ABSTRACT

The negative regulator of G-protein signalling 4 (Rgs4) is linked to several neurologic diseases, e.g. schizophrenia, addiction, seizure and pain perception. Consequently, Rgs4 expression is tightly regulated, resulting in high mRNA and protein turnover. The post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. Here, we show that in neurons the RBP HuR reduces endogenous Rgs4 expression by destabilizing Rgs4 mRNA. Interestingly, in smooth muscle cells, Rgs4 is stabilized by HuR, indicating tissue-dependent differences in HuR function. Using in vitro RNA-based pulldown experiments, we identify the functional AU-rich element (ARE) within the Rgs4 3ʹ-UTR that is recognized and bound by HuR. Bioinformatic analysis uncovered that this ARE lies within a highly conserved area next to a miR-26 binding site. We find that the neuronal-enriched miR-26 negatively influences Rgs4 expression in neurons. Further, HuR and miR-26 act synergistically in fluorescent reporter assays. Together, our data suggest a regulatory mechanism, in which an RBP selectively destabilizes a target mRNA in cooperation with a miRNA and the RISC machinery.

摘要

G蛋白信号传导4（Rgs4）的负调控因子与几种神经系统疾病有关，例如。精神分裂症，成瘾，癫痫发作和疼痛感。因此，Rgs4表达受到严格调节，从而导致高mRNA和蛋白质更新。基因表达的转录后控制是通过RNA结合蛋白（RBP）介导的，该蛋白以组合的方式与mRNA相互作用。在这里，我们显示，在神经元中，RBP HuR通过使Rgs4 mRNA不稳定来减少内源性Rgs4表达。有趣的是，在平滑肌细胞中，Rgs4被HuR稳定，表明HuR功能的组织依赖性差异。使用基于体外RNA的下拉实验，我们确定了功能丰富的AU富集元件（ARE）被HuR识别并绑定的Rgs4 3′-UTR。生物信息学分析发现，该ARE位于miR-26结合位点旁边高度保守的区域内。我们发现，富含神经元的miR-26对神经元中的Rgs4表达产生负面影响。此外，HuR和miR-26在荧光报告基因检测中具有协同作用。总之，我们的数据表明了一种调节机制，其中RBP与miRNA和RISC机制合作选择性地使目标mRNA不稳定。