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：
10551324
负责人：
ANITA H. CORBETT
金额：
$ 38.22万
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/10551324
关键词：
Affect Behavioral Binding Binding Proteins Binding Sites Biological Assay Biological Models Brain Cell Line Cells Central Nervous System Cis-Acting Sequence Clinical Consensus Coupled Data Defect Dendritic Spines Deposition Development Disease Drosophila genus Drosophila melanogaster Elements Event Exons Experimental Genetics FMR1 Female Foundations Genes Genetic Genetic Models Heritability Homologous Gene 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 Neurodevelopmental Disorder Neuronal Differentiation Neurons Nuclear Organ Orthologous Gene Pathway interactions Phenotype Play Polyadenylation Post-Transcriptional Regulation Proteins Public Health Publishing RNA RNA Splicing RNA-Binding Proteins Reader Regulation Reporter Role Site Specific qualifier value Testing Tissues Trans-Activators Transcript Translations Vertebrates Work Zinc Fingers activation-induced cytidine deaminase axon guidance cell type exon skipping fly human disease in vivo in vivo evaluation inhibitor insight mRNA Precursor model organism neurodevelopment novel polyadenosine protein function recruit sex sex determination tool transcriptome unpublished works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.

项目摘要 mRNA的转录后控制在通过RNA调节重要发展事件中起关键作用结合蛋白（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是证据，在神经系统中，它们的损失与数组有关疾病。了解单个RBP对组织特异性发育事件的贡献是如何的由于它们普遍普遍的表达和共识RBP结合位点的不足而复杂化预测体内占用或调节。这些观察结果暗示着不确定的机制在特定细胞类型中对RBP的要求。我们共同发现了遗传性神经发育疾病通过丧失无处不在的表达，锌指rbp ZC3H14并探测了该蛋白质的功能多种模型生物。 D. melanogaster中的ZC3H14 NAB2（核聚腺苷酸RNA结合）蛋白2），为探测分子和神经发育作用提供了可探讨的遗传模型。重要的是， ZC3H14/NAB2在所有细胞中均表达支持可行性，脑轴突指导，运动和嗅觉记忆。至关重要的是，人ZC3H14可以在蝇神经元中代替Nab2，表明这两个直系同源物具有分子角色和靶RNA。然而，ZC3H14/NAB2调节的RNA的身份以及提高ZC3H14/NAB2角色的机制神经元是限制对这种RBP在神经元中如何发挥关键作用的差距。我们最近的工作利用果蝇通过将Nab2识别为N6-的新型抑制剂，为填补这些空白提供了基础发育中的神经系统中mRNA子集的甲基腺苷（M6A）。 M6A以特异性沉积 M6A“作家” Mettl3在mRNA中的站点。 M6A在发育中的大脑中富集，在该大脑中扮演关键角色在神经发育中。我们对一个新鉴定的Nab2神经元靶标的分析，即编码的MRNA 性致命（SXL）性别确定因素，表明NAB2促进了开发的受调节外显子的关键通过结合富含A的内含子元件并抑制METTL3的M6A沉积，在SXL Pre-MRNA中跳过事件。至关重要的是，此剪接事件的NAB2调节是神经特异性的，并且通过多种NAB2表型营救并发的METTL3损失意味着NAB2-M6A高甲基化模型扩展到其他RNA靶标。我们假设NAB2/ZC3H14的神经发育要求是基于对集合的调节至少部分通过这种新颖的M6A抑制作用，编码关键神经元蛋白的mRNA的摄影作品。我们追求这一点三个目标的机械假设：目标1）利用SXL Pre-MRNA作为模型，以剖析NAB2抑制 M6A沉积；目标2）使用SXL剪接记者来解析NAB2和M6A调控的直接和直接效果剪接并确定Nab2-M6A途径中的其他因素；目标3）确定关键的神经发育 Nab2/ZC3H14的靶标在苍蝇和脊椎动物中。综上所述，这些方法将提供洞察力为什么丧失了普遍表达的RNA结合蛋白会导致大脑形态发生缺陷。