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
基本信息
- 批准号:10274187
- 负责人:
- 金额:$ 3.17万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:17p13.3Adaptor Signaling ProteinAffectAnimal ModelAreaBiogenesisBreast Epithelial CellsCRK geneCardiovascular systemCellsCleft PalateClustered Regularly Interspaced Short Palindromic RepeatsCo-ImmunoprecipitationsComplexCongenital AbnormalityCraniofacial AbnormalitiesDefectDevelopmentDiseaseDoctor of PhilosophyEmbryoEmbryonic DevelopmentEnzymesExcisionExhibitsGuanosineHela CellsHumanImmunofluorescence ImmunologicImmunoprecipitationIn Situ HybridizationIn Situ Nick-End LabelingInjectionsKnock-outKnockout MiceLaboratoriesLeadMCF10A cellsMandibulofacial DysostosisMeasuresMediatingMessenger RNAMiller-Dieker SyndromeModelingMolecularMorphologyMusMutationNeural CrestNeural Crest CellNorthern BlottingNoseOligonucleotidesOncoproteinsOptical Coherence TomographyOrganismOutputPathogenesisPathway interactionsPhenotypeProcessProductionProteinsPublishingRNARNA StabilityRanaRegulationRibosomal ProteinsRibosomesRoleSignal PathwaySignal TransductionSmall Interfering RNASmall Nucleolar RNAStainsStudy modelsTP53 geneTestingTimeTissuesWestern BlottingWorkXenopusbiological adaptation to stresscell motilitycell typecollaborative approachcraniofacialcraniofacial developmentdecapping enzymeexperimental studygenome-widehuman tissueinsightmRNA StabilitymRNA decappingmicrodeletionmigrationmolecular pathologymouse modelnovelproto-oncogene protein c-crkrRNA Precursorreceptortissue/cell culture
项目摘要
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.
项目概要/摘要
背景和总体假设:核糖体生物发生,蛋白质合成的过程
机械是所有细胞中产生的、必不可少的和必需的。核糖体生产所需因子的突变
引起一种称为核糖体病的疾病。这些疾病的惊人特征是
尽管核糖体在所有细胞类型中都发挥着重要作用,但仍存在组织特异性异常。颅面部特定缺陷,
发生在已确定的核糖体病中,例如特雷彻柯林斯综合征,从神经嵴细胞表现出来
胚胎发育过程中对核仁应激反应的敏感性。米勒-迪克无脑畸形
微缺失(17p13.3)引起的综合征也会导致颅面异常,但没有明确的定义
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
通过包含 CRK 进一步定义导致更严重的颅面缺陷。因此,CRK 在
使用表现出鼻发育不正常的 Crk (-/-) 缺失小鼠更精确地确定了发育
和腭裂。 CRK 是一种原癌衔接蛋白,存在于控制多种信号通路中
细胞输出,例如增殖和迁移。最近,Baserga 实验室确定了一个额外的角色
通过 MCF10A 乳腺全基因组 siRNA 筛选,发现 CRK 作为一种新型人类核糖体生物合成因子
上皮细胞(Farley-Barnes et al 2018)。我的初步结果表明 CRK 耗尽会导致抑制
通过控制必需 U8 的稳定性来研究核糖体生物合成中早期 rRNA 前体加工步骤
小核仁 RNA (snoRNA)。此外,我还发现了一种有前途的 CRK 介导的途径来控制
通过 DCP2 脱帽酶去除 U8 snoRNA 三甲基鸟苷帽来确定 U8 稳定性。我假设
新发现的 CRK 在核糖体生物发生中的作用有助于其在颅面部的重要性
发展。具体目标:我建议阐明 CRK 控制的分子机制
组织培养细胞中的人类核糖体生物合成。我将测试 U8 snoRNA 和 DCP2 蛋白的变化
CRK 耗尽时的定位和 U8 snoRNA 三甲基鸟苷帽丰度。然后我会测试是否
CRK 直接与含有脱帽复合物的 DCP2 相互作用。其次,我将确定范围
CRK 在颅面发育中的重要作用与其在核糖体生物发生中的作用有关
模式生物热带爪蟾。我将使用吗啉代寡核苷酸和 CRISPR 来耗尽和敲除 crk,以便
我可以观察胚胎形态、神经嵴发育、前 rRNA 加工和 p53 的变化
核仁应激反应诱导。我提出的工作将定义 CRK 在颅面部的分子作用
首次通过与核糖体生物发生的联系来发展。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
加工领域,人们仍然知之甚少。
项目成果
期刊论文数量(0)
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Mason McCool其他文献
Mason McCool的其他文献
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{{ truncateString('Mason McCool', 18)}}的其他基金
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:
10461110 - 财政年份:2020
- 资助金额:
$ 3.17万 - 项目类别:














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