Role of zinc finger protein ZC3H8 in ribosomal DNA transcription

Abstract

As a dedicated site for ribosome biogenesis, the nucleolus is one of the most resource-demanding organelles in eukaryotic cells. Over time, the nucleolus has adapted to integrate ribosome biogenesis with stress responses. Emerging evidence suggests that the nucleolus functions as a central hub for a variety of nuclear processes, achieved in part through the dynamic shuttling of proteins between the nucleolus and its surrounding nucleoplasm. As such, the nucleolus represents a highly dynamic entity, with both its morphology and function being sensitive to internal and external cues. Importantly, alterations in nucleolar structure and activity are characteristic of numerous human diseases, including cancer, neurodegeneration, and aging. Consequently, considerable effort has been dedicated to identifying compounds that target different aspects of ribosome biogenesis. Ribosomal DNA (rDNA), located within the nucleolus, is the most transcriptionally active area across the human genome. Due to its repetitive nature, rDNA is particularly susceptible to recombination events and, as a result, mutations. Moreover, high transcriptional activity at the rDNA loci also increases the likelihood of conflicts between DNA replication and transcription. Thus, the nucleolus represents a specialized genomic region that poses significant challenges for maintenance and preservation. In this study, we identified ZC3H8 (zinc finger CCCH-type containing 8) as a nucleolar protein that plays a crucial role in the regulation of rDNA transcription. We demonstrated that the nucleolar retention of ZC3H8 is tightly coupled with active rDNA transcription, and its loss results in significant impairment of rRNA synthesis. Furthermore, we identified DHX9 (RNA Helicase A), an R loop resolver involved in rRNA synthesis, as an interacting partner of ZC3H8, with both proteins shown to be functionally coordinated in rRNA synthesis. Loss of DHX9 led to the concentration of ZC3H8 within the nucleolus, accompanied by a drastic increase in nucleolar R loops. In vitro studies revealed that ZC3H8 preferentially binds single stranded nucleic acids. Based on these findings, we propose a model in which ZC3H8 regulates rDNA transcription by modulating R loop metabolism through binding to the protruding RNA strand within the RNA: DNA hybrid structure. This interaction likely stabilizes the DHX9 unwound intermediates, facilitating the complete resolution of R loops and preventing potential genomic instability.