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

控制关键发育途径所需的保守 RNA 结合蛋白

基本信息

批准号：
10391721
负责人：
ANITA H. CORBETT
金额：
$ 38.3万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10391721
关键词：
Affect Animal Model Behavioral Binding Binding Proteins Binding Sites Biological Assay Biological Models Brain Cell Line Cells Cis-Acting Sequence Clinical Consensus Coupled Data Defect Dendritic Spines Deposition Development Disease Drosophila genus Drosophila melanogaster Elements Event Exons FMR1 Female Foundations Genes Genetic Genetic Models Heritability Human Hypermethylation Individual Inherited Intellectual functioning disability Introns Link Locomotion Maps Mediating Memory Messenger RNA Modeling Modification Molecular Molecular Probes Morphogenesis Morphology Mus Mutation Nervous system structure Neuronal Differentiation Neurons Nuclear Organ Orthologous Gene Pathway interactions Phenotype Play Poly(A)+ RNA Post-Transcriptional Regulation Proteins Public Health Publishing RNA RNA Splicing RNA-Binding Proteins Reader Regulation Reporter Role Site Sum Testing Tissues Trans-Activators Transcript Translations Vertebrates Work Zinc Fingers activation-induced cytidine deaminase axon guidance base brain cell cell type exon skipping experimental study fly human disease in vivo in vivo evaluation inhibitor insight mRNA Precursor neurodevelopment novel polyadenosine protein function recruit sex sex determination tool transcriptome unpublished works

项目摘要

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.

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了解个体 RBP 如何促进组织特异性发育事件是很重要的。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暗示了尚未确定的提升机制。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。 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 作用的机制这些空白限制了我们对此类 RBP 如何在神经元中发挥关键作用的理解。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一部分 mRNA 上的甲基腺苷 (m6A) 沉积在特定的位置。 m6A“写入者”Mettl3 的 mRNA 位点在发育中的大脑中丰富，在大脑中发挥着关键作用。我们对一种新发现的 Nab2 神经元靶标（编码的前 mRNA）进行了分析。 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，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。我们确信 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，我们追求这一点。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 通路中的其他因素；目标 3) 确定关键的神经发育综上所述，这些方法将提供果蝇和脊椎动物中 Nab2/ZC3H14 的靶标。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。