Understanding RNA Polymerase III transcription in neural crest cell and craniofacial development

了解神经嵴细胞和颅面发育中的 RNA 聚合酶 III 转录

• 批准号: 10282921
• 负责人: Kristin Emily Noack Watt
• 金额: $ 10万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10282921
• 关键词: Affect; Award; Biochemical; Biogenesis; Budgets; Cartilage; Cell Culture Techniques; Cell Death; Cell Differentiation process; Cell Proliferation; Cell Survival; Cells; Communities; Congenital Abnormality; Connective Tissue; Craniofacial Abnormalities; Data; Dental; Development; Disease; Embryo; Embryonic Development; Etiology; Face; Fibroblasts; Foundations; Future; Genes; Genetic Transcription; Genomics; Goals; Head; Healthcare; Human; Hypodontia; Individual; Infant Mortality; Knowledge; Lead; Mandibulofacial Dysostosis; Mediating; Mentors; Modeling; Molecular; Mutation; Neural Crest Cell; Pathogenesis; Pathogenicity; Patients; Phase; Phenotype; Polymerase; Population; Prevalence; Prevention; Process; Protein Biosynthesis; RNA Polymerase I; RNA Polymerase III; RNA analysis; RNA chemical synthesis; RNA, Ribosomal, 5S; Regulation; Research; Resources; Ribosomal RNA; Ribosomes; Role; Small RNA; Specificity; Syndrome; System; Testing; Therapeutic Intervention; Tissues; Tooth structure; Training; Transcript; Transfer RNA; Translations; United States; Untranslated RNA; Variant; Work; Zebrafish; base; bone; craniofacial; craniofacial bone; craniofacial development; craniofacial disorder; developmental disease; differential expression; improved; in vivo; induced pluripotent stem cell; migration; mutant; neurodevelopment; novel; prevent; programs; ribosome profiling; spatiotemporal; stem cells; transcriptome sequencing

PROJECT SUMMARY Craniofacial anomalies account for one third of all birth defects and are a significant cause of infant mortality. Neural crest cells (NCC) give rise to the majority of craniofacial bone, cartilage, and connective tissue and un- derstanding their development is crucial for advancing the prevention of craniofacial birth defects. Disruptions in NCC development are known to underlie several craniofacial disorders including Treacher Collins syndrome, which is caused by mutations in TCOF1, POLR1B, POLR1C, and POLR1D. POLR1C and POLR1D are subu- nits of both RNA Polymerases (Pol) I and III and are important for transcription of ribosomal RNA. I previously demonstrated in polr1c and polr1d zebrafish models that ribosomal RNA transcription is reduced leading to Tp53-dependent cell death of NCC progenitors which results in craniofacial anomalies. However, how global disruptions in polr1c and polr1d specifically affect NCC development remains unresolved and the contribution of Pol III, which transcribes non-coding RNAs including 5S ribosomal RNA and transfer RNAs, to craniofacial development is not known. I hypothesize that in addition to disruption of Pol I transcription in the pathogenesis of Treacher Collins syndrome, Pol III transcription is also disrupted and contributes to the tissue-specific phe- notypes observed. Transcripts produced by Pol III, including tRNAs, have been shown in multiple systems to be tissue-specifically expressed. To generate a new understanding of the role of Pol III transcription specifically in NCC, I will receive training in profiling NCC for changes in Pol III transcripts and in evaluating the effect of these changes on translation. It has been postulated that distinct pathogenic variants in POLR3A, the largest subunit of Pol III, lead to differential effects on Pol III transcription. In order to test this hypothesis in a NCC- specific manner, I will use hiPSCs derived from patient fibroblasts with pathogenic variants in POLR3A and analyze them for proliferation, translation, differentiation, and Pol I and III transcription. Given the prevalence of dental anomalies in individuals with mutations in POLR3A, I expect to identify Pol III-specific effects in a subset of NCC derivatives. In the independent phase of this award, I will generate new zebrafish models to under- stand the role of specific variants in Pol III in a developmental context and assess NCC formation, migration, differentiation, and proliferation in combination with the effect on Pol I and III transcription. These models will provide new resources to the research community for the understanding of Pol III transcription. Altogether, I will receive the training necessary to analyze Pol III transcription and translation and model patient-specific var- iants in NCC which will form the foundation of my independent research program and further my goal of under- standing and preventing craniofacial birth defects.

项目摘要 颅面畸形占所有出生缺陷的三分之一，是婴儿死亡的一个重要原因。 神经嵴细胞（NCC）产生大部分颅面骨、软骨和结缔组织， 了解它们的发展对于促进颅面出生缺陷的预防至关重要。中断 在NCC发展中，已知是几种颅面疾病的基础， 这是由TCOF 1，POLR 1B，POLR 1C和POLR 1D突变引起的。POLR 1C和POLR 1D是subu- RNA聚合酶（Pol）I和III的nits，对核糖体RNA的转录很重要。我以前 在polr 1c和polr 1d斑马鱼模型中证明，核糖体RNA转录减少， 导致颅面畸形的NCC祖细胞的TP 53依赖性细胞死亡。然而，如何全球化 polr 1c和polr 1d的中断特别影响NCC的发展，这一问题尚未得到解决， Pol III转录非编码RNA，包括5S核糖体RNA和转移RNA， 发展尚不清楚。我推测，除了在发病机制中破坏Pol I转录外， 在特雷彻柯林斯综合征中，Pol III转录也被破坏，并导致组织特异性phe- 没有观察到任何类型。由Pol III产生的转录物，包括tRNA，已在多个系统中显示， 组织特异性表达。为了对Pol III转录的作用有一个新的认识， 在NCC，我将接受培训，在分析NCC的变化，在Pol III转录本和评估的影响， 这些变化在翻译上。据推测，POLR 3A中不同的致病性变体，最大的致病性变体， 亚基，导致对Pol III转录的差异效应。为了在NCC中验证这一假设- 具体而言，我将使用来源于患者成纤维细胞的hiPSC，其在POLR 3A和 分析它们的增殖、翻译、分化以及Pol I和III转录。鉴于普遍存在的 POLR 3A突变个体的牙齿异常，我希望在一个子集中确定Pol III特异性效应 NCC衍生品在该奖项的独立阶段，我将生成新的斑马鱼模型，以低于- 站在发展背景下Pol III中特定变体的作用，并评估NCC的形成，迁移， 分化和增殖以及对Pol I和III转录的影响。这些模型将 为研究界了解Pol III转录提供新的资源。总之，我 将接受必要的培训，以分析Pol III转录和翻译，并模拟患者特异性变异， 这将成为我独立研究计划的基础，并进一步实现我的目标， 站立和预防颅面出生缺陷。