Abstract 1125 STAT6 3' UTR Region with RNA-binding Protein Occupied Sites and Allergic Risk-Associated SNP Potentially Stabilizes STAT6 mRNA Expression

Overexpression of STAT6 caused by gain-of-function mutations promotes severe allergic phenotypes. Probabilistic Identification of Causal SNPs analysis implicates the single nucleotide polymorphism (SNP) rs1059513 to be highly associated with allergic disease risk. Preliminary data from the Ansel lab showed that it is also located in a region of the STAT6 3' UTR which is occupied by one or more RNA-binding proteins (RBP) in T cells, suggesting an RBP may bind here to influence transcript stability or translation. CRISPR deletion of this region in Jurkat T cells and human primary T cells reduced phosphor-STAT6 (pSTAT6) expression. As deletion of this region reduced pSTAT6 expression, we hypothesized that the region has a stabilizing effect on the STAT6 transcript. To evaluate this, a Dual Luciferase reporter construct was created containing the STAT6 3' UTR region of interest. This plasmid was transfected into Jurkat T cells through electroporation and dual reporter expression was performed to assess the activity of this regulatory element. The insert significantly increased reporter activity compared with an empty control vector, showing that the STAT6 3' UTR region with RBP-occupied sites and a disease-risk-associated SNP has functional activity. The Dual Luciferase assay results also suggest that the region can stabilize the STAT6 transcript and subsequent protein expression. Taken together, these results establish the region as a potential stabilizing element for the expression of STAT6 and indicate it as a potential target to remediate allergic disease. This work was supported by the U.S. National Institutes of Health (NIH) (R25GM144250, R01HL109102, P01HL107202) and the Sandler Asthma Basic Research Center.