Phase separation drives X-chromosome inactivation: a hypothesis

Cerase, Andrea; Armaos, Alexandros; Neumayer, Christoph; Avner, Philip; Guttman, Mitchell; Tartaglia, Gian Gaetano

doi:10.1038/s41594-019-0223-0

Cited by 111 publications

(123 citation statements)

References 72 publications

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“…In line with previous reports 9,16 , our analysis of protein interactions indicates that other PrLDs are recruited into condensates. Thus, PrLD interactions could be essential to seed condensates formation 82 and once a critical mass of interactions is reached, the condensate would gather additional molecules establishing a large network of proteinprotein (PPIs) and protein-RNA (PRIs) interactions 68 . One could also hypothesize the intriguing possibility that PrLDs might establish weak interactions with nucleic acids to promote condensation.…”

Section: Discussionmentioning

confidence: 99%

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RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

Gotor

Armaos

Calloni

et al. 2020

Preprint

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Biomolecular condensates are membrane-less organelles mainly composed of proteins and RNAs that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to phase separate is encoded in their sequences, yet it is still unknown which domains drive the process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting biomolecular condensation. Using advanced computational models to predict the phase separation propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-Binding Domains (RBDs). We found that while just one PrLD is sufficient to drive protein condensation, multiple RBDs are needed to modulate the dynamicity of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation.

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Section: Discussionmentioning

confidence: 99%

“…A characteristics associated to phase-separation (long, RNA-binding and highly interacting) 20,68 are intimately connected with the presence of PrLD in Pub1.…”

Section: Incubation Time Sample Analysismentioning

confidence: 99%

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

Gotor

Armaos

Calloni

et al. 2020

Preprint

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“…Acting as a scaffold for a multitude of RNA binding proteins and potentially forming a phase‐separated compartment, Xist recruits members of different gene‐silencing pathways, orchestrating chromosome‐wide gene repression. [ 6–10 ] A small subset of genes however can resist silencing and thus escape X inactivation. [ 11,12 ]…”

Section: Introductionmentioning

confidence: 99%

“…Acting as a scaffold for a multitude of RNA binding proteins and potentially forming a phase-separated compartment, Xist recruits members of different gene-silencing pathways, orchestrating chromosome-wide gene repression. [6][7][8][9][10] A small subset of genes however can resist silencing and thus escape X inactivation. [11,12] All placental mammals studied so far express Xist from one out of two X chromosomes in female somatic cells, indicating that the outcome of XCI is similar across species.…”

Section: Introductionmentioning

confidence: 99%

Dosage Sensing, Threshold Responses, and Epigenetic Memory: A Systems Biology Perspective on Random X‐Chromosome Inactivation

Mutzel

Schulz

2020

BioEssays

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X‐chromosome inactivation ensures dosage compensation between the sexes in mammals by randomly choosing one out of the two X chromosomes in females for inactivation. This process imposes a plethora of questions: How do cells count their X chromosome number and ensure that exactly one stays active? How do they randomly choose one of two identical X chromosomes for inactivation? And how do they stably maintain this state of monoallelic expression? Here, different regulatory concepts and their plausibility are evaluated in the context of theoretical studies that have investigated threshold behavior, ultrasensitivity, and bistability through mathematical modeling. It is discussed how a twofold difference between a single and a double dose of X‐linked genes might be converted to an all‐or‐nothing response and how mutually exclusive expression can be initiated and maintained. Finally, candidate factors that might mediate the proposed regulatory principles are reviewed.

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“…These data suggest that protein interaction networks in the E region contribute to formation of a phase-separated particle, thus playing a role in XIST compartmentalization in vivo. Similar roles for Xist repeats, including in the E region, have recently been proposed based on analysis of Xist particle shape and composition 63 . Importantly, RNP-MaP directly identified hubs of conserved function in the absence of additional information and for RNAs lacking extensive sequence conservation.…”

Section: Discussionmentioning

confidence: 83%

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

Weidmann¹,

Mustoe²,

Jariwala³

et al. 2020

Preprint

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RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but these networks are currently impossible to examine in a comprehensive way.We developed a live-cell chemical probing strategy for mapping protein interaction networks in any RNA with single-nucleotide resolution. This RNP-MaP strategy (RNP network analysis by mutational profiling) simultaneously detects binding by and cooperative interactions involving multiple proteins with single RNA molecules. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein interaction network hubs identify function-critical sites in these RNAs. RNP-MaP identified numerous protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and distinguished communities of proteins that network together on XIST. RNP-MaP data show that the Xist E region is densely networked by protein interactions and that PTBP1, MATR3, and TIA1 proteins each interface with the XIST E region via two distinct interaction modes; and we find that the XIST E region is sufficient to mediate RNA foci formation in cells. RNP-MaP will enable discovery and mechanistic analysis of protein interaction networks across any RNA in cells. RESULTS RNP-MaP ValidationTo comprehensively map protein interaction networks of an RNA of interest, we identified a cellpermeable reagent, NHS-diazirine (SDA), that can rapidly label RNA nucleotides at sites of protein binding. SDA has two reactive moieties: a succinimidyl ester and a diazirine ( Fig. 1a).Succinimidyl esters react to form amide bonds with amines such as those found in lysine side chains. When photoactivated with long-wavelength ultraviolet light (UV-A, 365 nm), diazirines form carbene or diazo intermediates 10 , which are broadly reactive to nucleotide sugar and base moieties. The two-step reaction of SDA thus crosslinks protein lysine residues with RNA with a distance governed by SDA linker length (4 Å) and lysine flexibility (6 Å). Lysine is one of the most prevalent amino acids in RNA-binding domains 11 , and photo-intermediate lifetimes are short; thus, SDA is expected to crosslink short-range RNA-protein interactions relatively independently of local RNA structure or protein properties. To perform crosslinking, live cells are treated with SDA for a short time and then exposed to UV-A light.To detect SDA-mediated RNA-protein crosslinks, we use the MaP reverse transcription technology ( Fig. 1b). SDA-treated cells are lysed and crosslinked proteins are digested to short peptide adducts. Adduct-containing RNA is then used as a template for relaxed-fidelity reverse transcription 12,13 . Under MaP conditions, reverse transcriptase reads through adduct-containing nucleotides and incorporates non-templated nucleotides into the product DNA at the site of RNAprotein crosslinks. Importantly, because transcription reads through adducts, RNP-MaP detects multiple pro...

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Phase separation drives X-chromosome inactivation: a hypothesis

Cited by 111 publications

References 72 publications

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

RNA-Binding and Prion Domains: The Yin and Yang of Phase Separation

Dosage Sensing, Threshold Responses, and Epigenetic Memory: A Systems Biology Perspective on Random X‐Chromosome Inactivation

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

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