Makorin RING zinc-finger protein 1-dependent translational control of dendritic mRNAs in mammalian neurons

在神经细胞中，不同形式的学习和记忆需要新的蛋白质合成，这可以通过翻译驻留在树突中的特定信使核糖核酸物种在突触局部发生。这些新的蛋白质允许突触特异性的重塑以响应突触的激活。然而，到目前为止，控制树突转化的分子机制还知之甚少。在哺乳动物的脑神经元中，Poly(A)结合蛋白的相互作用伙伴Makorin环锌指蛋白1(MKRN1)与几个树突定位的转录本相关。在大鼠大脑中，它专门在激活的突触处蓄积。此外，功能分析表明，MKRN1刺激神经元中报告mRNAs的翻译启动。这一点和其他证据表明，MKRN1调节突触上树突状mRNAs的活性依赖的翻译。我们的目标是描述MKRN1介导的神经元中的翻译控制机制。特别是，我们将解决以下问题：i.我们将确定不同的突触信号级联是否调制MKRN1的活动。具体地说，我们想分析神经元中个别信号成分的选择性刺激或抑制如何影响MKRN1介导的报告mRNAs的翻译控制。二、翻译是一个高度复杂的过程，涉及的因素很多。我们打算表征在结构和功能上将MKRN1与翻译机器联系起来的分子。含有MKRN1的细胞复合体将被生化提纯，单个成分将通过质谱学进行鉴定。随后，将使用小干扰(Si)RNA技术评估单个蛋白质的生理意义。三、MKRN1是一种真正的RNA结合蛋白。通过对蛋白质/RNA复合体进行体内紫外光交联后的免疫沉淀，我们希望在啮齿类动物的脑中识别MKRN1的mRNA靶点。之后，我们将调查MKRN1是否调控这些mRNAs的翻译。我们希望我们的结果有助于更好地理解作用于树突状mRNAs的活性依赖的翻译控制机制。这将为研究哺乳动物学习和记忆以及认知缺陷的分子过程提供新的见解。

In nerve cells, distinct forms of learning and memory require new protein synthesis, which can occur locally at synapses by translation of select mRNA species residing in dendrites. These new proteins permit synapse-specific remodelling in response to synaptic activation. However, to date the molecular mechanisms controlling dendritic translation are only sparsely understood. In mammalian brain neurons, makorin RING zinc-finger protein 1 (MKRN1), an interaction partner of poly(A)-binding protein, associates with several dendritically localized transcripts. In vivo in the rat brain, it specifically accumulates at activated synapses. Moreover, functional assays show that MKRN1 stimulates translation initiation of reporter mRNAs in neurons. This and additional evidence indicate that MKRN1 regulates activity-dependent translation of dendritic mRNAs at synapses. Our goal is to characterize the MKRN1-mediated translational control mechanisms in neurons. In particular, we will address the following issues: i. We will determine whether distinct synaptic signaling cascades modulate the activity of MKRN1. Specifically, we want to analyze how the selective stimulation or inhibition of individual signaling components in neurons influences MKRN1-mediated translational control of reporter mRNAs. ii. Translation is a highly complex process with numerous factors involved. We intend to characterize molecules that structurally and functionally link MKRN1 to the translation machinery. Cellular complexes harbouring MKRN1 will be biochemically purified and individual components will be identified by mass spectrometry. Subsequently, the physiological significance of individual proteins will be assessed using small interfering (si)RNA technology. iii. MKRN1 is a bona fide RNA-binding protein. By performing immunoprecipitations after in vivo UV-crosslinking of protein/RNA complexes we want to identify MKRN1 mRNA targets in rodent brain. Afterwards we will investigate if MKRN1 regulates translation of these mRNAs. We expect our results to be relevant for a better understanding of activity-dependent translational control mechanisms operating on dendritic mRNAs. This will provide new insights into the molecular processes underlying learning and memory as well as cognitive deficits in mammals.