The role of Polr1D in development and disease: insights from Drosophila melanogaster

Abstract

In eukaryotes, two DNA-dependent RNA polymerases (Pols), Pols I and III, are responsible for the bulk of cellular transcription. Pols I and III transcribe the rRNAs incorporated into ribosomes, in a process known as ribosome biogenesis. POLR1D is a shared subunit of Pol I and III, POLR1D heterodimerizes with POLR1C an essential interaction for the function of both Pols. Mutations in POLR1D cause Treacher Collins Syndrome (TCS), a craniofacial disorder that arises from impaired ribosome biogenesis in a neuronal stem cell population. We found that RNAi knockdown of Polr1D in several non-neural Drosophila melanogaster tissues caused developmental defects. Here, we identified that a Drosophila endocrine tissue is especially sensitive to disruptions in Polr1D during development. This work broadens the role of Polr1D, suggesting that disruptions in human Polr1D might impact additional cell types during development. In addition, we utilized a Drosophila model carrying a clinically relevant mutation in Polr1D to evaluate chemical compounds which suppress TCS phenotypes. We experimentally validated the phenotypic rescue of our Drosophila Polr1D mutant larvae with a known chemical suppressor and provided two treatment approaches for high-throughput screening. Our findings highlight the utility of Drosophila as a scalable, genetically tractable platform that bridges the gap between cell-based assays and resource-intensive mammalian models for therapeutic discovery. Finally, we provide bioinformatic and in vivo evidence for three Drosophila POLR1D (dPOLR1D) splice isoform transcripts. Structural modeling indicated that the protein isoforms are completely conserved apart from their N-termini, which vary in length and sequence. In vitro assays showed that longer N-terminal isoforms exhibit reduced binding to dPOLR1C and decrease heterodimer stability. Importantly, we found that the human POLR1D (hPOLR1D) gene also encodes multiple splice variants resulting in unique N-terminal lengths and sequences. hPOLR1D isoforms with longer N-termini showed reduced hPOLR1C binding and decreased heterodimer stability, mirroring the pattern observed in Drosophila and supporting a conserved role for POLR1D isoform N-termini in modulating heterodimer function. Together, these results broaden our understanding of Polr1D in developmental biology, disease modeling, and heterodimer interactions.NAUpstate Medical UniversityBiochemistry & Molecular BiologyPh