Proteogenomic Approach to UTR Peptide Identification.

Area of Science:

• Genomics
• Proteomics
• Molecular Biology

Background:

• Recent advances in sequencing highlight the translation of untranslated regions (UTRs), but direct evidence of these translated products in cells is limited.
• Identifying UTR-translated peptides is challenging due to the lack of corresponding sequences for standard peptide spectrum matching in proteogenomic analyses.

Purpose of the Study:

• To develop and validate a proteogenomic approach for identifying peptides translated from UTRs at the proteome level.
• To investigate alternative translation initiation mechanisms, including non-AUG start codons and stop codon readthrough, within UTRs.

Main Methods:

• Construction of a translated UTR (tUTR) database encompassing hypothetical sequences from UTRs, considering non-AUG initiation and stop codon readthrough.
• Application of the tUTR database to analyze mass spectrometry (MS/MS) data from the H1299 cell line.
• Validation of identified UTR peptides using high spectral similarity with synthetic peptides.

Main Results:

• Detection of 52 5'-UTR peptides from 45 genes and 9 3'-UTR peptides from 9 genes using the tUTR database approach.
• Identification of alternative translation initiation sites utilizing non-AUG start codons within 5'-UTRs, consistent with Kozak context.
• Confirmation of amino acid insertion during stop codon readthrough for the MDH1 gene.

Conclusions:

• The developed proteogenomic approach enables the detection of translated amino acid sequences from UTRs, providing direct evidence of UTR translation.
• This method offers a significant advancement over ribosome profiling by identifying the actual translated products, not just translation evidence.
• The findings expand the understanding of the functional proteome by including products derived from UTRs.