Defining the neuronal function of the polyadenosine RNA-binding protein ZC3H14

描述(申请人提供)：智力残疾，以前被称为精神发育迟滞，是一种以认知功能显著延迟和适应行为受限为特征的障碍。虽然智力残疾的原因是高度不同的，但一些遗传性智力残疾的发生背后的遗传缺陷已经开始被发现。在与智力残疾有关的基因中，编码RNA结合蛋白的基因与许多病例有关，包括最常见的遗传性智力残疾形式--脆性X综合征。最近，我们小组报道了ZC3H14基因的失活突变，该基因编码一种进化上保守的、普遍表达的多聚腺苷RNA结合蛋白ZC3H14。尽管ZC3H14蛋白普遍表达，但突变的ZC3H14患者表现出非综合征的智能障碍，只有大脑功能受损。这一发现表明，ZC3H14在大脑中具有关键的、专门的作用。对果蝇的研究揭示了一个关键作用，特别是在神经元中。神经元与许多其他类型细胞的一个主要区别是需要高度调控和局部的翻译控制，特别是在轴突延伸中。虽然ZC3H14的稳态定位是核的，但我们最近的研究表明，ZC3H14存在于初级海马神经元的轴突中。此外，我们还发现ZC3H14与80S核糖体结合。这些结果表明，ZC3H14具有类似于其他调节翻译的RNA结合蛋白的细胞质作用。事实上，对果蝇的初步研究已经确定了ZC3H14的果蝇同源物和另外两个调节翻译脆性X智力低下蛋白(FMRP)和Aaxin-2的RNA结合蛋白的同源物之间的功能相互作用。由于FMRP和Aaxin-2都调节翻译，这些功能相互作用表明ZC3H14也可以调节翻译。根据我们的初步发现，我们假设ZC3H14与特定的RNA调节因子协同工作，以确保神经元的局部翻译。在这个建议中，我们通过以下互补目的研究ZC3H14的分子功能来验证我们的假设。在目标1中，我们通过操纵ZC3H14的水平和检查蛋白质合成，从全局方法转向更有针对性的方法，评估了ZC3H14在调节翻译方面的需求。在目标2中，我们将测试ZC3H14与候选翻译调节子之间的物理相互作用。这些研究将利用我们最近开发的ZC3H14基因敲除小鼠，提供一个评估ZC3H14缺失的功能和分子后果的最佳系统。其广泛的长期目标是深入了解ZC3H14与其他相互作用的蛋白质如何影响转录后事件，并确保适当的高阶大脑功能。了解ZC3H14的功能是阐明RNA结合蛋白在控制基因表达的时空调控中的复杂作用的关键一步。

DESCRIPTION (provided by applicant): Intellectual disability, previously termed mental retardation, is a disorder characterized by a significant delay in cognitive function and limitatios in adaptive behaviors. Although the causes of intellectual disability are highly heterogeneous, genetic defects that underlie some occurrences of inherited intellectual disability have begun to be identified. Among the genes linked to intellectual disability, genes encoding RNA- binding proteins have been implicated in a number of cases, including the most common form of inherited intellectual disability, Fragile X Syndrome. Recently, our group reported inactivating mutations in the ZC3H14 gene, which encodes an evolutionarily conserved, ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14. Despite ubiquitous expression of the ZC3H14 protein, the patients with mutant ZC3H14 display non- syndromic, intellectual disability where only brain function is impaired. This finding suggests that ZC3H14 has a critical, specialized role in the brain. Studies in Drosophila reveal a key role specifically in neurons. A major difference between neurons and many other cell types is the need for highly regulated and localized translational control particularly in the neurite extensions. While the steady-state localization of ZC3H14 is nuclear, our recent studies reveal that a population of ZC3H14 is present in axons of primary hippocampal neurons. In addition, we also find that ZC3H14 associates with 80S ribosomes. These results suggest a cytoplasmic role for ZC3H14 that is similar to that of other RNA-binding proteins that regulate translation. Indeed, preliminary studies in Drosophila have identified functional interactions between the Drosophila orthologue of ZC3H14 and Drosophila orthologues of two other RNA-binding proteins that regulate translation Fragile X Mental Retardation Protein (FMRP) and Ataxin-2. As both FMRP and Ataxin-2 regulate translation, these functional interactions suggest that ZC3H14 could also modulate translation. Given our preliminary findings, we hypothesize that ZC3H14 works cooperatively with specific RNA regulatory factors to ensure local translation in neurons. In this proposal, we test our hypothesis by investigating the molecular functions of ZC3H14 through the following complementary aims. In Aim 1, we assess the requirement for ZC3H14 in regulating translation by manipulating levels of ZC3H14 and examining protein synthesis, moving from global approaches to more targeted approaches. In Aim 2, we will test for physical interactions between ZC3H14 and candidate translation regulators. These studies will exploit a ZC3H14 knockout mouse we have recently developed, providing an optimal system to assess the functional and molecular consequences of loss of ZC3H14. The broad long-term goal is to provide insight into how ZC3H14, along with other interacting proteins, influences post-transcriptional events and ensures proper higher order brain function. Understanding the function of ZC3H14 is a critical step in elucidating the complex roles of RNA-binding proteins in controlling spatial and temporal regulation of gene expression.