Ubiquitin-dependent regulation of ribosome function in cell fate determination

 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，使多能干细胞采用特定的命运。分化通常由染色质结构或转录网络的全局变化引起，并且如最近发现的，也可以通过核糖体组成和功能的重塑由调节的翻译驱动。然而，这些“特化核糖体”的形成机制及其在调节分化细胞的翻译输出中的作用仍然难以捉摸。 沃纳博士 鉴定了多亚基泛素E3连接酶CRL 3及其脊椎动物特异性底物衔接子KBTBD 8（CRL 3KBTBD 8）作为细胞命运的重要调节因子。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是必需的。在hESC的神经诱导过程中，CRL 3 KBTBD 8单倍化核糖体生物发生调节因子NOLC 1及其partial TCOF 1，其突变是Treacher柯林斯综合征的基础，Treacher柯林斯综合征是一种颅面发育障碍，其特征在于颅神经嵴细胞的损失。NOLC 1和TCOF 1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，CRL3KBTBD 8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，以引导向特定细胞命运分化。 在这个项目中，Werner博士将建立在这些结果的基础上，并提出（目标1）确定泛素依赖性核糖体特化的分子机制，（目标2）解剖控制这种动态调节的信号网络，（目标3）确定KBTBD8等位基因缺失的小鼠模型中泛素依赖性核糖体调节在颅面发育中的作用。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，从而为治疗Treacher柯林斯综合征的治疗方法的发展提供分子基础。 在他的导师Rape教授的指导下，Werner博士在生物化学、基于质谱的方法和人类胚胎干细胞技术方面获得了广泛的知识，这对成功进行拟议的研究至关重要。与此同时，Werner博士将需要在其申请的指导阶段进行RNA测序和核糖体分析实验、CRISPR介导的基因组编辑和小鼠遗传学方面的额外培训。他将接受顾问和合作者的培训，他们是这些技术的专家和各自研究领域的领导者。除了他的主要导师外，这些顾问和合作者还将参加Werner博士的“导师委员会”，该委员会是他创建的，旨在接收有关他的实验和职业发展指导的反馈。辅导阶段的进一步培训活动将针对Werner博士的专业教育（即国际会议和讲习班，参加机构期刊俱乐部和研讨会）和发展他的辅导技能（即继续监督轮换学生和技术助理，参加讲习班）。 总之，沃纳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的，技术多样的，创新的研究方向，重点是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研究员奠定坚实的基础。

 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.