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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要颅面异常占所有出生缺陷的三分之一，是婴儿死亡的重要原因。神经嵴细胞 (NCC) 产生大部分颅面骨、软骨和结缔组织以及非了解它们的发育对于推进颅面出生缺陷的预防至关重要。干扰众所周知，NCC 的发展是多种颅面疾病的基础，包括特雷彻·柯林斯综合征 (Treacher Collins Syndrome)、这是由 TCOF1、POLR1B、POLR1C 和 POLR1D 突变引起的。 POLR1C 和 POLR1D 是子 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 polr1c 和 polr1d 斑马鱼模型中证明核糖体 RNA 转录减少导致 Tp53 依赖性 NCC 祖细胞死亡，导致颅面异常。然而，全球如何 polr1c 和 polr1d 的中断具体影响 NCC 的发展仍未解决，其贡献 Pol III，将包括 5S 核糖体 RNA 和转移 RNA 在内的非编码 RNA 转录至颅面发展情况不得而知。我推测除了发病机制中 Pol I 转录的破坏之外在 Treacher Collins 综合征中，Pol III 转录也被破坏，并导致组织特异性 phe- 未观察到任何类型。 Pol III 产生的转录本（包括 tRNA）已在多个系统中被证明可以组织特异性表达。对 Pol III 转录的作用产生新的认识在 NCC，我将接受有关分析 NCC Pol III 成绩单变化以及评估效果的培训这些翻译上的变化。据推测，POLR3A（最大的 Pol III 的亚基，导致对 Pol III 转录的不同影响。为了在 NCC 中检验这一假设具体方式，我将使用源自患者成纤维细胞的 hiPSC，其中 POLR3A 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 POLR3A 突变个体的牙齿异常，我希望在一个子集中识别 Pol III 特异性效应 NCC 衍生品。在这个奖项的独立阶段，我将生成新的斑马鱼模型来- 站在发育背景下 Pol III 中特定变异的作用，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提供新的资源以了解 Pol III 转录。总而言之，我将接受必要的培训来分析 Pol III 转录和翻译以及对患者特异性变量进行建模 NCC 的专家，这将构成我的独立研究计划的基础，并进一步推进我的目标：站立并预防颅面出生缺陷。