5′ UTR length shapes alternative N-terminal protein isoforms across cancers and in rare disease

The 5' untranslated region (5' UTR) of an mRNA is classically viewed as a regulatory region that controls the amount of protein production, but not the resulting protein sequence. Here, we demonstrate that 5' UTR length plays a direct role in alternative N-terminal protein isoform production by controlling start codon selection. We find that very short 5' UTRs enhance leaky ribosome scanning, thereby promoting the production of truncated alternative N-terminal protein isoforms. We also show that endogenous changes in 5' UTR length due to alternative transcription initiation can tune the relative abundance of alternative N-terminal isoforms from the same gene. In addition, we identify mutations in rare genetic diseases that alter 5' UTR length, including a deletion in the VHL 5' UTR in von Hippel-Lindau disease that shifts translation toward the shorter VHLp19 isoform. Together, our results implicate 5' UTR length as a determinant of alternative N-terminal isoform production and reveal an underappreciated mechanism by which noncoding changes can reshape the proteome.