Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Newer technologies have led to the recent discovery of hundreds to thousands of new microproteins. The biological functions of a few microproteins have been elucidated, and these microproteins have fundamental roles in biology ranging from limb development to muscle function, highlighting the value of characterizing these molecules. Additionally, a majority of smORFs are found upstream of long open reading frames, called the uORFs, that have been shown to play critical role in regulating translation of the main ORF.

The identification of microprotein-protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of non-specific interactions for microproteins. Here, we test and apply an in-situ proximity tagging method that relies on an engineered ascorbate peroxidase (APEX) to elucidate MPIs. The results demonstrate that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX-tagging to an uncharacterized microprotein called C11orf98 revealed that this microprotein interacts with nucleolar proteins nucleophosmin and nucleolin, demonstrating the ability of this approach to identify novel hypothesis-generating MPIs.

We also characterize the protein-interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while elevated levels of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this process.

Lastly, we report a smORF present upstream and partially overlapping with the Steroidogenic Acute Regulatory (StAR) gene, that encodes a 109 amino acid long StAR microprotein (StAR-MP). Expression studies at transcript and peptide level show highly tissue-specific expression of the StAR microprotein correlating with StAR protein expression in sites of steroidogenesis. Genetic analysis of the smORF start codons revealed translational repression of downstream StAR ORF (dORF) due to the presence of upstream smORF, pointing towards an additional mode of StAR protein regulation.