MicroRNA directed pathway discovery in allergy and asthma

PROJECT SUMMARY Asthma is a highly prevalent chronic inflammatory airway disease that is genetically and mechanistically linked to allergy and dysregulated cytokine-mediated communication between the airway epithelial barrier and tissue-infiltrating immune cells. Th2 responses, insufficiently checked by Tregs, drive chronic allergic inflammation through the release of cytokines that signal through STAT6. Discovering novel regulatory circuits that control this process is a research opportunity with high promise for therapeutic application. In the proposed research, we will investigate RNA circuits that regulate Treg function and the pathobiology of allergy and asthma. Strong preliminary data implicate specific microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and mRNA cis-regulatory sequences as nodes in critical circuits that impact asthma incidence and pathogenesis. The proposed work is organized into 2 scientific aims. Aim 1 focuses on a circuit composed of the abundant miRNA family miR-15/16, its large network of target mRNAs, and the lncRNA MALAT1. miR-15/16 is required in Tregs to safeguard their suppressive function and inhibit allergic lung inflammation. We will functionally characterize the empirically determined target network of miR-15/16 in Tregs, and investigate the role of miR-15/16 interaction with MALAT1. Both miR-15/16 and Malat1 are extremely abundant in T cells, and their physical interaction prevents miR-15/16 from fully suppressing its coding gene targets. We will test the importance of this interaction in allergic lung inflammation using novel transgenic mice with a precise CRISPR-mediated mutation in the miR-15/16 interaction site. Aim 2 probes the biological functions of noncoding mRNA sequences implicated in asthma susceptibility by genetic studies, and implicated as cis-regulatory elements involved in RNA regulation by their association with RNA binding proteins. This work is guided by GCLiPP, a biochemical technique that we developed to map RNA:protein interactions transcriptome-wide. We will interrogate the function of protein-occupied RNA cis-regulatory elements through GCLiPP- directed CRISPR dissection, and conduct a deep analysis of cis-regulatory elements in the STAT6 3’ UTR using conventional reporter assays and CRISPR-directed base editing technology. If successful, the proposed research will illuminate novel RNA regulatory circuits active in allergy and asthma immunopathogenesis, and supply mechanistic insights to their molecular functions.