RNA-Dependent Protein Assemblies in Epidermal Homeostasis - RNA-DEPENDENT PROTEIN ASSEMBLIES IN EPIDERMAL HOMEOSTASIS Project Summary/Abstract Dysfunction of epidermal homeostasis can result in a plethora of diseases, such as psoriasis, atopic dermatitis, and skin cancer. The high incidence of skin diseases in about 25% of Americans highlights the need to expand our understanding of epidermal homeostasis mechanisms. Previous studies have shown that RNA-binding proteins (RBPs) are required to control keratinocyte proliferation and differentiation. However, how RBPs organize and collaborate in cis on RNA molecules to form functional ribonucleic protein complexes (RNPs) and how additional layers of regulation, such as post-translational modifications (PTMs), affect their roles in epidermal homeostasis, is currently unexplored. To address this, I have developed, in collaboration with postdoc fellow Dr. Brian Zarnegar, two new crosslinking immunoprecipitation (CLIP)-based methods, termed irCLIP-RNP and Re-CLIP. Both methods leverage the infrared-coupled 3’biotin adapter modification of the original irCLIP protocol to greatly enrich for RNA-dependent cis-RBP assemblies (irCLIP-RNP) and for RNA targets that are simultaneously co-bound by two RBPs of interest (Re-CLIP). These approaches represent a unique framework for studying RBP assembly code and its PTMs on co-bound RNA targets central to epidermal differentiation. Thus, this K99/R00 application aims to characterize the roles of RBP assemblies in epidermal homeostasis. In Aim I, we will test the model of RBP code in epidermal differentiation by comparing two RBPs, HNRNPC and HNRNPU, which we found essential for progenitor maintenance. Preliminary irCLIP-RNP data indicates distinct RBP codes for either HNRNPC or HNRNPU. Aim I.A will test the consequences of knocking out these distinct RBPs on progenitor RNA lifecycles. Aim I.B will analyze the extent to which these distinct combinatorial assemblies collaborate to affect the fate of co-bound RNA targets. In Aim II, we will test the RBP assembly model in untranslated regions (UTRs) in epidermal functions and how PTMs affect this process. Specifically, we will analyze the RBP code of TARDBP, a highly modified RBP, which we found essential in regulating progenitor RNA expression by binding their 3’UTRs. Aim II.A will define the identity, assembly dependencies, and functions of TARDBP and co-associated RBPs to regulate progenitor RNAs through 3’UTR binding. Aim II.B will determine how the increased phosphorylation of TARDBP during epidermal differentiation affects its functional recruitment on target RNA 3’UTRs. My long-term goal is to lead an academic research group focused on skin homeostasis mechanisms. I aim to study RNA-protein regulatory networks in epidermal differentiation, building on my expertise in RNA biology, RBP biochemistry, and bioinformatics. While training at Stanford's Department of Dermatology, I plan to gain new skills in machine learning, proteomics, genomics, and statistics. I will also participate in leadership and management seminars. This comprehensive research and training plan, supported by my mentor and collaborators, will provide me with a broad skillset crucial for a successful transition to independent investigator.
