1, 2). There was a step-wise decrease in luciferase reporter activity in rASBT3′-luciferase constructs containing increasing sized fragments of the rat ASBT

3′UTR (Fig. 2A). In order to determine whether the effect on luciferase reporter activity was mediated by changes in mRNA stability or translation, mRNA half-life was assessed for the various rASBT-βglobin constructs. Half-life was progressively shorter in constructs containing larger fragments of the rat ASBT 3′UTR (Fig. 2B; Supporting Fig. 3). Basal half-life of the β-globin core construct was 36 hours and decreased to approximately 50 minutes in constructs that incorporated the full 3′UTR of rat ASBT. click here The observed profound destabilizing effect from the 3′UTR of rASBT mRNA well accounts for the repression of luciferase activity by the rat ASBT 3′UTR. As an initial investigation of the potential RNA binding proteins mediating this effect, gel shift assays were

performed using the entire rat or human ASBT 3′UTR (Fig. 2C; Supporting Fig. 4). Four well-characterized RNA binding proteins were investigated to initiate PCI-32765 chemical structure analysis of the mechanisms involved in regulating ASBT mRNA stability; gel shift was observed with extracts from IEC-6 (for rat) or Caco-2 (for human) cells and antibodies directed against HuR and TTP but not Auf-1 or KSRP (Fig. 2C; Supporting Fig. 4). All four of the RNA binding proteins are expressed in ileum and kidney cells and tissues (Fig. 2D). The effect of alterations in HuR expression were first studied using the rASBT3′-luciferase constructs. HuR loss of function was accomplished using HuR siRNA, whereas gain of function was achieved with a wildtype expression vector. Alterations in HuR expression had no effect on the basal luciferase activity

of the reporter construct (Fig. 3A). Luciferase activity of all of the rASBT3′-luciferase constructs was significantly increased when HuR was overexpressed, whereas it was significantly reduced when HuR was knocked down by siRNA (Fig. 3A). In contrast, silencing TTP had the opposite effect on the luciferase activity for the rASBT3′-0.3kb construct (Fig. 3B). Luciferase activity was markedly increased when TTP was silenced, whereas it was reduced when HuR was silenced. The specific effect of HuR silencing was assessed using an rASBT-βglobin construct which incorporated Alanine-glyoxylate transaminase 0.3 kb of the rASBT 3′UTR. This short construct was chosen because the half-life of the longer constructs was too brief for this analysis. Silencing HuR did not alter the half-life of the basal globin reporter construct, but did lead to a more than 75% reduction in the half-life of the construct containing 0.3 kb of the rat ASBT 3′UTR (Fig. 3C). The effects of silencing HuR and TTP on endogenous ASBT mRNA stability were examined in Caco-2 cells because they have been shown to express all of these genes.19 Silencing HuR diminished the stability of ASBT, whereas the opposite effect was seen with TTP (Fig. 3D).