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Ubiquitin-dependent regulation of ribosome function in cell fate determination

细胞命运决定中核糖体功能的泛素依赖性调节

基本信息

批准号：
9109623
负责人：
Achim Werner
金额：
$ 12.26万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-13 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9109623
关键词：
Adopted Alleles Architecture Binding Biochemistry Biogenesis Biological Assay Biological Models Biology Brain CUL3 gene Cell model Cell physiology Cells Cephalic Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Core Protein Defect Detection Development Development Plans Disease Educational workshop Event Feedback Forcible intercourse Foundations Genetic Genome engineering Global Change Health Institution International Journals Knockout Mice Knowledge Laboratories Lead Ligase Mandibulofacial Dysostosis Mass Spectrum Analysis Mediating Mentors Mentorship Modification Molecular Molecular Machines Mus Mutation Neural Crest Neural Crest Cell Neuraxis Organism Output Pattern Phase Phenotype Pluripotent Stem Cells Post-Translational Protein Processing Process Professional Education Protein Biosynthesis Proteins Recombinant DNA Regulation Research Research Personnel Ribosomal Proteins Ribosomal RNA Ribosomes Role Rotation Signal Transduction Stem cells Students Supervision Techniques Time Tissues Training Training Activity Translations Ubiquitin Work base career development craniofacial craniofacial development developmental disease differential expression genome editing human embryonic stem cell human embryonic stem cell line innovation insight mouse model novel therapeutic intervention paralogous gene programs reconstitution relating to nervous system research study ribosome profiling skills symposium therapeutic development tool ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Metazoan development requires the precise execution of highly coordinated differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation is frequently brought about by global changes to chromatin architecture or transcriptional networks, and, as only recently discovered, can also be driven by regulated translation via remodeling of ribosome composition and function. However, mechanisms underlying the formation of these "specialized ribosomes" and their roles in regulating the translational output of differentiating cells have remained elusive. Dr. Werner has identified the multi-subunit ubiquitin E3 ligase CRL3 with its vertebrate-specific substrate adaptor KBTBD8 (CRL3KBTBD8) as an important regulator of cell fate. Specifically, CRL3KBTBD8 is essential for the formation of neural crest cells in a human embryonic stem cell (hESC) model. During neural induction of hESCs, CRL3KBTBD8 monoubiquitylates ribosome biogenesis regulator NOLC1 and its paralog TCOF1, a protein whose mutation underlies Treacher Collins Syndrome, a disorder of craniofacial development characterized by loss of cranial neural crest cells. Ubiquitylation of NOLC1 and TCOF1 promotes interaction of the rDNA synthesis and rRNA modification machinery, thereby altering the translational program of newly produced ribosomes to direct differentiation from a central nervous system fate towards a neural crest stem cell program. Thus, the mechanism of action of CRL3KBTBD8 provides compelling evidence that hESCs employ a ubiquitin-dependent mechanism to regulate ribosome function to guide differentiation towards a specific cell fate. In this project, Dr. Werner will build on thes results and proposes (Aim 1) to determine the molecular mechanisms of ubiquitin-dependent ribosome specification, (Aim 2) to dissect signaling networks controlling this dynamic regulation, and (Aim 3) to determine the role of ubiquitin-dependent ribosome regulation in craniofacial development in a mouse model with deleted KBTBD8 alleles. The results of these studies will uncover fundamental principles of ribosome regulation during cell fate determination, thus providing the molecular basis for the development of therapeutic approaches for treatment of Treacher Collins Syndrome. Under the guidance of his mentor, Prof. Rape, Dr. Werner has gained extensive knowledge in biochemistry, mass spectrometry-based approaches, and human embryonic stem cell techniques, which will be crucial for the successful conduct of the proposed research. At the same time, Dr. Werner will require additional training in performing and analyzing RNA sequencing and ribosomal profiling experiments, CRISPR-mediated genome editing, and mouse genetics during the mentored phase of his application. He will receive this training from advisors and collaborators, who are experts in these techniques and leaders of their respective research fields. In addition to his primary mentor, these advisors and collaborators will also participate in Dr. Werner's "mentorship committee", which he has created to receive feedback on his experimentation and guidance for his career development. Further training activities during the mentored phase will be geared at Dr. Werner's professional education (i.e. international conferences and workshops, participation in institutional journal clubs and seminars) and development of his mentorship skills (i.e. continued supervision of rotation students and technical assistants, participation in workshops). In conclusion, Dr. Werner's strong research application as well as training and career development plan will enable him to launch a vigorous, technically diverse, and innovative research direction with focus on ribosomal and craniofacial biology. This will provide him with a strong foundation to successfully transition into an independent investigator at an academic institution in the US.

