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

项目总结 颅面畸形占所有出生缺陷的三分之一，是婴儿死亡的重要原因。 神经脊细胞(NCC)是颅面骨、软骨和结缔组织的主要来源，非成骨细胞是颅面骨、软骨和结缔组织的主要来源。 了解它们的发育对于推进颅面部出生缺陷的预防至关重要。中断 已知在NCC的发展过程中是几种头面部疾病的基础，包括Treacher Collins综合征， 这是由TCOF1、POLR1B、POLR1C和POLR1D突变引起的。POLR1C和POLR1D是Subu- RNA聚合酶(Pol)I和III的Nits对于核糖体RNA的转录都是重要的。我之前 在polr1c和polr1d斑马鱼模型中证明核糖体rna转录减少导致 导致头面部畸形的NCC祖细胞依赖于TP53的细胞死亡。然而，全球化程度 Polr1c和polr1d中的中断具体影响NCC开发的问题仍未解决，其贡献 Pol III转录包括5S核糖体RNA在内的非编码RNA并转移RNAs到颅面部 发展情况尚不清楚。我推测，除了Pol I转录中断在发病机制中 在Treacher Collins综合征中，Pol III转录也被干扰，并有助于组织特异性Phe- 没有观察到任何类型。POL III产生的转录本，包括tRNAs，已经在多个系统中显示出来，以 是组织特异性表达的。对Pol III转录的作用产生新的理解 在NCC，我将接受培训，了解NCC对Pol III成绩单的变化情况，并评估 翻译上的这些变化。据推测，POLR3A中最大的致病变异 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- 具体来说，我将使用来自具有POLR3A和POLR3A致病变异的患者成纤维细胞的HiPSCs 分析它们的增殖、翻译、分化以及Pol I和Pol III转录。鉴于……的流行 POLR3A基因突变个体的牙齿异常，我希望在一个子集中识别Pol III特有的效应 NCC衍生品。在这个奖项的独立阶段，我将产生新的斑马鱼模型来- 了解POLIII特定变异体在发育背景下的作用，评估NCC的形成、迁移、 分化、增殖以及对Pol I和III转录的影响。这些型号将 为研究界了解POLIII转录提供新的资源。总而言之，我 将接受必要的培训，以分析POL III转录和翻译，并模拟患者特定的变量- 这将成为我独立研究计划的基础，并进一步实现我的目标-- 站立和预防头面部出生缺陷。

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.