DNA Damage Triggers a New Phase in Neurodegeneration
Authors: Fabio Pessina, Ubaldo Gioia, Ornella Brandi, Stefania Farina, Marta Ceccon, Sofia Francia, Fabrizio d’Adda di Fagagna
Journal: Trends in Genetics (2020)

Intracellular compartments can assemble as membraneless organelles through a demixing process known as 'liquid–liquid phase separation' (LLPS).DNA damage response foci are membraneless structures fueled by LLPS of some DNA damage response factors and are modulated by noncoding transcripts synthesized at DNA damage sites.Several forms of neurodegeneration are associated with, and possibly caused by, dysfunctional LLPS events, ultimately leading to the accumulation of toxic solid-like structures.Emerging evidence links factors involved in LLPS events and neurodegeneration with the cellular response to DNA damage. Subcellular compartmentalization contributes to the organization of a plethora of molecular events occurring within cells. This can be achieved in membraneless organelles generated through liquid–liquid phase separation (LLPS), a demixing process that separates and concentrates cellular reactions. RNA is often a critical factor in mediating LLPS. Recent evidence indicates that DNA damage response foci are membraneless structures formed via LLPS and modulated by noncoding transcripts synthesized at DNA damage sites. Neurodegeneration is often associated with DNA damage, and dysfunctional LLPS events can lead to the formation of toxic aggregates. In this review, we discuss those gene products involved in neurodegeneration that undergo LLPS and their involvement in the DNA damage response. Subcellular compartmentalization contributes to the organization of a plethora of molecular events occurring within cells. This can be achieved in membraneless organelles generated through liquid–liquid phase separation (LLPS), a demixing process that separates and concentrates cellular reactions. RNA is often a critical factor in mediating LLPS. Recent evidence indicates that DNA damage response foci are membraneless structures formed via LLPS and modulated by noncoding transcripts synthesized at DNA damage sites. Neurodegeneration is often associated with DNA damage, and dysfunctional LLPS events can lead to the formation of toxic aggregates. In this review, we discuss those gene products involved in neurodegeneration that undergo LLPS and their involvement in the DNA damage response. In response to a stimulus or an insult, molecular interactions and enzymatic reactions have to occur promptly within the cell. Equally quickly, once the stimulus ends, interactions have to disengage. Thus, cells constantly face the demanding task of organizing a variety of simultaneous molecular reactions, often in close spatial proximity, yet preserving their selectivity. To address this challenge, cells evolved compartments to facilitate spatiotemporal control of biological reactions. It is emerging that, in addition to confining specific events within organelles delimited by a lipid membrane, cells also exploit the properties of membraneless organelles (MLOs), coherent structures that can compartmentalize and concentrate selected molecules, thus favoring reactions. In the past few years, MLOs have been referred to with various terms, such as 'condensates' or 'droplets,' but essentially they are the result of demixing events due to liquid–liquid phase separation (LLPS) (see Glossary). The formation of MLOs relies on dynamic interactions among little structured proteins and/or nucleic acids, held together by weak intermolecular bonds, which generate a surface tension sufficient to induce phase separation [1.Titus A.R. et al.Interfacial tension and mechanism of liquid–liquid phase separation in aqueous media.Phys. Chem. Chem. Phys. 2020; 22: 4574-4580Crossref PubMed Google Scholar]. Compared with a lipid membrane, surface tension between two phases has the advantage of insulating MLO content from molecules with different biophysical properties, yet granting transience and reversibility [2.Hyman A.A. et al.Liquid–liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar]. Protein components of MLOs generally contain intrinsically disordered regions (IDRs) or low-complexity domains (LCDs), which are more prone to phase separate through their flexibility, lack of predetermined structure, and ability to engage multivalent weak interactions [2.Hyman A.A. et al.Liquid–liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar]. Although there is ample evidence that LCDs are able per se to phase separate in vitro, they are usually embedded as part of proteins also containing structured domains [2.Hyman A.A. et al.Liquid–liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar], raising the question whether such structured regions modulate their LLPS properties. This has recently been investigated for hnRNPA1, an RNA-binding protein (RBP) composed of a folded, structured module and an LCD [3.Milkovic N.M. et al.Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation.bioRxiv. 2020; (Published online May 16, 2020. https://www.biorxiv.org/content/10.1101/2020.05.15.096966v1)Google Scholar]. Differently from the isolated LCD that undergoes LLPS at high salt concentrations, the full-length protein phase separates only at low ionic concentrations, and thanks to the interaction of its LCD with the RNA recognition motifs (RRMs), suggesting that folded regions in a protein contribute to modulation of LLPS [3.Milkovic N.M. et al.Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation.bioRxiv. 2020; (Published online May 16, 2020. https://www.biorxiv.org/content/10.1101/2020.05.15.096966v1)Google Scholar]. Recently, a molecular grammar of phase separation has been proposed, indicating that the percentage and position of specific amino acids in the protein sequence can predict the ability of a protein to undergo LLPS more accurately than its disordered structure [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar]. For example, tyrosine residues in prion-like domains (PrLDs) and arginine residues in folded RNA-binding domains establish multivalent interactions; thus, proteins with structured domains enriched in specific amino acids can be more prone to LLPS than a disordered region devoid of them [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar]. Examples of MLOs in the nucleus are paraspeckles, bodies formed by the ubiquitously expressed long noncoding RNA NEAT1, involved in gene expression regulation [5.Fox A.H. et al.Paraspeckles: where long noncoding RNA meets phase separation.Trends Biochem. Sci. 2018; 43: 124-135Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar]; Cajal bodies, centers of assembly and modification of spliceosomal small nuclear RNPs [6.Neugebauer K.M. Special focus on the Cajal body.RNA Biol. 2017; 14: 669-670Crossref PubMed Scopus (5) Google Scholar]; nucleoli, sites of ribosomal RNA transcription and assembly [7.Latonen L. Phase-to-phase with nucleoli – stress responses, protein aggregation and novel roles of RNA.Front. Cell. Neurosci. 2019; 13: 151Crossref PubMed Scopus (19) Google Scholar]; histone locus bodies, where histone mRNAs are transcribed [8.Frottin F. et al.The nucleolus functions as a phase-separated protein quality control compartment.Science. 2019; 365: 342-347Crossref PubMed Scopus (79) Google Scholar]; and promyelocytic leukemia bodies, involved in multiple processes of genome homeostasis, including homologous recombination at telomeres, in some cancer cell lines [9.Bernardi R. Pandolfi P.P. Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies.Nat. Rev. Mol. Cell Biol. 2007; 8: 1006-1016Crossref PubMed Scopus (635) Google Scholar]. MLOs in the cytoplasm include processing bodies (P-bodies), condensates of enzymes involved in mRNA decay and microRNA (miRNA)-induced mRNA silencing, and stress granules (SGs), which function mainly as reservoirs of untranslated mRNA (Box 1). Some MLOs are constitutive, such as nucleoli, whereas others are transient and need to be efficiently resolved to avoid pathological stabilization, as, for instance, SGs in neurodegeneration [10.Alberti S. Dormann D. Liquid–liquid phase separation in disease.Annu. Rev. Genet. 2019; 53: 171-194Crossref PubMed Scopus (76) Google Scholar, 11.Babinchak W.M. Surewicz W.K. Liquid–liquid phase separation and its mechanistic role in pathological protein aggregation.J. Mol. Biol. 2020; 432: 1910-1925Crossref PubMed Scopus (14) Google Scholar, 12.Elbaum-Garfinkle S. Matter over mind: liquid phase separation and neurodegeneration.J. Biol. Chem. 2019; 294: 7160-7168Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. Although the number of reported examples of MLOs constantly increases, a need for more rigorous analyses and a careful choice of the techniques used to identify and define LLPS in the cell has recently been invoked [13.