Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Human transfer RNA (tRNA) anticodon variants can lead to translation errors.
• A specific tRNA variant (G35A) in serine tRNA (tRNASerAGA-2-3) is present in 2% of the human population.
• This variant causes serine to be incorrectly incorporated at phenylalanine codons.

Purpose of the Study:

• To develop and validate a method for quantifying mistranslation caused by tRNA variants in live cells.
• To investigate the molecular characteristics and mistranslation levels associated with the endogenous human tRNA variant (G35A).
• To assess the implications of this natural tRNA mutation for human translation fidelity.

Main Methods:

• Development of a dual fluorescent reporter system for quantifying misincorporation in live human and murine cells.
• Validation of mistranslation levels using mass spectrometry.
• Analysis of β-lymphocytes from the 1000 genomes project for genotype and tRNA characteristics (sequencing, modification, fragmentation).
• Utilizing nanoparticle delivery of the reporter system in pan-genome cell lines.

Main Results:

• The dual fluorescent reporter system successfully quantified serine misincorporation.
• Cells with the A35 minor allele confirmed the expected genotype.
• Endogenous mutant tRNASerAAA showed expression, C32 hypo-modification, and 5'-fragmentation.
• Serine misincorporation was confirmed in pan-genome cell lines via reporter delivery.

Conclusions:

• A naturally occurring human tRNA mutant (G35A) causes mistranslation in cells from healthy individuals.
• This study provides a quantitative method to assess tRNA-mediated mistranslation.
• Findings highlight the impact of genetic variation in tRNA on protein synthesis and suggest potential therapeutic avenues using missense suppressor tRNAs.