A Conserved RNA Binding Protein Required for Control of Key Developmental Pathways

项目摘要 mRNA的转录后调控在通过RNA调控重要发育事件中起着关键作用 结合蛋白（RBP）调节RNA命运的关键方面，包括剪接，稳定性和翻译。 RBP的发育作用在神经系统中尤其明显，在神经系统中，它们的损失与一系列 疾病。了解个体RBP如何促进组织特异性发育事件， 复杂的是它们普遍存在的表达和缺乏共识的RBP结合位点， 预测体内占有或调节。这些观察结果意味着尚未确定的机制， 特定细胞类型中的RBP要求。我们共同发现了一种遗传性神经发育疾病 通过丢失广泛表达的锌指RBP ZC 3 H14，并探测了这种蛋白在 多种模式生物ZC 3 H14在D.黑腹，Nab 2（核多聚腺苷酸RNA结合 蛋白2），提供了一个易于处理的遗传模型，以探测分子和神经发育的作用。重要的是， ZC 3 H14/Nab 2在所有细胞中表达，但在发育中的神经系统的神经元中特异性地需要， 支持生存能力、脑轴突引导、运动和嗅觉记忆。重要的是，人类ZC 3 H14可以 替代了果蝇神经元中的Nab 2，表明这两个直系同源物共享分子作用和靶RNA。 然而，ZC 3 H14/Nab 2调节的RNA的特性，以及提高ZC 3 H14/Nab 2在细胞内作用的机制， 神经元，是限制了解这些RBP如何在神经元中发挥关键作用的差距。我们最近的工作 利用果蝇为填补这些空白提供了基础，将Nab 2鉴定为N6- 甲基腺苷（m6 A）对发育中的神经系统中的mRNA子集的影响。m6 A在特定的温度下沉积 由m6 A 'writer' Mettl 3在mRNA内定位。m6 A在发育中的大脑中富集，在那里它起着关键作用。 在神经发育中。我们分析了一个新发现的Nab 2神经元靶点，即编码Nab 2的前体mRNA。 性致死（Sxl）性别决定因子，表明Nab 2促进一个关键的发育调控外显子- 通过结合富含A的内含子元件并通过Mettl 3抑制m6 A沉积，在Sxl前mRNA中发生跳跃事件。 重要的是，这种剪接事件的Nab 2调节是神经元特异性的，并且通过以下方式拯救多种Nab 2表型： 同时发生的Mettl 3丢失意味着Nab 2-m6 A超甲基化模型扩展到其他RNA靶点。 我们假设Nab 2/ZC 3 H14的神经发育需求是基于对一组 的mRNA编码的关键神经元蛋白，至少部分，通过这种新的m6 A抑制作用。我们寻求这项 1）利用Sxl前体mRNA作为模型来剖析Nab 2抑制， 目的2）使用Sxl剪接报告基因解析Nab 2和m6 A调节的直接和直接效应 目的3）确定Nab 2-m6 A通路中的关键神经发育因子 Nab 2/ZC 3 H14在果蝇和脊椎动物中的靶标。综合起来，这些方法将提供洞察力， 为什么一种普遍表达的RNA结合蛋白的缺失会导致大脑的形态发生缺陷。

Project Summary Post-transcriptional control of mRNAs plays a key role in regulating important developmental events via RNA binding proteins (RBPs) that modulate key aspects of RNA fate, including splicing, stability, and translation. Developmental roles of RBPs are particularly evident in the nervous system, where their loss is linked to an array of diseases. Understanding how individual RBPs contribute to tissue-specific developmental events is complicated by their generally ubiquitous expression and by the insufficiency of consensus RBP-binding sites to predict in vivo occupancy or regulation. These observations imply as yet undefined mechanisms that elevate requirements for RBPs in specific cell types. We co-discovered an inherited neurodevelopmental disease caused by loss of the ubiquitously expressed, zinc finger RBP ZC3H14 and have probed function of this protein in multiple model organisms. The ZC3H14 ortholog in D. melanogaster, Nab2 (nuclear polyadenylated RNA binding protein 2), provides a tractable genetic model to probe molecular and neurodevelopmental roles. Importantly, ZC3H14/Nab2 is expressed in all cells but required specifically in neurons of the developing nervous system to support viability, brain axon guidance, locomotion, and olfactory memory. Critically, human ZC3H14 can substitute for Nab2 in fly neurons, indicating that the two orthologs share molecular roles and target RNAs. However, the identity of ZC3H14/Nab2-regulated RNAs, and mechanisms that elevate the ZC3H14/Nab2 role in neurons, are gaps that limit understanding of how such RBPs play key roles in neurons. Our recent work exploiting Drosophila provides a foundation to fill these gaps by identifying Nab2 as a novel inhibitor of N6- methyladenosine (m6A) on a subset of mRNAs in the developing nervous system. m6A is deposited at specific sites within mRNAs by the m6A ‘writer’ Mettl3. m6A is enriched in the developing brain, where it plays key roles in neurodevelopment. Our analysis of one newly identified Nab2 neuronal target, the pre-mRNA encoding the Sex Lethal (Sxl) sex determination factor, suggests that Nab2 promotes a key developmentally regulated exon- skipping event in Sxl pre-mRNA by binding an A-rich intronic element and inhibiting m6A deposition by Mettl3. Critically, Nab2-regulation of this splicing event is neuron-specific and rescue of multiple Nab2 phenotypes by concurrent Mettl3 loss implies that the Nab2-m6A hypermethylation model extends to additional RNA targets. We hypothesize that the neurodevelopmental requirement for Nab2/ZC3H14 is based on regulating on a set of mRNAs encoding key neuronal proteins, at least in part, via this novel m6A inhibitory role. We pursue this mechanistic hypothesis in three aims: Aim 1) Exploit the Sxl pre-mRNA as a model to dissect Nab2 inhibition of m6A deposition; Aim 2) Use a Sxl splicing reporter to parse direct and direct effects of Nab2 and m6A-regulation of splicing and identify additional factors in the Nab2-m6A pathway; and Aim 3) Identify key neurodevelopmental targets of Nab2/ZC3H14 in both flies and in vertebrates. Taken together, these approaches will provide insight into why loss of a ubiquitously expressed RNA binding protein causes morphogenesis defects in the brain.