The Protein Coded by a Short Open Reading Frame, Not by the Annotated Coding Sequence, Is the Main Gene Product of the Dual-Coding Gene MIEF1 .

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 a same gene is unknown. MIEF1 , a gene recently identified as a dual-coding gene, harbors 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 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 incorporating altORFs and small proteins in functional annotations.