Nucleation by rRNA Dictates the Precision of Nucleolus Assembly

Falahati, Hanieh; Pelham-Webb, Bobbie; Blythe, Shelby A.; Wieschaus, Eric

doi:10.1016/j.cub.2015.11.065

Cited by 169 publications

(174 citation statements)

References 42 publications

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“…For instance, the presence of actively transcribed RNA can modulate the demixing behavior of certain phase-separating proteins (7,16). In addition, posttranslational modifications, such as phosphorylation of proteins, can regulate their localization to membraneless organelles, potentially by changing intermolecular interactions that govern LLPS (17)(18)(19).…”

mentioning

confidence: 99%

Independent active and thermodynamic processes govern the nucleolus assembly in vivo

Falahati

Wieschaus

2017

Proc. Natl. Acad. Sci. U.S.A.

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104

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Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision.liquid-liquid phase separation | intracellular phase transition | membrane-less organelle | RNA granule | Drosophila nucleologenesis M embraneless organelles are highly concentrated assemblies of proteins and RNAs that provide specialized microenvironments for particular cellular functions (1). Recent studies suggest that such organelles may form via a liquid-liquid phase separation (LLPS) process, in which the constituent components spontaneously assemble on reaching a critical concentration at a given temperature (2-5). LLPS provides an attractive energyefficient mechanism for cells to organize different biochemical reactions spatially, whereas the liquid nature of the emerging organelles, such as P granules and nucleoli (2, 6), allows for rapid exchange of molecules. The role of LLPS has been supported by studies in which the purified RNA binding proteins that localize to such subcellular bodies in vivo also self-assemble in vitro (3-11). However, because of the complexity of living cells, our current understanding of the role of LLPS in membraneless organelle assembly is by far limited to in vitro studies. Particularly, membraneless organelles are multicomponent, and the interactions between different components can enhance or diminish the ability of individual proteins to phase separate inside living cells (12). Therefore, the behavior of the individua...

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mentioning

confidence: 99%

Independent active and thermodynamic processes govern the nucleolus assembly in vivo

Falahati

Wieschaus

2017

Proc. Natl. Acad. Sci. U.S.A.

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104

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“…62,63 In several instances, most notably the nucleolus, the seeding event is transcription of a specific RNA (e.g., rRNA), although other types of seeding events are possible. 62,[64][65][66][67] Identifying seeding events is key to understanding assembly of particular nuclear bodies during development and in specific cell types. The third concept postulates that nuclear body assembly results in a phase transition characterized by liquid-liquid demixing.…”

Section: Hlb Assemblymentioning

confidence: 99%

Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

2017

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Metazoan replication-dependent (RD) histone genes encode the only known cellular mRNAs that are not polyadenylated. These mRNAs end instead in a conserved stem-loop, which is formed by an endonucleolytic cleavage of the pre-mRNA. The genes for all 5 histone proteins are clustered in all metazoans and coordinately regulated with high levels of expression during S phase. Production of histone mRNAs occurs in a nuclear body called the Histone Locus Body (HLB), a subdomain of the nucleus defined by a concentration of factors necessary for histone gene transcription and premRNA processing. These factors include the scaffolding protein NPAT, essential for histone gene transcription, and FLASH and U7 snRNP, both essential for histone pre-mRNA processing. Histone gene expression is activated by Cyclin E/Cdk2-mediated phosphorylation of NPAT at the G1-S transition. The concentration of factors within the HLB couples transcription with pre-mRNA processing, enhancing the efficiency of histone mRNA biosynthesis. KEYWORDSCell cycle; Drosophila; histone genes; mRNA processing; nuclear bodyProper utilization of genetic information in eukaryotes requires extensive three-dimensional organization of the genome and its associated proteins within the nucleus. The basic unit of genome organization is the nucleosome, an octamer of two molecules of each of the four core histone proteins (H2A, H2B, H3 and H4) that packages »147 base pairs of DNA. In mammalian cells, approximately 4£10 8 molcules of each core histone must be synthesized during S phase of the cell cycle to package the newly replicated DNA. Defects in this process result in genome instability that can contribute to human pathologies like cancer. Histone protein synthesis results from a large increase in production at the beginning of S phase of equal amounts of mRNAs encoding the four core nucleosomal histones, as well as histone H1. This highly coordinated gene expression event is performed by a set of transcription and pre-mRNA processing factors unique to replication dependent (RD) histone mRNA biosynthesis that assemble into a nuclear body tightly associated with RD histone genes called the histone locus body (HLB). HLB formation involves a combination of ordered and stochastic assembly steps, and cell cycle regulated changes in the HLB occur to activate histone gene expression during S phase. Here we describe the history of how the HLB was discovered followed by a discussion of HLB assembly mechanism and how the HLB functions in RD histone mRNA biosynthesis.

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“…In Drosophila, fibrillarin positive bodies form during cleavage stages, but these are variable in size and number before stabilization by transcription. 5 In zebrafish, fibrillarin foci -spatially distinct from CBsform much later during gastrulation, reflecting the late onset of rDNA transcription and likely nucleolus formation. Taken together, these observations suggest that transcription of histone and rDNA genes drives the nucleation of specific nuclear bodies at their respective chromosomal sites.…”

Section: Model For the Formation And Maintenance Of Discrete Nuclear mentioning

confidence: 99%

“…For example, nucleoli -the sites of rDNA transcription and rRNA processing -arise as rDNA transcription is activated, and it is well-known that multiple small nucleoli fuse into a bigger nucleolus as the cell cycle proceeds. 5,6 However, nucleoli do not fuse over time with other distinct nuclear bodies. How does specificity among membraneless compartments arise?…”

Section: Introductionmentioning

confidence: 99%

Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos

et al. 2016

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Nuclear bodies are cellular compartments that lack lipid bilayers and harbor specific RNAs and proteins. Recent proposals that nuclear bodies form through liquid-liquid phase separation leave the question of how different nuclear bodies maintain their distinct identities unanswered. Here we investigate Cajal bodies (CBs), histone locus bodies (HLBs) and nucleoli -involved in assembly of the splicing machinery, histone mRNA 3 0 end processing, and rRNA processing, respectively -in the embryos of the zebrafish, Danio rerio. We take advantage of the transcriptional silence of the 1-cell embryo and follow nuclear body appearance as zygotic transcription becomes activated. CBs are present from fertilization onwards, while HLB and nucleolar components formed foci several hours later when histone genes and rDNA became active. HLB formation was blocked by transcription inhibition, suggesting nascent histone transcripts recruit HLB components like U7 snRNP. Surprisingly, we found that U7 base-pairing with nascent histone transcripts was not required for localization to HLBs. Rather, the type of Sm ring assembled on U7 determined its targeting to HLBs or CBs; the spliceosomal Sm ring targeted snRNAs to CBs while the specialized U7 Sm-ring localized to HLBs, demonstrating the contribution of protein constituents to the distinction among nuclear bodies. Thus, nucleolar, HLB, and CB components can mix in early embryogenesis when transcription is naturally or artificially silenced. These data support a model in which transcription of specific gene loci nucleates nuclear body components with high specificity and fidelity to perform distinct regulatory functions.

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Nucleation by rRNA Dictates the Precision of Nucleolus Assembly

Cited by 169 publications

References 42 publications

Independent active and thermodynamic processes govern the nucleolus assembly in vivo

Independent active and thermodynamic processes govern the nucleolus assembly in vivo

Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos

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