Deciphering the Interactome and Functions of Disordered Microproteins

PROJECT SUMMARY The human genome project was expected to define all protein-coding genes, but many blind spots still persist. Microproteins are a recently uncovered class of proteins composed of less than 100-150 amino acids encoded by small open reading frames (smORFs). Previous studies from our group discovered thousands of unannotated microproteins encoded on transcript regions previously assumed to be non-coding, including on annotated long non-coding RNAs and 5’- and 3’-untranslated regions (UTRs). While the majority of microproteins have yet to be functionally characterized, many have been found to regulate fundamental processes, including DNA repair, RNA metabolism, ER stress, and translation, underscoring their significance and diverse biological roles. Our long-term vision is to develop strategies for identifying functions for the remaining thousands of microproteins, which will ultimately expand our understanding of many areas of biology and potentially reveal new therapeutic approaches for different diseases. In this proposal, our goal is to leverage the unique biochemical properties of microproteins to rationally screen for which pathways and processes they participate in. First, microproteins frequently function by interacting with and regulating the activity of larger proteins and protein complexes. In addition, most microproteins are highly disordered and enriched for short linear motifs (SLiMs) and other binding motifs that would allow for interactions between their intrinsically disordered regions (IDRs) and other biomolecules. Based on decades of research on larger annotated proteins, we know that IDRs are essential to many cellular processes, including cell signaling, RNA metabolism, and subcellular organization. We also know that many microproteins harbor the same motifs as annotated proteins involved in these specific processes. We will therefore use a combination of high- throughput screens and chemical tools to identify protein interaction partners for curated sets of disordered microproteins that are likely to function in these processes. Then for each validated microprotein interaction uncovered, we will characterize the microprotein’s role in regulating the activity of the interacting protein(s) and the particular cellular process it acts on. Altogether, these studies will uncover new functional microproteins that will expand our understanding of basic biology and provide new insights into health and disease.