Destabilization of axonal mRNAs by KHSRP complexes during axon regeneration

摘要 这个应用程序询问局部的mrna稳定性如何改变轴突再生能力，重点是 RNA结合蛋白[RBP]KHSRP的贡献。神经系统广泛地利用后遗症。 调节细胞蛋白质组的转录机制以响应细胞外刺激和 发育、功能和对轴突损伤反应的生理环境。因为有一个mRNA 可以被翻译成蛋白质很多倍，一个给定的mRNA在多长时间内可用于翻译影响 从该信使核糖核酸中产生的蛋白质的量。MRNAs的稳定性确实受到调控，与 与限制性商业惯例的相互作用稳定和破坏不同的mRNAs，以及与microRNAs的相互作用 针对一些目标进行降级。MRNAs的翻译显然支持轴突再生，但我们 对轴突mRNAs的稳定性是如何局部调节的知之甚少。我们已经证明了限制性商业惯例 HUD(也称为ELAVL4)和KHSRP(也称为FUBP2、MARTA1和ZBP2)竞争与神经元的结合 含有富AU元素的mRNAs，其中HUD相互作用稳定而KHSRP相互作用破坏靶的稳定 MRNAs。在分子水平上，这种相互作用受到mRNA与HUD或HUD结合亲和力的影响 KHSRP。我们在01-05年间的工作表明，KHSRP的缺失会增加KHSRP的目标mRNA水平，原因是 轴突和树突的过度生长，损害了海马和前额叶皮质的记忆巩固， 并增加前额叶皮质和海马区的突触前活动。KHSRP表示为 成年后，我们惊讶地发现周围神经轴突KHSRP水平在 受伤。轴突KHSRP的这种增加是通过轴突内翻译其编码的mRNA来实现的。我们的 初步数据显示，KHSRP基因敲除小鼠表现出加速的神经再生，这表明 轴突-KHSRP在再生中的内在功能。基于这些观察，我们假设 轴突KHSRP通过调节局部mRNA的稳定性来控制轴突再生的速度。我们 我将通过以下具体目标来验证这一假说：1)KHSRP是否调节三叉神经节轴突再生 通过神经元内在机制？2)轴突KHSRP增加是否通过以下方式限制轴突再生 编码再生相关蛋白的不稳定轴突mRNAs？3)KHSRP的蛋白质 交互作用对轴突内功能有影响吗？在这里完成的研究将开始填补这一点 通过关注轴突启动的RNA-蛋白质相互作用，可以影响信使核糖核酸的存活，从而形成知识鸿沟。 这将提供第一次RBP结构域特异性RNA调节子的亚细胞分析，并将带来第一次 系统评估RNA存活对周围神经再生的贡献。

SUMMARY This application asks how localized mRNA stability modifies axonal regeneration capacity, focusing on contributions of the RNA binding protein [RBP] KHSRP. The nervous system makes extensive use of post- transcriptional mechanisms to regulate cellular proteomes in response to extracellular stimuli and physiologic environments during development, function, & in response to axonal injury. Since one mRNA can be translated into protein many times over, how long a given mRNA is available for translation impacts the amount of protein generated from that mRNA. Stability of mRNAs is indeed regulated, with interactions with RBPs stabilizing & destabilizing different mRNAs, as well as interactions with microRNAs targeting some targets for degradation. Translation of mRNAs clearly supports axon regeneration, but we have little knowledge for how stability of axonal mRNAs is locally regulated. We have shown that the RBPs HuD (also called ELAVL4) and KHSRP (also called FUBP2, MARTA1, & ZBP2) compete for binding to neuronal mRNAs with AU-rich elements, where HuD interaction stabilizes and KHSRP interaction destabilizes target mRNAs. At the molecular level, this interaction is impacted by an mRNA’s affinity for binding to HuD or KHSRP. Our work over years 01-05 show that loss of KHSRP increases KHSRP target mRNA levels, causes excessive axonal and dendritic growth, impairs memory consolidation in hippocampus & prefrontal cortex, and increases presynaptic activity in prefrontal cortex and hippocampus. KHSRP is expressed into adulthood, and we surprisingly find that axonal KHSRP levels rapidly increase in peripheral nerves after injury. This increase in axonal KHSRP occurs through intra-axonal translation of its encoding mRNA. Our preliminary data indicate that KHSRP knockout mice show accelerated nerve regeneration pointing to axon-intrinsic functions for KHSRP in regeneration. Based on these observations, we hypothesize that axonal KHSRP controls the rates of axon regeneration through regulation of localized mRNA stability. We will test this hypothesis with the following specific aims: 1) Does KHSRP regulate PNS axon regeneration through a neuron intrinsic mechanism? 2) Does increased axonal KHSRP limit axon regeneration by destabilizing axonal mRNAs encoding regeneration-associated proteins? and 3) Does KHSRP’s protein interactome influence its intra-axonal functions? Completion of the studies here will begin to fill this knowledge gap by focusing on RNA-protein interactions initiated in axons that can affect mRNA survival. This will provide the first subcellular analyses of RBP domain-specific RNA regulons and will bring the first systematic assessment for contributions of RNA survival to peripheral nerve regeneration.