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (81) Google Scholar].Box 1Stress GranulesStress granules (SGs) are cytoplasmic membraneless organelles (MLOs) with the main function of slowing down mRNA translation thought to guarantee cell survival upon different insults, such as viral infections, oxidative stress, heat shock, and increased osmolarity [64.Protter D.S.W. Parker R. Principles and properties of stress granules.Trends Cell Biol. 2016; 26: 668-679Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar]. Fundamental constituents of SGs are polyadenylated RNAs, stalled translation complex, and various RNA-binding proteins (RBPs) which bear intrinsically disordered regions (IDRs) that promote extensive weak intermolecular interactions [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. The RBPs T cell internal antigen 1 (TIA1) and RAS GTP-activating protein-binding protein 1 (G3BP-1) are constitutive SG components, intrinsically able to undergo liquid–liquid phase separation (LLPS) and considered the minimal set of factors needed to nucleate the formation of these MLOs in response to stress [140.Aulas A. Vande Velde C. Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS?.Front. Cell. Neurosci. 2015; 9: 423Crossref PubMed Google Scholar,141.Panas M.D. et al.Mechanistic insights into mammalian stress granule dynamics.J. Cell Biol. 2016; 215: 313-323Crossref PubMed Scopus (140) Google Scholar].SG nucleation is initiated upon polysome dissolution following translation arrest. This exposes mRNA to TIA1 and G3BP-1 and favors their accumulation on the RNA [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. SG then recruits additional intrinsically disordered RBPs, ultimately promoting phase separation [14,29]. Importantly, this process is reversible, and, once stress is resolved, recovery of translation causes an ATP-dependent SG disassembly [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar,64.Protter D.S.W. Parker R. Principles and properties of stress granules.Trends Cell Biol. 2016; 26: 668-679Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar,141.Panas M.D. et al.Mechanistic insights into mammalian stress granule dynamics.J. Cell Biol. 2016; 215: 313-323Crossref PubMed Scopus (140) Google Scholar].Beyond their physiological relevance, SGs are becoming objects of interest due to a proposed connection with the pathogenesis of various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. Affected tissues of the ALS/FTLD spectrum often harbor TDP-43- and FUS-positive inclusions colocalizing with SG components [10.Alberti S. Dormann D. Liquid–liquid phase separation in disease.Annu. Rev. Genet. 2019; 53: 171-194Crossref PubMed Scopus (76) Google Scholar,63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar,140.Aulas A. Vande Velde C. Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS?.Front. Cell. Neurosci. 2015; 9: 423Crossref PubMed Google Scholar]. However, although SGs devoid of TDP-43 are reversible, TDP-43 incorporation makes them more persistent after stress removal. It has been reported that TIA1 mutations, in a subset of patients with ALS, are accompanied by less dynamic TDP-43-containing SGs [142.Mackenzie I.R. et al.TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.Neuron. 2017; 95: 808-816.e9Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar]. A recent study found that mitoxantrone, a planar compound known to interact with RNAs, prevents the inclusion of TDP-43 into SGs, thus representing a potential therapeutic strategy for ALS/FTD [143.Fang M.Y. et al.Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD.Neuron. 2019; 103: 802-819.e11Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar].Also, the microtubule-associated protein tau was observed to colocalize with TIA1 at SGs in both animal models and postmortem brain tissues of subjects with Alzheimer disease [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. It has been proposed that interactions between SGs and MAPT, possibly due to the high RNA content present within SGs, stimulate MAPT LLPS and aggregations [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. Stress granules (SGs) are cytoplasmic membraneless organelles (MLOs) with the main function of slowing down mRNA translation thought to guarantee cell survival upon different insults, such as viral