Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3’ untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN , APC , B2M , CASP8 , CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions. Nonstop extension mutations in cancer remain poorly characterized. Here, the authors test the impact of 2335 tumor-derived C-terminal nonstop extensions on protein expression and find the majority to be suppressive; they also analyze their biochemical properties and relation to evolutionary selection patterns.