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

• The study combines polysomal profiling and translatome analysis by RNA-sequencing, with a PRM proteomics workflow to investigate instances of allele-specific mRNA translation driven by heterozygous coding variants. • Allelic imbalance in polysomal RNA led us to nominate 52 coding variants in HCT116 cells associated with allele-specific mRNA translation, of which 16 are nonsynonymous. • A proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring (PRM) using isotope-labeled peptide standards. • The alternative rs1554710467 allele was over-represented in polysomal RNA fractions and associated with a higher abundance of the corresponding peptide. The study combines polysomal profiling and translatome analysis by RNA-sequencing, with a PRM proteomics workflow to investigate instances of allele-specific mRNA translation driven by heterozygous coding variants. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants in HCT116 cells associated with allele-specific mRNA translation, of which 16 are nonsynonymous. A proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring (PRM) using isotope-labeled peptide standards. The alternative rs1554710467 allele was over-represented in polysomal RNA fractions and associated with a higher abundance of the corresponding peptide. To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116 colorectal adenocarcinoma-derived cells, computing allelic fractions from both 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 proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring (PRM) using isotope-labeled peptide standards. Using this approach, we provide proof-of-concept validation of the heterozygous G>A, R183H missense SNV rs1554710467 in the EIF4H gene. The variant is present in two EIF4H alternative spliced variants, which showed equivalent translation efficiency in HCT116 cells but differ in abundance. The alternative peptide containing H183 was significantly more abundant than the corresponding reference peptide 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 subpolysomal localization. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels by 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 regulation that could reveal inter-individual differences in disease-relevant phenotypes.