infections, oxidative stress, heat shock, and increased osmolarity [64.Protter D.S.W. Parker R. Principles and properties of stress granules.Trends Cell Biol. 2016; 26: 668-679Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar]. Fundamental constituents of SGs are polyadenylated RNAs, stalled translation complex, and various RNA-binding proteins (RBPs) which bear intrinsically disordered regions (IDRs) that promote extensive weak intermolecular interactions [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. The RBPs T cell internal antigen 1 (TIA1) and RAS GTP-activating protein-binding protein 1 (G3BP-1) are constitutive SG components, intrinsically able to undergo liquid–liquid phase separation (LLPS) and considered the minimal set of factors needed to nucleate the formation of these MLOs in response to stress [140.Aulas A. Vande Velde C. Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS?.Front. Cell. Neurosci. 2015; 9: 423Crossref PubMed Google Scholar,141.Panas M.D. et al.Mechanistic insights into mammalian stress granule dynamics.J. Cell Biol. 2016; 215: 313-323Crossref PubMed Scopus (140) Google Scholar]. SG nucleation is initiated upon polysome dissolution following translation arrest. This exposes mRNA to TIA1 and G3BP-1 and favors their accumulation on the RNA [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. SG then recruits additional intrinsically disordered RBPs, ultimately promoting phase separation [14,29]. Importantly, this process is reversible, and, once stress is resolved, recovery of translation causes an ATP-dependent SG disassembly [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar,64.Protter D.S.W. Parker R. Principles and properties of stress granules.Trends Cell Biol. 2016; 26: 668-679Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar,141.Panas M.D. et al.Mechanistic insights into mammalian stress granule dynamics.J. Cell Biol. 2016; 215: 313-323Crossref PubMed Scopus (140) Google Scholar]. Beyond their physiological relevance, SGs are becoming objects of interest due to a proposed connection with the pathogenesis of various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. Affected tissues of the ALS/FTLD spectrum often harbor TDP-43- and FUS-positive inclusions colocalizing with SG components [10.Alberti S. Dormann D. Liquid–liquid phase separation in disease.Annu. Rev. Genet. 2019; 53: 171-194Crossref PubMed Scopus (76) Google Scholar,63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar,140.Aulas A. Vande Velde C. Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS?.Front. Cell. Neurosci. 2015; 9: 423Crossref PubMed Google Scholar]. However, although SGs devoid of TDP-43 are reversible, TDP-43 incorporation makes them more persistent after stress removal. It has been reported that TIA1 mutations, in a subset of patients with ALS, are accompanied by less dynamic TDP-43-containing SGs [142.Mackenzie I.R. et al.TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.Neuron. 2017; 95: 808-816.e9Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar]. A recent study found that mitoxantrone, a planar compound known to interact with RNAs, prevents the inclusion of TDP-43 into SGs, thus representing a potential therapeutic strategy for ALS/FTD [143.Fang M.Y. et al.Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD.Neuron. 2019; 103: 802-819.e11Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Also, the microtubule-associated protein tau was observed to colocalize with TIA1 at SGs in both animal models and postmortem brain tissues of subjects with Alzheimer disease [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. It has been proposed that interactions between SGs and MAPT, possibly due to the high RNA content present within SGs, stimulate MAPT LLPS and aggregations [63.Wolozin B. Ivanov P. Stress granules and neurodegeneration.Nat. Rev. Neurosci. 2019; 20: 649-666Crossref PubMed Scopus (59) Google Scholar]. Identification and analysis of MLOs in cells is in fact not always an easy task, and the simple model of a homogeneous MLO has proved to be inadequate to capture the variety of cellular MLOs. Indeed, some MLOs may exhibit multilayered structures with distinct coexisting liquid phases, thanks to different surface tensions, generating 'droplet within droplet' architectures [2.Hyman A.A. et al.Liquid–liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar,14.Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357eaaf4382Crossref PubMed Scopus (740) Google Scholar], with some having partially solid-like portions [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar]. A recent report that condensate can undergo a structural transition from a droplet-like to a hollow vesicle-like form, characterized by a rim and an internal lumen, seems consistent with this notion [15.Alshareedah I. et al.Interplay between short-range attraction and long-range repulsion controls reentrant liquid condensation of ribonucleoprotein–RNA complexes.J. Am. Chem. Soc. 2019; 141: 14593-14602Crossref PubMed Scopus (0) Google Scholar]. Given the potentially metastable nature of phase-separated MLOs, their liquid-like reversible condensed state can eventually evolve toward a more rigid, less dynamic state. This is a consequence of the formation of stronger ionic interactions between molecules, often organized in intermolecular beta sheets, leading to the formation of stable aggregates [16.Patel A. et al.A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.Cell. 2015; 162: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar,17.Hughes M.P. et al.Atomic structures of low-complexity protein segments reveal kinked beta sheets that assemble networks.Science. 2018; 359: 698-701Crossref PubMed Scopus (0) Google Scholar]. These phenomena have a relevant impact on pathologies caused by proteinopathies [10.Alberti S. Dormann D. Liquid–liquid phase separation in disease.Annu. Rev. Genet. 2019; 53: 171-194Crossref PubMed Scopus (76) Google Scholar]. Intriguingly, an important role in the assembly of MLOs and in the regulation of their stability has emerged for RNA molecules. Local synthesis or recruitment of RNA molecules often controls condensation of MLOs [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar,18.Saha S. Hyman A.A. RNA gets in phase.J. Cell Biol. 2017; 216: 2235-2237Crossref PubMed Scopus (9) Google Scholar]. Even before the realization that MLOs result from LLPS events, several of them were reported to assemble in an RNA-dependent manner [19.Drino A. Schaefer M.R. RNAs, Phase separation, and membrane-less organelles: are post-transcriptional modifications modulating organelle dynamics?.Bioessays. 2018; 40e1800085Crossref PubMed Scopus (3) Google Scholar]. Nucleolar RBPs associated with RNA of transcriptionally active rDNA loci were known to form the nucleation point for nucleoli in vivo [20.Berry J. et al.RNA transcription modulates phase transition-driven nuclear body assembly.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5237-E5245Crossref PubMed Google Scholar]. Indeed, transcriptional inhibition has been shown to result in the loss of nucleolar structural organization and delocalization of several nucleolar components [21.Shav-Tal Y. et al.Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.Mol. Biol. Cell. 2005; 16: 2395-2413Crossref PubMed Scopus (233) Google Scholar]. Conversely, triggering ectopic transcription or concentrating RNA locally is sufficient to favor the formation of nuclear bodies, including paraspeckles, histone locus bodies, Cajal bodies, and nuclear stress bodies [22.Mao Y.S. et al.Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs.Nat. Cell Biol. 2011; 13: 95-101Crossref PubMed Scopus (302) Google Scholar,23.Shevtsov S.P. Dundr M. Nucleation of nuclear bodies by RNA.Nat. Cell Biol. 2011; 13: 167-173Crossref PubMed Scopus (221) Google Scholar]. Still, several observations also point to an RNA-mediated increase in fluidity of MLOs, which counteracts a more unsafe liquid-to-solid phase transition [10.Alberti S. Dormann D. Liquid–liquid phase separation in disease.Annu. Rev. Genet. 2019; 53: 171-194Crossref PubMed Scopus (76) Google Scholar,24.Pessina F. et al.Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.Nat. Cell Biol. 2019; 21: 1286-1299Crossref PubMed Scopus (44) Google Scholar]. This suggests that RNA can modulate the viscoelasticity of MLOs, inducing LLPS but preventing their more rigid stabilization. How this occurs remains to be fully understood [25.Rhine K. et al.RNA droplets.Annu. Rev. Biophys. 2020; 49: 247-265Crossref PubMed Scopus (12) Google Scholar]. As mentioned before, the forces driving the formation of LLPS are a set of intermolecular weak interactions that, acting as pulling forces, induce the demixing of a subset of molecules from a homogeneous solution [2.Hyman A.A. et al.Liquid–liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar,4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar,14.Shin Y. Brangwynne C.P. Liquid phase condensation in cell physiology and disease.Science. 