Regulation of rRNA transcription in mammalian tissues

University Of Pennsylvania, Philadelphia PA

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Abstract

Abstract / Project Summary Ribosomal RNAs (rRNAs) comprise >80% of cellular RNA, and their transcription from rDNA repeats by RNA Polymerase I (Pol I) accounts for a bulk of all transcription. The bodies of complex eukaryotes have different ribosome production rates in different cell types. Precise control of rRNA transcription rates is important for normal physiology, and its dysregulation leads to disease. However, though regulation of Pol II activity and the transcription of mRNAs has been dissected in detail in virtually every organ system, the transcriptional control of the most abundant RNA in the cell has been largely ignored, creating a fundamental knowledge gap. Using MIRA R35 funding, we have created new bioinformatic and technical tools for mammalian rDNA and rRNA studies, quantified rRNA transcription dynamics in a complex organ system, mapped ~2200 ChIP-Seq tracks for ~250 transcription factors (TFs) and chromatin proteins to assemble a TF-rDNA atlas, demonstrated that a cell-type-specific TF directly binds and regulates Pol I occupancy and rRNA transcription, and developed a protocol to directly edit rDNA repeats. Our central hypothesis is that normal cellular identity and functioning requires precise rRNA levels, fine-tuned in each cell type through a combination of control mechanisms. We will interrogate this model in the next period of funding through the following projects: PROJECT 1: Defining levels of rRNA regulation across cell types: We will use ChIP-Seq, FISH-Flow, and metabolic labeling to quantify rRNA dynamics in defined primary mouse cell types in homeostatic and stress-recovery states. The goal of this project will be to assemble a foundational map of the key steps in rRNA transcription, processing, or lifespan that are differentially regulated across different mammalian cell types. PROJECT 2: Testing the ‘Ribosome Concentration Hypothesis’: We will use a targeted degradation approach to degrade Pol I and reduce rRNA transcription in a dose-dependent fashion, allowing us to achieve any arbitrary ribosome number between 100% and 30% of normal. We will use this system to test the effects of altered rRNA levels on selective mRNA translation and cell fates in cultured cells and in live animals. PROJECT 3: Editing rDNA repeats to dissect promoters and regulatory regions: rDNA repeats have several unique and poorly understood features, including the unusual structure of their promoters and a cluster of cell-type-specific TFs whose conserved rDNA binding we recently identified. We will use Cas9-guided base editors to systematically edit hundreds of rDNA repeats to dissect promoter sequences and TF motifs. Our approach will rigorously test the model that the combinatorial binding of cell-type-specific TFs to regulatory regions on rDNA controls Pol I occupancy and rRNA transcription in a cell-type-specific manner. The long-term goal of this work is to gain a detailed understanding of how the universal process of rRNA transcription has been customized to meet diverse tissue needs in complex organisms, a question of fundamental importance to normal and disordered eukaryotic biology.