Regulators of rDNA array morphology in fission yeast

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions. Cells devote significant energy and resources to produce ribosomes, large protein complexes that carry out protein synthesis. To ensure that ribosome production matches cellular needs, activation of ribosome assembly is regulated by many inputs. One key regulatory target is transcription of the tandemly repeated rRNA genes in the ribosomal DNA arrays. A broad understanding of how cells regulate the organization of the rDNA under different cellular conditions and transcriptional states has been difficult to assess with current tools and model systems. We present a new imaging reporter, a fluorescently tagged DNA-binding protein called GapR, that highlights rDNA spatial organization in fission yeast, a model system that shares similar regulatory networks with mammals. Using GapR, we found that the 3D organization of the rDNA arrays changes with the cell cycle, glucose starvation, RNA pol I inhibition, and TOR signaling. We surveyed hundreds of yeast mutants to identify factors that regulate rDNA spatial organization, discovering that the deletion of ribosome protein genes causes rDNA array expansion. Further experiments indicated that ribosome protein insufficiency activates conserved regulatory signaling pathways as part of a compensatory response. Our work presents the first broad investigation for regulators of rDNA spatial organization in any organism. Importantly, we identify a compensatory response to compromised ribosome biogenesis that could be conserved in mammals.