2017; 357eaaf4382Crossref PubMed Scopus (740) Google Scholar]. These interactions are often favored by polymeric molecules such as RNA or DNA and by the similarly highly negatively charged poly(ADP-ribose) (PAR) chains [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar,26.Leung A.K.L. Poly(ADP-ribose): a dynamic trigger for biomolecular condensate formation.Trends Cell Biol. 2020; 30: 370-383Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. Thanks to their repetitive and charged nature, these polymers act as concentrating agents and nucleation points that initiate phase separation of proteins [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar,26.Leung A.K.L. Poly(ADP-ribose): a dynamic trigger for biomolecular condensate formation.Trends Cell Biol. 2020; 30: 370-383Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar]. In the case of SGs (Box 1), LLPS is strictly dependent on RNA. In particular, single-stranded, ≥250-nt-long RNA molecules were shown to boost Ras GTPase-activating protein-binding protein 1 (G3BP-1) phase transition in SGs, apparently regardless of any sequence specificity [27.Yang P. et al.G3BP1 is a tunable switch that triggers phase separation to assemble stress granules.Cell. 2020; 181: 325-345.e28Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. In more general terms, proteins containing IDRs often bear RNA-binding domains, too, leading to a cooperative effect favoring LLPS [4.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (1069) Google Scholar]. RNA promotes phase separation in a dose-dependent manner and, intriguingly, sometimes in a sequence-specific fashion, suggesting that LLPS condensates might bear specific densities based on the sequence of the RNA retained [28.Langdon E.M. et al.mRNA structure determines specificity of a polyQ-driven phase separation.Science. 2018; 360: 922-927Crossref PubMed Scopus (152) Google Scholar]. RNA concentration acts by lowering viscosity inside droplets, thus promoting more dynamic interactions and liquidity [10.Alberti S. Dormann D. Liquid–liquid phase separation in disease.Annu. Rev. Genet. 2019; 53: 171-194Crossref PubMed Scopus (76) Google Scholar]. At super-stoichiometric ratios, RNA can trigger the formation of vesicle-like condensate with a lumen inside [15.Alshareedah I. et al.Interplay between short-range attraction and long-range repulsion controls reentrant liquid condensation of ribonucleoprotein–RNA complexes.J. Am. Chem. Soc. 2019; 141: 14593-14602Crossref PubMed Scopus (0) Google Scholar]. Nonetheless, it appears that there are exceptions, and in vitro studies with the P-granule RBP LAF-1 demonstrated that its phase separation is controlled by the combination of length, number of ionic interactions per molecule, and concentration of RNA molecules. Indeed, short RNA molecules decrease LAF-1 droplet viscosity, whereas the same mass of a longer RNA increases it [29.Wei M.T. et al.Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.Nat. Chem. 2017; 9: 1118-1125Crossref PubMed Google Scholar]. Importantly, long G/C-rich transcripts have been found to trigger the formation of solid aggregates with toxic biological properties [30.Jain A. Vale R.D. RNA phase transitions in repeat expansion disorders.Nature. 2017; 546: 243-247Crossref PubMed Scopus (246) Google Scholar]. Beyond RNA avidity, other aspects determinant for LLPS are protein–protein interactions and multimerization. Nevertheless, the hierarchy of these components during LLPS is still unknown. A recent study characterized networks of RNA–protein interactions in reconstituted cytoplasmic SGs and P-bodies and contributed to better identifying what defines the composition and miscibility of MLOs [31.Sanders D.W. et al.Competing protein-RNA interaction networks control multiphase intracellular organization.Cell. 2020; 181: 306-324.e28Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. It was observed that the IDR of G3BP-1 is dispensable for LLPS of SG (Box 1), whereas its dimerization and RNA-binding domains are essential. Upon RNA exposure, specific protein interaction networks involving G3BP-1 act as nodes of nucleation for phase sepa