First evidence that a protein coded by a short ORF, not by the annotated CDS is the main gene product

Proteogenomics and ribosome profiling concurrently show that genes may code for both a large and one or more small proteins translated from annotated coding sequences (CDSs) and unannotated alternative open reading frames (named alternative ORFs or altORFs), respectively, but the stoichiometry between large and small proteins translated from the same gene is unknown. \textit{MIEF1}, a gene recently identified as a dual-coding gene, harbours a CDS and a newly annotated and actively translated altORF located in the 5’UTR . Here, we use absolute quantification with stable isotope-labeled peptides and parallel reaction monitoring to determine levels of both proteins in two human cells lines and in human colon. We report that the main \textit{MIEF1} translational product is not the canonical 463 amino acid MiD51 protein but the small 70 amino acid alternative MiD51 protein (altMiD51). These results demonstrate the inadequacy of the single CDS concept and provide a strong argument for modernizing functional annotations of genes.