Abstract 1464 Developing a Novel Multiplexed Screening Assay for Low-Complexity Domains

The function of proteins is traditionally inferred from their structures. However, many proteins contain low-complexity domains (LCDs) that do not fold to form stable structures. In the past decade, there has been a growing interest in how this "dark proteome" contributes to the function of proteins through forming protein-protein interactions. Many RNA binding proteins (RBPs), in particular, contain low complexity domains, yet the molecular determinants of their interactions are not fully understood. The RNA-binding Fox (Rbfox) proteins are a highly conserved family of RBPs with critical roles in regulating alternative splicing in multiple physiological processes. In the chromatin compartment of the nucleus, Rbfox is bound to a large assembly of splicing regulators (LASR), which include hnRNP M, hnRNP C, hnRNP H, Matrin3, NF110, NF45, and DDX5. The Rbfox/LASR complex can self-assemble into higher-order structures through Rbfox's C-terminal domain (CTD). This CTD contains an LCD characterized by repetitive tyrosine residues essential for its self-assembly and ability to activate splicing. However, other sequences within the CTD that mediate either its self-assembly or splicing activity are not defined. We are developing a novel multiplexed binding assay to determine the molecular drivers of Rbfox self-assembly. This method would allow the testing of hundreds of variants by linking each variant with a peptide barcode. As proof of concept, we have shown that proteins encoding peptide barcodes can be expressed, purified, and detected via mass spectrometry. In addition, a co-immunoprecipitation assay can distinguish binding between wildtype and mutant variants of the CTD. We are currently working on coupling the co-immunoprecipitation assay with the peptide barcode readout to test more mutants. This method can provide insight into the biochemical mechanism behind Rbfox's homotypic interactions and should be applicable to studies of other protein-protein interactions. We gratefully acknowledge the support of the National Institutes of Health Maximizing Access to Research Careers (NIH-MARC) program, the National Science Foundation California Alliance for Minority Participation (NSF-CAMP), and the National Institute of General Medical Sciences (NIGMS) for their invaluable funding that made this research possible.