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) 的负调节因子与多种神经系统疾病有关，例如。精神分裂症、成瘾、癫痫发作和痛觉。因此，Rgs4 表达受到严格调控，导致高 mRNA 和蛋白质周转。基因表达的转录后控制是通过以组合方式与 mRNA 相互作用的 RNA 结合蛋白 (RBP) 介导的。在这里，我们表明在神经元中，RBP HuR 通过破坏Rgs4 mRNA的稳定性来降低内源性 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。使用体外基于 RNA 的下拉实验，我们确定了功能性富含 AU 的元素 (ARE)被 HuR 识别和约束的Rgs4 3ʹ-UTR。生物信息学分析发现，该 ARE 位于靠近 miR-26 结合位点的高度保守区域内。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，选择性地破坏目标 mRNA 的稳定性。