Identification of microproteins with transactivation activity by polyalanine motif selection

Microproteins are an emerging class of proteins that are encoded by small open reading frames (smORFs) less than or equal to 100 amino acids. The functions of several microproteins have been illuminated through phenotypic screening or protein-protein interaction studies, but thousands of microproteins remain uncharacterized. The functional characterization of microproteins is challenging due to a lack of sequence homology. Here, we demonstrate a strategy to enrich microproteins that contain specific motifs as a means to more rapidly characterize microproteins. Specifically, we used the fact that polyalanine motifs are associated with nuclear proteins to select 58 candidate microproteins to screen for transactivation function. We identified three microproteins with transactivation activity when tested as GAL4-fusions in a cell-based luciferase assay. The results support the continued use of the motif selection strategy for the discovery of microprotein function.