Eukaryotic initiation factor 5A hypusine as a negative regulator of adenosine 2B receptor (A2bAR) through interaction with stem loop sequences within the A2bAR 3′-untranslated region

Purpose: The aim of this study was to investigate whether eIF5A hypusine (eIF5A^Hyp) reduces adenosine 2b receptor (A2bAR) gene expression through interaction with highly structured stem-loop sequences within the A2bAR 3′UTR. Methods and Results: Based on real-time PCR and western blotting, expression of A2bAR mRNA was significantly decreased upon treatment with eIF5A^Hyp in mouse embryonic fibroblasts of eIF5A (eIF5A-MEF) and 3T3-L1 cells. Target Scan software and RNAfold web server predicted two different structures formed by stem-loop sequences with overlapping microRNA 27 seed sequences and mutations. The EMSA results showed significantly impaired formation of the wild type (WT) biotin-labeled A2bAR probe (27 base) containing stem loop sequences—eIF5A^Hyp complex by mutation of stem-loop sequences or by eIF5A non-hypusine (eIF5A^Lys). The luciferase reporter assay showed that GC7-induced eIF5A^Lys accumulation increased the activity of pMIR-A2bAR WT containing the same stem-loop sequence in 3T3-L1 cells, whereas the activity with pMIR-A2bAR Mut was increased compared to WT control without dependence on GC7. Oil Red O staining showed that suppression of A2bAR expression (A2bAR siRNA and eIF5A^Hyp) increased the amount of lipid droplet formation and the mRNA levels of lipid droplet-related genes (C/EBP-β, PPAR-γ, FABP4, SREBP-1, and Perilipin). In contrast, overexpression of A2bAR (A2bAR vector, eIF5A^Lys vector, and GC7) significantly decreased the expression of lipid droplet-associated genes and lipid droplet formation. Conclusions: eIF5A^Hyp acts as a negative regulator of A2bAR gene expression through stem loop sequences in A2bAR 3′UTR, allowing differentiation of adipocytes.