RNA transcription modulates phase transition-driven nuclear body assembly

Abstract

Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid-liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of "extranucleolar droplets" (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism.RNA/protein droplets | intracellular phase separation | Brownian coalescence | Ostwald ripening | Flory-Huggins regular solution theory L iving cells are composed of complex and spatially heterogeneous materials that partition into functional compartments called organelles. Many organelles are vesicle-like structures with an enclosing membrane. However, a large number of RNA and protein-rich bodies maintain a dynamic but coherent structure even in the absence of a membrane. These so-called RNA/ protein granules are found in the cytoplasm and in the nucleus, where they are referred to as nuclear bodies. The mechanisms by which such structures form and stably persist are not well understood. However, recent evidence suggests that these membraneless organelles, such as P granules (1, 2), nucleoli (3), and stress granules (4), are liquid-phase droplets that may assemble by intracellular phase separation (5, 6). This concept is supported by work on synthetic systems, including repetitive protein domains that form droplets in vitro and in the cytoplasm (7) and intrinsically disordered protein domains that show signatures of liquid-liquid phase separation when expressed in the cell nucleus (8).By examining the cell size-dependent assembly of nucleoli in early Caenorhabditis elegans embryos, we previously showed that nucleolar assembly is controlled by a concentration-dependent phase transition (9). Using a simple model based on the de...

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“…Phase transitions have recently been shown to mediate cytoplasmic organization at the subcellular level. The assembly and disassembly of nucleoli and other nuclear bodies cycle between liquid-phase droplets and solid-phase condensations and can be modulated by rRNA transcription (33). Phase transition of the microtubule-associated zinc finger protein plays an essential role in the assembly of the spindle apparatus and its associated components (34).…”

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

“…[8][9][10] Furthermore, a mechanism has been proposed according to which this type of liquid-liquid phase separation involves unstructured domains in RNA-binding proteins. 16 The latter are referred to as low complexity domains or intrinsically disordered domains.…”

“…In support of this view, recent in vitro work has shown the ability of fibrillarin to phase-separate at lower concentrations if yeast RNA was present. 10 It was also demonstrated that the RNA-binding domains and the GAR domain of NCL are important for its localization to the nucleolus. 33,34 This could mean that those domains are important for the protein to phase-separate in an RNA-dependent manner.…”

“…[8][9][10] This description accounts for the selfassembly of the nucleolus as well as that of PML and Cajal bodies, splicing speckles and other nuclear bodies. The partitioning of the nucleus into organelles with specific activities cannot be explained by viewing the nucleus simply as a membrane-confined container for the nucleoplasm -an aqueous solution with high concentrations of proteins and nucleic acids macromolecules.…”

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…These reversible droplets represent physiologically active protein or protein‐nucleic acid “bioreactors” (Hyman et al , 2014), which form through a process known as LLPS (Brangwynne et al , 2015). Such transient membrane‐less organelles have multiple cellular functions, such as p‐granule formation to establish intracellular gradients of RNA transcription (Brangwynne et al , 2009), the enrichment of RNA binding proteins in stress granules (Lin et al , 2015; Molliex et al , 2015), concentrating transcription factors in nucleoli (Berry et al , 2015), and the initiation of microtubule spindle formation (Jiang et al , 2015). However, the functional phase separation of nuclear proteins was shown to be disrupted by C9orf72 GR/PR dipeptide repeats (Lee et al , 2016) and is related to protein aggregation in neurodegeneration (Schmidt & Rohatgi, 2016).…”