Polysomal Profiling Coupled to Allele-Specific Proteomics Reveals an EIF4H TranSNP Allele Possessing Higher mRNA Translation Potential

TL;DR

This study uses genetic and proteomic methods to find variants that influence how efficiently mRNA is translated into proteins.

Contribution

A novel proteomics workflow validates allele-specific mRNA translation using heterozygous coding variants.

Findings

52 coding variants in HCT116 cells show allele-specific polysome association.

The EIF4H R183H variant allele shows higher mRNA and protein levels in polysomes.

A ribosome-stalling assay confirms the variant allele's enhanced translation potential.

Abstract

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 using isotope-labeled peptide standards. Using this approach, we provide proof-of-concept validation of the heterozygous G>A, R183H missense single-nucleotide variant rs1554710467 in the eukaryotic initiation factor 4H (EIF4H) gene. The variant is present in two EIF4H alternatively 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 over-representation 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 interindividual differences in disease-relevant phenotypes.

•Polysomal RNA-seq and PRM proteomics map allele-specific mRNA translation instances.•RNA-seq reveals 52 HCT116 coding variants with allele-specific polysome association.•Label-free, high-pH fractionated PRM workflow quantifies allele-specific peptides.•EIF4H rs1554710467 alternative allele has higher polysomal RNA and peptide levels.

Polysomal RNA-seq and PRM proteomics map allele-specific mRNA translation instances.

RNA-seq reveals 52 HCT116 coding variants with allele-specific polysome association.

Label-free, high-pH fractionated PRM workflow quantifies allele-specific peptides.

EIF4H rs1554710467 alternative allele has higher polysomal RNA and peptide levels.

This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein level, exploiting heterozygous coding variants to identify genetic variants that affect mRNA translation potential.

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