Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts

Abstract

The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression.

Keywords: Alu repeat-containing RNA; RNA-protein droplets-like structures; RNA-protein interactions; cell cycle; intracellular phase separation; nuclear bodies.

Figure 1.

Cell cycle-dependent structural changes of the nucleolus. Confocal laser scanning microscopy (CLSM) images showing the nucleolar marker proteins nucleolin (NCL, red, stably expressed RFP-NCL) and nucleophosmin (NPM, green, immunofluorescence) with DNA (DAPI, blue) counterstaining in U2OS cells at different stages of the cell cycle. As evident from the NCL and NPM distribution, nucleoli are still completely disrupted during telophase and fully assembled during interphase. Scale bars, 10 µm.

Figure 2.

Impaired post mitosis nucleolus assembly through Pol II inhibition. (A) CLSM images showing NCL (red, stably expressed RFP-NCL), NPM (green, immunofluorescence) and DNA (DAPI) in U2OS cells treated with nocodazole (100 ng/ml) and either actinomycin D (50 ng/ml), α-amanitin (50 µg/ml) or cycloheximide (50 µg/ml) for 5 h. (B) Cells 60 min after they were released from the nocodazole block. (C) Cells 180 min after they were released from the nocodazole block. Scale bars, 10 µm.

Figure 3.

Model for RNA and cell cycle-dependent (dis)assembly of the nucleolus. (A) Scheme of RNA-driven liquid-liquid phase separation of the nucleolus. The interaction of nucleolar proteins like NCL or NPM (blue and green respectively) with aluRNAs might trigger a conformational change of their unstructured regions that drives the assembly of nucleolar subdomains via a liquid-liquid phase separation process into larger domains representing functional nucleoli. Depletion of aluRNAs induces a change of this equilibrium back into the dispersed state with small nucleolar droplets. (B) As illustrated in the enclosed scheme, assembly and disassembly of the nucleolus are dependent on the interactions between RNA transcripts (aluRNAs) and proteins (NCL, NPM). The RNA-protein assemblies form nucleolar domains that associate with rDNA and efficiently support Pol I transcriptional activity. Protein-RNA interactions could be dependent on the cell cycle state, which affects posttranslational modifications of the proteins, changes in their concentration and/or exposition to competing interacting species, like for example binding of importins to their NLS. Thereby, the formation of RNA-protein assemblies could be coupled to specific cell cycle phases.