Abstract 395: Identification of mRNA-Binding Proteins that Regulate the Stability of LDL Receptor mRNA through the AU-rich Elements
The messenger RNA stability plays an important role in human LDL receptor (LDLR) gene expression in liver cells. An increase in LDLR mRNA half-life has been identified as the underlying mechanism for the reduced plasma LDL-cholesterol levels in hypercholesterolemic patients that were treated with the new cholesterol-lowering drug berberine (BBR). We have previously demonstrated that BBR increases LDLR mRNA stability through effects on the 905 bp proximal section of the LDLR mRNA 3′untranslated region (UTR) that contains 3 AU-rich elements (AREs). However, it is totally unknown what mRNA binding proteins (MBPs) are involved in the control of LDLR mRNA stability at the constitutive level and upon BBR treatment. To identify these regulatory proteins, we have conducted a comprehensive investigation. First, we established HepG2-derived stable cell lines that express a reporter gene carrying the entire LDLR 3′UTR at the end of firefly luciferase coding region. Second, we constructed a library of MBP specific siRNAs capable of individually knocking down 56 known human MBPs. Thirdly, using biotin-labeled mRNA probe and cytoplasmic S100 fractions of untreated and BBR-treated HepG2 cells we performed RNA-pull down assays, followed by SDS-PAGE and mass spectrometry. We now demonstrate that the luciferase activity and the endogenous LDLR mRNA expression are differentially affected by individually siRNA knockdown of 19 MBPs in HepG2 cells. Among them, reduced expressions of 3 MBPs KSRP, hnRNPD0, and hnRNPI are strongly associated with elevated expressions of the LDLR mRNA and the luciferase activity. Moreover, knockdown of these specific MBPs also abolished the BBR effects. Experiments using truncated and ARE-mutated 3′UTR RNA probes demonstrate that KSRP, hnRNPI, and hnRNPD0 all bind to the ARE-region of 3′UTR and their bindings to LDLR 3′UTR are diminished by BBR treatment. These new findings demonstrate that LDLR mRNA stability is controlled by a group of ARE-binding proteins including KSRP, hnRNPI, and hnRNPD0. Specific interactions of these trans-acting factors with the ARE motifs are regulated by BBR, leading to altered LDLR mRNA half-life. This work provides an insight for understanding the regulation of LDLR expression at the posttranscriptional level.