Comprehensive discovery and functional characterization of the noncanonical proteome

Area of Science:

• Genomics and Proteomics
• Cancer Biology
• Molecular Cell Biology

Background:

• Understanding the human genome requires identifying all translation products, including novel peptides.
• Noncanonical translation products, particularly those from noncoding RNAs, represent a largely unexplored proteomic landscape.

Purpose of the Study:

• To systematically identify and functionally characterize novel peptides, especially those originating from noncoding RNAs.
• To investigate the role of these novel peptides in cancer cell proliferation, metabolism, and clinical prognosis.

Main Methods:

• Construction of a comprehensive peptide sequencing reference library (11,668,944 open reading frames).
• Ultrafiltration tandem mass spectrometry for novel peptide discovery.
• CRISPR screening to identify peptides involved in tumor cell proliferation.
• Artificial intelligence structure prediction and peptide-protein interaction network analysis.
• Experimental validation including Flag-knockin assays and xenograft models.

Main Results:

• Discovery of 8,945 previously unannotated peptides, with nearly half derived from noncoding RNAs.
• Identification of 1,161 peptides influencing tumor cell proliferation.
• Functional characterization of specific peptides (pep1-nc-OLMALINC, pep5-nc-TRHDE-AS1, pep-nc-ZNF436-AS1, pep2-nc-AC027045.3) involved in mitochondrial and cholesterol metabolism.
• Demonstration that pep5-nc-TRHDE-AS1 and pep2-nc-AC027045.3 significantly affect tumor growth.
• Correlation of dysregulated peptides with clinical prognosis.

Conclusions:

• This study provides a comprehensive map of the noncanonical proteome, revealing numerous novel peptides.
• These newly identified peptides play critical roles in cancer biology, impacting proliferation, metabolism, and tumor progression.
• The findings highlight the therapeutic and diagnostic potential of noncanonical peptides in oncology.