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

描述（由申请人提供）：智力残疾，以前称为精神发育迟滞，是一种以认知功能显著延迟和适应行为限制为特征的疾病。虽然智力残疾的原因是高度异质性的，遗传缺陷的基础上发生的一些遗传性智力残疾已开始被确定。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，我们的研究小组报告了ZC 3 H14基因的失活突变，该基因编码一种进化上保守的、普遍表达的聚腺苷RNA结合蛋白ZC 3 H14。尽管ZC 3 H14蛋白普遍表达，但具有突变ZC 3 H14的患者显示出非综合征性智力残疾，其中仅脑功能受损。这一发现表明ZC 3 H14在大脑中具有关键的专门作用。对果蝇的研究揭示了一个关键作用，特别是在神经元中。神经元和许多其他细胞类型之间的主要区别是需要高度调节和局部化的翻译控制，特别是在神经突延伸中。虽然ZC 3 H14的稳态定位是核，但我们最近的研究表明，ZC 3 H14的群体存在于原代海马神经元的轴突中。此外，我们还发现ZC 3 H14与80 S核糖体结合。这些结果表明ZC 3 H14的细胞质作用与调节翻译的其他RNA结合蛋白相似。事实上，在果蝇中的初步研究已经确定了ZC 3 H14的果蝇直向同源物与调节翻译的其他两种RNA结合蛋白质脆性X智力迟钝蛋白（FMRP）和共济失调蛋白-2的果蝇直向同源物之间的功能相互作用。由于FMRP和Ataxin-2都调节翻译，这些功能相互作用表明ZC 3 H14也可以调节翻译。鉴于我们的初步研究结果，我们假设ZC 3 H14与特定的RNA调控因子合作，以确保在神经元中的局部翻译。在这个提议中，我们通过以下互补目标研究ZC 3 H14的分子功能来验证我们的假设。在目标1中，我们通过操纵ZC 3 H14水平和检查蛋白质合成来评估ZC 3 H14在调节翻译中的需求，从全局方法转向更有针对性的方法。在目标2中，我们将测试ZC 3 H14和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ZC 3 H14敲除小鼠，提供最佳系统来评估ZC 3 H14丢失的功能和分子后果。广泛的长期目标是提供深入了解如何ZC 3 H14，沿着与其他相互作用的蛋白质，影响转录后事件，并确保适当的高阶脑功能。了解ZC 3 H14的功能是阐明RNA结合蛋白在控制基因表达的空间和时间调节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.