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.

项目摘要/摘要 背景和总体假设：核糖体生物发生，蛋白质合成的过程 机械是生产出来的，在所有细胞中都是必不可少的，也是必不可少的。核糖体生产所需因子的突变 会导致一系列称为核糖体疾病的疾病。令人惊讶的是，这些疾病的特点是 尽管核糖体在所有细胞类型中发挥重要作用，但仍存在组织特异性异常。头面部特有的缺陷， 发生在已确定的核糖体疾病中，如Treacher Collins综合征，表现为神经脊细胞 胚胎发育过程中对核仁应激反应的敏感性。Miller-Dieker无脑畸形 由微缺失(17p13.3)引起的综合征也会导致头面部异常，但没有明确的定义 到目前为止与核糖体生物发生的分子机制或联系。这些微缺失表型是 进一步定义为包括CRK，导致更严重的头面部缺陷。因此，CRK在 Crk(-/-)缺失的小鼠表现出鼻部发育不正常，这一点得到了更准确的证实 还有腭裂。Crk是一种原致癌适配蛋白，存在于多种信号通路中，控制 细胞输出，如增殖和迁移。最近，巴塞加实验室确定了另一个角色 在MCF10A乳腺中通过全基因组siRNA筛选CRK作为一种新的人类核糖体生物发生因子 上皮细胞(Farley-Barnes等人，2018年)。我的初步结果表明，CRK的耗尽会导致抑制 通过控制必需的U8的稳定性来研究核糖体生物发生中的早期前rRNA加工步骤 小核仁RNA(SnoRNA)。此外，我还发现了一种很有前景的CRK介导的调控途径 U8稳定性通过U8 snoRNA三甲基鸟苷通过DCP2去解酶去除。我假设 这种新发现的CRK在核糖体生物发生中的作用有助于它在颅面部的重要性 发展。具体目的：我建议阐明CRK控制的分子机制 人核糖体在组织培养细胞中的生物发生。我会检测U8 snoRNA和DCP2蛋白的变化 CRK耗竭时的定位和U8 snoRNA三甲基鸟苷帽丰度。然后我会测试一下 Crk直接与含有脱壳复合体的DCP2相互作用。其次，我将确定在多大程度上 哪个CRK在头面部发育中的重要作用与其在核糖体生物发生中的作用有关 热带模式生物非洲爪哇。我将使用吗啉寡核苷酸和CRISPR来耗尽和敲除Crk So 我可以观察到胚胎形态、神经脊发育、前rRNA处理和p53的变化 核仁应激反应诱导。我提议的工作将定义CRK在颅面部的分子作用 首次通过其与核糖体生物发生的联系来进行发育。此外，我的工作将提供 核糖体病组织特异性缺陷的分子基础及Pre-rRNA的信号调控 加工，仍然知之甚少的领域。