The rDNA is biomolecular condensate formed by polymer–polymer phase separation and is sequestered in the nucleolus by transcription and R-loops

• Published in: Nucleic acids research 2021-05, Vol.49 (8), p.4586-4598
• Main Authors: Lawrimore, Josh, Kolbin, Daniel, Stanton, John, Khan, Muznah, de Larminat, Solenn C, Lawrimore, Colleen, Yeh, Elaine, Bloom, Kerry
• Format: Article
• Language: English
• Subjects: Cell Cycle - drug effects; Cell Cycle - genetics; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Nucleolus - genetics; Cell Nucleolus - metabolism; Clinical Trials, Phase I as Topic; DNA, Ribosomal - genetics; DNA, Ribosomal - metabolism; G1 Phase - drug effects; G1 Phase - genetics; G2 Phase Cell Cycle Checkpoints - drug effects; G2 Phase Cell Cycle Checkpoints - genetics; Hydro-Lyases - metabolism; Kinetics; Microtubule-Associated Proteins - metabolism; Molecular Biology; Nuclear Proteins - metabolism; Polymers - chemistry; Polymers - metabolism; Protein Tyrosine Phosphatases - genetics; Protein Tyrosine Phosphatases - metabolism; R-Loop Structures; Ribonuclease H - genetics; Ribonuclease H - metabolism; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; Ribonucleoproteins, Small Nuclear - metabolism; RNA Polymerase I - genetics; RNA Polymerase I - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sirolimus - pharmacology; Up-Regulation; Water - chemistry; Water - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab229

Abstract The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer–polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14’s nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA–binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA–DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer–polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.