CRK regulation of ribosome biogenesis and craniofacial development

CRK 对核糖体生物发生和颅面发育的调节

• 批准号: 10274187
• 负责人: Mason McCool
• 金额: $ 3.17万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-29 至 2023-09-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10274187
• 关键词: 17p13.3; Adaptor Signaling Protein; Affect; Animal Model; Area; Biogenesis; Breast Epithelial Cells; CRK gene; Cardiovascular system; Cells; Cleft Palate; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Congenital Abnormality; Craniofacial Abnormalities; Defect; Development; Disease; Doctor of Philosophy; Embryo; Embryonic Development; Enzymes; Excision; Exhibits; Guanosine; Hela Cells; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Situ Hybridization; In Situ Nick-End Labeling; Injections; Knock-out; Knockout Mice; Laboratories; Lead; MCF10A cells; Mandibulofacial Dysostosis; Measures; Mediating; Messenger RNA; Miller-Dieker Syndrome; Modeling; Molecular; Morphology; Mus; Mutation; Neural Crest; Neural Crest Cell; Northern Blotting; Nose; Oligonucleotides; Oncoproteins; Optical Coherence Tomography; Organism; Output; Pathogenesis; Pathway interactions; Phenotype; Process; Production; Proteins; Publishing; RNA; RNA Stability; Rana; Regulation; Ribosomal Proteins; Ribosomes; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Small Nucleolar RNA; Stains; Study models; TP53 gene; Testing; Time; Tissues; Western Blotting; Work; Xenopus; biological adaptation to stress; cell motility; cell type; collaborative approach; craniofacial; craniofacial development; decapping enzyme; experimental study; genome-wide; human tissue; insight; mRNA Stability; mRNA decapping; microdeletion; migration; molecular pathology; mouse model; novel; proto-oncogene protein c-crk; rRNA Precursor; receptor; tissue/cell culture

PROJECT SUMMARY/ABSTRACT Background and Overall Hypothesis: Ribosome biogenesis, the process by which the protein synthesizing machinery is produced, is essential and required in all cells. Mutations in factors required for ribosome production give rise to a subset of diseases called ribosomopathies. These diseases are surprisingly characterized by tissue-specific abnormalities despite the essential role of ribosomes in all cell types. Craniofacial specific defects, occurring in established ribosomopathies such as Treacher Collins syndrome, manifest from neural crest cell sensitivity to the nucleolar stress response during embryonic development. Miller-Dieker lissencephaly syndrome caused by microdeletion (17p13.3) also leads to craniofacial abnormalities, but with no defined molecular mechanism or connection to ribosome biogenesis thus far. These microdeletion phenotypes are further defined by inclusion of CRK resulting in more severe craniofacial defects. Accordingly, CRK’s role in development has been more precisely established with Crk (-/-) null mice exhibiting improper nasal development and cleft palate. CRK is a protooncogenic adapter protein that is present in several signaling pathways controlling cellular outputs such as proliferation and migration. Recently, the Baserga laboratory identified an additional role for CRK as a novel human ribosome biogenesis factor through a genome-wide siRNA screen in MCF10A breast epithelial cells (Farley-Barnes et al 2018). My preliminary results indicate that CRK depletion leads to inhibition of early pre-rRNA processing steps in ribosome biogenesis through controlling the stability of the essential U8 small nucleolar RNA (snoRNA). Furthermore, I have identified a promising CRK-mediated pathway for controlling U8 stability by U8 snoRNA trimethyl guanosine cap removal via the DCP2 decapping enzyme. I hypothesize that this newly identified role of CRK in ribosome biogenesis contributes to its importance in craniofacial development. Specific Aims: I propose to elucidate the molecular mechanisms underlying CRK’s control of human ribosome biogenesis in tissue culture cells. I will test for changes in U8 snoRNA and DCP2 protein localization and U8 snoRNA trimethyl guanosine cap abundance upon CRK depletion. Then I will test whether CRK directly interacts with the DCP2 containing decapping complex. Secondly, I will determine the extent to which CRK’s essential role in craniofacial development is connected to its role in ribosome biogenesis in the model organism Xenopus tropicalis. I will use morpholino oligos and CRISPR to deplete and knockout crk so that I can observe changes in embryonic morphology, neural crest development, pre-rRNA processing, and p53 nucleolar stress response induction. My proposed work will define CRK’s molecular role in craniofacial development through its connection to ribosome biogenesis for the first time. Additionally, my work will provide insight into the molecular basis of ribosomopathy tissue-specific defects and signaling control of pre-rRNA processing, areas that remain poorly understood.

项目总结/摘要 背景和总体假设：核糖体生物合成，蛋白质合成的过程 机器是产生的，是所有细胞所必需的。核糖体产生所需因子的突变 引起一种叫做核糖体病的疾病令人惊讶的是，这些疾病的特点是 组织特异性异常，尽管核糖体在所有细胞类型中起着重要作用。颅面特定缺陷， 发生于确定的核糖体病，如Treacher柯林斯综合征，表现为神经嵴细胞 胚胎发育过程中对核仁应激反应的敏感性。Miller-Dieker二氏无脑畸形 由微缺失（17p13.3）引起的综合征也会导致颅面畸形，但没有明确的 迄今为止的分子机制或与核糖体生物发生的联系。这些微缺失表型是 进一步定义为包括CRK，导致更严重的颅面缺损。因此，CRK在以下方面的作用 用Crk（-/-）缺失小鼠表现出不适当的鼻发育， 和腭裂CRK是一种原癌衔接蛋白，存在于多种信号通路中， 细胞输出，如增殖和迁移。最近，Baserga实验室确定了一个额外的作用， CRK作为一种新的人核糖体生物合成因子，通过全基因组siRNA筛选MCF 10A乳腺癌 上皮细胞（Farley-Barnes et al 2018）。我的初步结果表明CRK缺失导致抑制 核糖体生物发生中的早期前rRNA加工步骤，通过控制必需的U8的稳定性 核仁小RNA（snoRNA）。此外，我已经确定了一个有前途的CRK介导的途径，用于控制 通过DCP 2脱帽酶去除U8 snoRNA三甲基鸟苷帽的U8稳定性。我假设 CRK在核糖体生物发生中的新发现的作用有助于其在颅面神经系统中的重要性， 发展具体目标：我建议阐明CRK控制的分子机制， 组织培养细胞中人核糖体生物发生。我会检测U8 snoRNA和DCP 2蛋白的变化 定位和CRK耗竭后U8 snoRNA三甲基鸟苷帽丰度。然后我会测试 CRK直接与含有脱帽复合物的DCP 2相互作用。其次，我将确定 CRK在颅面发育中的重要作用与其在核糖体生物合成中的作用有关， 模式生物热带爪蟾。我将使用吗啉寡核苷酸和CRISPR来耗尽和敲除crk， 我可以观察到胚胎形态的变化，神经嵴的发育，前rRNA的加工，和p53 核仁应激反应诱导我的工作计划将确定CRK在颅面神经中的分子作用， 第一次通过它与核糖体生物发生的联系来促进发育。此外，我的工作将提供 深入了解核糖体病组织特异性缺陷的分子基础和前rRNA的信号控制 加工，仍然知之甚少的领域。