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

项目摘要 转录后调控在通过RNA调节重要的发育事件中起着关键作用 结合蛋白(RBPs)，调节RNA命运的关键方面，包括剪接、稳定性和翻译。 限制性商业惯例的发育作用在神经系统中尤其明显，在神经系统中，限制性商业惯例的缺失与阵列有关 疾病的威胁。了解个体限制性商业惯例如何对组织特异性发育事件做出贡献 由于它们普遍普遍表达，以及共识RBP结合位点不足而变得复杂 在体内预测占有率或调节。这些观察结果暗示了一种尚未明确的机制 对特定细胞类型的限制性商业惯例的要求。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，并探讨了该蛋白在 多种模式生物。黑腹盘藻中的ZC3H14同源基因NaB_2(核多腺化RNA结合 蛋白质2)，提供了一个易于处理的遗传模型来探索分子和神经发育的作用。重要的是 ZC3H14/Nab2在所有细胞中都有表达，但仅在发育中的神经系统的神经元中表达 支持生存能力、脑轴突引导、运动和嗅觉记忆。关键是，人类ZC3H14可以 替代果蝇神经元中的NaB2，表明这两个同源基因具有共同的分子作用和靶向RNA。 然而，ZC3H14/NaB2调控的RNA的同一性，以及提升ZC3H14/NaB2在 神经元，是限制对这种限制性商业惯例如何在神经元中发挥关键作用的理解的缺口。我们最近的工作 利用果蝇为填补这些空白提供了基础，通过确定NaB2是一种新的N6- 甲基腺苷(M6A)在发育中的神经系统中的mRNAs子集上。M6A存放在特定位置 M6A‘编写者’METTL3在mRNAs内的位点。M6A在发育中的大脑中含量丰富，在那里它发挥着关键作用 在神经发育方面。我们对一个新发现的NAB2神经元靶点的分析，即编码 性致死(SXL)性别决定因子，表明NaB2促进了一个关键的发育调节外显子- 通过结合富含A的内含子元件并抑制METTL3的m6A沉积来跳过SXL前-mRNA中的事件。 关键的是，这种剪接事件的NaB2调节是神经元特异性的，并通过 并发的METTL3缺失意味着NAB2-m6A超甲基化模型扩展到更多的RNA靶标。 我们假设，对NaB2/ZC3H14的神经发育需求是基于对集合的调节 编码关键神经元蛋白的mRNAs，至少部分是通过这种新的m6A抑制作用。我们追求这一点 三个目的的机制假说：目的1)利用SXL前-mRNA作为模型来剖析NaB_2抑制 目的2)使用SXL剪接报告来解析NaB2和m6A-调控的直接和直接影响 以及目标3)确定关键的神经发育 Nab2/ZC3H14在果蝇和脊椎动物中的靶标。综合起来，这些方法将提供洞察力 为什么无处不在表达的RNA结合蛋白的丢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.