygous cells, suggesting the two alleles exhibit a difference in translational efficiency. CRISPR/Cas9-mediated knock-in experiments confirmed that T homozygous clones exhibited significantly higher DDIT4 protein levels compared to A homozygous ones, particularly under stress conditions such as endoplasmic reticulum stress or p53 activation by Nutlin. Moreover, functional analyses revealed that TT clones repressed mTORC1 activity more efficiently during stress, while the AA clones exhibited increased proliferation and potential tumorigenic features in competition assays and zebrafish xenografts in vivo experiments. Mechanistically, we identified RBMX as an important trans-acting factor mediating the observed differences in the rs1053639-mediated allele-specific DDIT4 translation efficiency. These findings establish rs1053639 as a functional tranSNP with implications for stress responses and cancer progression. The second study investigates rs1554710467, a missense variant in the coding region of the eIF4H gene, which encodes a translation initiation factor involved in unwinding structured 5'UTRs. Polysomal profiling followed by RNA-seq or Sanger sequencing revealed higher translational efficiency for the alternative allele. We used this variant to provide a proof of concept for using proteomics approaches to measure allele-specific differences in protein expression in cells heterozygous for a nonsynonymous variant. Interestingly, targeted proteomics using synthetic isotope-labeled peptides confirmed a pronounced imbalance favoring the alternative rs1554710467 EIF4H allele also at the protein levels. Cycloheximide chase experiments coupled to allele-specific proteomics revealed that the amino acid substitution does not impact protein stability. Sucrose gradient fractionation further revealed that both alleles localize similarly to ribonucleoprotein particles (RNPs) and 40S ribosomal subunits, suggesting functional similarity. Notably, the alternative allele produced higher levels of functional eIF4H protein, suggesting the rs1554710467 as a potential gain-of-function variant. Taken collectively, this thesis demonstrates the power of integrating RNA-seq data with polysomal profiling and proteomics to identify, annotate, and validate instances of allele-specific translational regulation. The findings provide novel insights into how subtle hidden genetic variation impacts mRNA translation and protein expression, offering potential implications for understanding cancer biology and informing future prognostic and therapeutic strategies.