The human genome project was supposed to have identified all protein-coding genes in the human genome, but recent work has revealed that gene finding algorithms had a blind spot; they failed to recognize protein-coding small open reading frames (smORFs). By definition, smORFs contain less than 150 codons and encode peptides or small proteins, collectively referred to as microproteins. Recent advances in the Mass Spectrometry and the Ribosome Profiling technology has led to the discovery of hundreds to thousands of these microproteins. The functional characterization of the microproteins is essential to understand their biological and cellular importance. 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. Bioactive microproteins operate through microprotein-protein interactions (MPIs) and I’ll talk about the application of an in situ proximity tagging method that relies on an engineered ascorbate peroxidase (APEX) to elucidate MPIs. The APEX tagging method has been shown to be superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX-tagging to uncharacterized microproteins called C11orf98 and MIEF revealed their interaction with protein complexes in nucleus and mitochondria respectively, thus, demonstrating the ability of this approach to identify novel hypothesis-generating MPIs.
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