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

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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.