描述（由申请人提供）：后生动物的发育需要精确执行高度协调的分化程序，以允许多能干细胞采取特定的命运。分化通常是由染色质结构或转录网络的整体变化引起的，并且最近才发现，分化也可以通过核糖体组成和功能的重塑而受到调节翻译的驱动。然而，这些“特殊核糖体”形成的机制及其在调节分化细胞翻译输出中的作用仍然难以捉摸。维尔纳博士有鉴定出多亚基泛素 E3 连接酶 CRL3 及其脊椎动物特异性底物接头 KBTBD8 (CRL3KBTBD8) 是细胞命运的重要调节剂。具体而言，CRL3KBTBD8 对于人胚胎干细胞 (hESC) 模型中神经嵴细胞的形成至关重要。在 hESC 的神经诱导过程中，CRL3KBTBD8 单泛素化核糖体生物合成调节因子 NOLC1 及其旁系同源物 TCOF1，TCOF1 是一种蛋白质，其突变是 Treacher Collins 综合征的基础，Treacher Collins 综合征是一种以颅神经嵴细胞缺失为特征的颅面发育障碍。 NOLC1 和 TCOF1 的泛素化促进 rDNA 合成和 rRNA 修饰机制的相互作用，从而改变新产生的核糖体的翻译程序，以指导从中枢神经系统命运向神经嵴干细胞程序的分化。因此，CRL3KBTBD8 的作用机制提供了令人信服的证据，证明 hESC 采用泛素依赖性机制来调节核糖体功能，以指导向特定细胞命运的分化。在该项目中，Werner 博士将在这些结果的基础上提出（目标 1）确定泛素依赖性核糖体规范的分子机制，（目标 2）剖析控制这种动态调节的信号网络，以及（目标 3）确定泛素依赖性核糖体调节在删除 KBTBD8 等位基因的小鼠模型中颅面发育中的作用。这些研究的结果将揭示细胞命运决定过程中核糖体调节的基本原理，从而为开发特雷彻柯林斯综合征的治疗方法提供分子基础。在导师 Rape 教授的指导下，Werner 博士获得了生物化学、基于质谱的方法和人类胚胎干细胞技术方面的广泛知识，这对于拟议研究的成功进行至关重要。与此同时，Werner 博士在其申请的指导阶段将需要接受额外的培训，以执行和分析 RNA 测序和核糖体分析实验、CRISPR 介导的基因组编辑以及小鼠遗传学。他将从顾问和合作者那里接受培训，他们是这些技术的专家和各自研究领域的领导者。除了他的主要导师之外，这些顾问和合作者还将参加维尔纳博士的“导师委员会”，该委员会是他创建的，旨在接收他的实验反馈和职业发展指导。指导阶段的进一步培训活动将针对维尔纳博士的专业教育（即国际会议和研讨会、参加机构期刊俱乐部和研讨会）以及他的指导技能的发展（即持续监督轮换学生和技术助理、参加研讨会）。总之，Werner 博士强大的研究应用以及培训和职业发展计划将使他能够启动一个充满活力、技术多样化和创新的研究方向，重点关注核糖体和颅面生物学。这将为他成功转型为美国学术机构的独立调查员奠定坚实的基础。