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

项目概要 mRNA 的转录后控制在通过 RNA 调节重要发育事件中发挥着关键作用 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，它们的丢失与一系列因素有关 的疾病。了解个体 RBP 如何促进组织特异性发育事件是 由于它们普遍存在的表达以及共有 RBP 结合位点的不足，使情况变得复杂。 预测体内占据或调节。这些观察结果表明，目前尚未明确的机制可以提高 特定细胞类型对 RBP 的要求。我们共同发现了一种遗传性神经发育疾病 通过丢失普遍表达的锌指 RBP ZC3H14，并探究了该蛋白在 多种模式生物。黑腹果蝇中的 ZC3H14 直向同源物，Nab2（核聚腺苷酸化 RNA 结合 蛋白 2)，提供了一个易于处理的遗传模型来探测分子和神经发育作用。重要的是， ZC3H14/Nab2 在所有细胞中表达，但在发育中的神经系统的神经元中特别需要 支持活力、脑轴突引导、运动和嗅觉记忆。至关重要的是，人类 ZC3H14 可以 替代果蝇神经元中的 Nab2，表明这两个直向同源物具有相同的分子作用和靶标 RNA。 然而，ZC3H14/Nab2 调节的 RNA 的身份以及提升 ZC3H14/Nab2 作用的机制 神经元中存在的空白限制了人们对 RBP 如何在神经元中发挥关键作用的理解。我们最近的工作 利用果蝇将 Nab2 识别为 N6- 的新型抑制剂，为填补这些空白奠定了基础。 发育中的神经系统中 mRNA 子集上的甲基腺苷 (m6A)。 m6A 沉积在特定的 m6A“作家”Mettl3 在 mRNA 中的位点。 m6A 在发育中的大脑中丰富，发挥着关键作用 在神经发育方面。我们对一种新发现的 Nab2 神经元靶标（编码 Sex Lethal (Sxl) 性别决定因子，表明 Nab2 促进关键的发育调节外显子 通过结合富含 A 的内含子元件并通过 Mettl3 抑制 m6A 沉积来跳过 Sxl pre-mRNA 中的事件。 重要的是，Nab2 对该剪接事件的调节是神经元特异性的，并且通过以下方式拯救多种 Nab2 表型： 同时发生的 Mettl3 丢失意味着 Nab2-m6A 超甲基化模型扩展到其他 RNA 靶标。 我们假设 Nab2/ZC3H14 的神经发育需求是基于一组调节 编码关键神经元蛋白的 mRNA，至少部分是通过这种新的 m6A 抑制作用。我们追求这个 三个目标的机制假设： 目标 1) 利用 Sxl pre-mRNA 作为模型来剖析 Nab2 抑制 m6A沉积；目标 2) 使用 Sxl 剪接报告基因解析 Nab2 和 m6A 调节的直接影响 剪接并确定 Nab2-m6A 途径中的其他因素；目标 3）确定关键的神经发育 Nab2/ZC3H14 在果蝇和脊椎动物中的靶标。总的来说，这些方法将提供洞察力 解释为什么普遍表达的 RNA 结合蛋白的缺失会导致大脑形态发生缺陷。

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.