A Nucleolar Paradigm for Pluripotency of Human Embryonic Stem Cells

SUMMARY This project aims to establish a novel mechanism of human stem cell pluripotency control through nucleolar structure integrity for functional ribosome biogenesis (RiBi) and proper genome organization/gene expression, regulated by the nucleolar DEAD-box (DDX) RNA helicase DDX18. This paradigm contrasts with the extensively studied direct transcription and translation control by well-established transcription factors and translation regulators in the nucleoplasm and cytoplasm. The nucleolus has mystified the research community for centuries due to the elusive biophysical principles governing the well-known subcompartmental organization and limited molecular tools interrogating their nuclear functions. Much work on the nucleolus has focused on its role in regulating RNA polymerase I (RNA Pol I) transcription and ribosome biogenesis; however, emerging evidence points to the nucleolus as an organizing hub for many nuclear functions, accomplished via the shuttling of proteins and nucleic acids between the nucleolus and the nucleoplasm. As a result, we know very little about how the nucleolus maintains its membraneless structural integrity necessary to control stem cell-specific RiBi and translation rates, as well as genome organization and gene expression. With the new technology that is becoming available in studying biomolecular condensates and the realization of the roles of DDX RNA helicases in cellular RNP condensates and RNA metabolism, this project will address novel nucleolar functions in controlling conventional hESC pluripotency. Notably, nucleolar functions beyond the RiBi, such as the regulation of nuclear architectures and genome stability in shaping cellular identities, have just begun to be appreciated, providing a window of opportunity to explore a paradigm of pluripotency control beyond the well-established nuclear transcription and cytoplasmic translation machinery. DDX18 is unique among many RNA helicases as it is strictly nucleolus-specific, and its depletion alters gene expression through the impairment of the nucleolus integrity pericentromeric heterochromatin reorganization, providing new evidence supporting the functions of the nucleolus as an inter-chromosomal hub for 3D chromatin organization within ESCs. We hypothesize that DDX18 interacts with specific nucleolar partner proteins and snoRNAs/rRNAs to maintain nucleolar integrity and genome architecture for controlling the pluripotency of hESCs. We propose two aims to test this hypothesis. 1) Define the molecular traits of DDX18 in modulating LLPS for nucleolar structure integrity in hESCs. 2) Establish the nucleolar functions of DDX18 in the organization of chromatin and gene expression to maintain hESC pluripotency. 3) Dissect the roles of nucleolar DDX18 protein partners and RNA targets in maintaining nucleolar structure integrity and hESC pluripotency. Upon the completion of this project, we will establish a novel paradigm in which a nucleolar RNA helicase, namely DDX18, coordinates phase separation and chromatin organization through its unique biophysical properties and functional partnerships with nucleolar cofactors and RNAs in controlling human pluripotency.