Regulation And Mechanism Of Cpg15 Expression By Hud

Regulation and mechanism of cpg15 expression by HuDCandidate plasticity related gene 15(cpgl5) plays important roles in neurite outgrowth and synaptic plasticity. The expression of cpg15 is induced by various stimuli, such as light stimulation, neural injury and ischemia. To understand the mechanisms underlying the regulation of cpg15 expression, we identified an AU-rich element (ARE) in the 3'untranslated region (3'UTR) by sequence analysis of rat cpg15 mRNA. This element is known to be important in post-transcription regulation via interaction with RNA binding proteins (RBPs). HuD is one of the neuron-specific RBPs playing important roles in neuronal differentiation through modulating multiple processes of RNA metabolism.Our previous studies have shown that in mouse transient global ischemia-reperfusion model, both cpg15 and HuD expression in the hippocampus were parallely induced in a similar pattern during the recovery process after the injury, suggesting a relationship between cpgl5 and HuD expression. To address this possibility, we characterized the effect of HuD over-expression on cpg15 expression in cultured cells. The results demonstrated that both cpg15 mRNA and protein were up-regulated in cells with HuD over-expression, supporting that cpgl5 expression is likely regulated by HuD. However, the cpg15 expression was not significantly regulated in cells with HuD silence, which could be attributed to the functional replacement by other proteins of Hu family.In order to examine the role of the ARE in HuD-regulated cpg15 expression, we determined the effect of HuD on the expression of reporter gene (GFP) bearing or lacking the ARE. The expression of the reporter gene bearing the cpg15 3'UTR was up-regulated by HuD in SH-SY5Y cells, however, the expression of the reporter gene lacking the ARE was not regulated by HuD. Moreover, HuD was unable to regulate the expression of the cpg15 lacking the ARE. The results suggest that HuD-regulated cpg15 expression requires the presence of the ARE. Next, we performed immunoprecipitation to study the importance of the ARE in cpgl5 mRNA-HuD interaction or binding. The results indicate that the ARE mediates the binding, although it seems that other elements may also attribute to the binding. In addition, we also characterized the structure-activity relation of HuD protein in regulation of cpgl5 expression. The results demonstrated that either loss of RRM1 or RRM2 (both domains are important for regulating the stability of targeted mRNA) or the hinge region (for initiation of translation) or RRM3 (for the binding to poly(A)), attenuates the function of HuD in enhancing the cpg15 expression. Furthermore, a double loss of Hinge region and RRM3 significantly reduces the function of HuD. The results indicate that HuD protein regulates the cpg15 expression via the ARE in its 3'-UTR with regard to HuD-mediated cpg15 mRNA stability and translation.Taken together, our results suggest that HuD regulates the cpg15 expression via the 3'-UTR-mediated mechanism, which reguires the presence of the ARE in 3'-UTR is an important for the regulation. In addition, HuD likely plays its roles by regulating the cpg15 mRNA stability and translation.