3.1 T-cell receptor and B-cell receptor immune signaling

Immune signaling pathways alter the conformation of immune receptors by ligand binding of pathogenic stimuli, inducing spatial reorganization and, thus information transfer. LLPS is found in a variety of immune signaling pathways, including the T-cell receptor (TCR)⁴⁹ and the B-cell receptor (BCR)⁵⁰ pathway, as well as cytoplasmic signaling pathways, such as cGAS–STING.⁵¹ Normally, LLPS is recognized as 3D structures formed in the cytoplasm and nucleus, but 2D membrane-associated condensates formed along the cell membrane are well represented in immune signaling.⁵² Membrane LLPS restricts protein movement to higher concentrations and induces a lower protein concentration threshold in condensates than in membraneless organelles.⁵³ On the surface of the cell membrane of immune cells, there are membrane clusters⁵⁴ that LLPS regulates and thus proceed the immune signaling cascade.^{55, 56} To decipher how LLPS drives the formation of immune signaling condensates to establish immunity could contribute to developmental research and disease treatment.

The TCR signaling pathway depends on T cell microclusters on the plasma membrane,⁵⁷ which contain multiple transmembrane receptors.⁵⁸ Components of clusters typically have high densities and low mobilities. High densities allow more frequent contact between molecules within the cluster and low mobilities mean less time for interactions to occur.⁵⁹ In addition to the effects of microclusters, the combined effects between proteins and lipids drive LLPS between TCR-associated proteins in the proximal part of the membrane. For example, cholesterol enhances protein condensate TCR clustering, whereas downstream TCR triggering TCR clustering promotes cholesterol LLPS.^{60, 61} It has been shown that LAT, GRB2, and SOS1 can form oligomers through multivalent interactions.⁶² In the signal transduction system of the T-cell junction protein LAT, stimulation induces phosphorylation of specific residues to generate multiple binding sites to promote LLPS of the signal transduction protein.^{49, 63, 64} Transmembrane proteins and their cytoplasmic binding partners (GRB2 of LAT) induce LLPS, which in turn prompts the multivalent SH3 structural domain-PRM to bind to a downstream binding partner (SOS1 of GRB2).^{55, 65} In addition, the dependence of LLPS on phosphorylation by harmful feedback regulatory mechanisms means that it will be significantly reduced or eliminated without a stimulus.⁶⁶ LAT condensate promotes tyrosine phosphorylation and marks the activation of the TCR signaling pathway. However, LAT can be bypassed in the microcluster formation of chimeric antigen receptors (CARs) by establishing multivalent interactions between CARs and the LAT-binding chaperone GADS.⁶⁷ Distinct physical and biochemical compartments LLPS create can facilitate different TCR signaling.

The scaffolding protein SLP65 (also known as BLNK) possesses a critical function in the BCR signaling pathway in driving LLPS, similar to LAT in the TCR signaling pathway (Figure 2). SLP65 with its binding partner CIN85, undergoes LLPS via multivalent interactions between the SH3 structural domain of CIN85 and the proline motifs of SLP65.⁶⁸ Liposomes play a crucial role in facilitating cohesion formation. SLP65 contains an amino-terminal lipid-binding domain that binds to small, highly curved vesicles.⁵⁰ SLP65 mutants lacking the N-terminal vesicle-binding domain fail to undergo LLPS at the cytoplasm or plasma membrane, leading to defects in BCR signaling, such as calcium inward flow.⁵⁰ Higher-order assembly provides a theme in sensitivity control, signal transduction, innate immunity and adaptive. BCR downstream has a ternary complex called CBM signalosome, generated by CARM1, BCL10, and MALT1 assembly, which mediates NF-κB activation.^69-71 These higher-order assemblies with solid-like behaviors or phase-separated liquid-like droplets can inhibit unnecessary immune activation and enable activation of proximity-driven protein and spatial control of immune signaling.

3.2 cGAS–STING signaling

In response to stimulation by pathogens or damaged cytoplasmic dsDNA, cGAS would be activated and binds to dsDNA.⁷² Cyclic GMP–AMP (cGAMP) synthesizes and activates STING, which transduces downstream signals and induces the expression of proinflammatory cytokines.⁷³ Sensing DNA, cGAS–STING signaling triggers innate immune responses and is an important axis of autoimmunity, sterile inflammatory response and cellular senescence.⁷⁴ Besides the role of immune defense, the GAS–STING DNA-sensing pathway has also been shown to be connected with antitumor immunity, exhibiting paradoxical function in both immune surveillance for tumor inhibition and immune-suppressive tumor microenvironment for metastasis-promoting.

cGAS consists of a positively charged disordered N-terminal and a structured C-terminal region. Depending on the positively charged residue at the N-terminal and the DNA-binding site in the C-terminal, cGAS can induce LLPS upon DNA binding.^{48, 75, 76} Long DNA strands, free zinc ions and DNA-binding structural domains in the catalytic core of cGAS are significant factors contributing to LLPS formation.⁷⁷ Factors affecting the occurrence of LLPS in cGAS have now been found to be multiple.^{51, 78} It has been reported that the binding of streptavidin to cGAS enhances the interaction of cGAS with DNA and thus promotes the formation of its condensation complex.⁷⁹ However, ORF52/VP22-type tegument proteins, as a family of inhibitors against cGAS–DNA phase separation, restrict cGAS–DNA LLPS. ORF52/VP22-mediated restriction of cGAS–DNA LLPS is implemented by self-condensation with viral genomic DNA without strong direct interaction between viral proteins and cGAS.⁸⁰ This method of altering one's structure to undergo LLPS with DNA to inhibit phase-separated polymers cleverly evades head-on counteracting cGAS–STING immunization. Also, it demonstrates the impact of LLPS on the way biomolecules interact with each other. It has been shown that the percentage of cells containing cGAS condensates is significantly lower in G3BP1-deficient cells^{81, 82}; however, the underlying mechanism remains obscure. LLPS could also modulate cGAS–STING cascades. For example, LLPS of the oncogene neurofibromin 2 (NF2) mutant inhibits the cGAS–STING pathway.⁸³ NF2 promotes innate immune-sensing of cytoplasmic nucleic acids and has a missense mutant NF2m. NF2m aggregates into dynamic condensates in the cytoplasm induced by IRF3 in its activated state. Aggregates of NF2m can restore average tumor growth in a mouse model of STING-initiated antitumor immunity and hinder STING-initiated antitumor immunity.

3.3 Wnt/β-Catenin signaling

The Wnt/β-Catenin pathway regulates the cellular transcriptional program and plays a vital role in the organism's homeostasis and development.^{84, 85} APC and Axin are key multidomain scaffolding proteins in the signaling pathway. The G protein signaling domain of Axin has an intrinsically disordered intermediate region that contains multiple kinases and APC binding sites.^{50, 86, 87} When Axin is expressed in different cell types, it spreads into a “dot” to increase its concentration, recruiting APCs and other destructive complex proteins.^{88, 89} There is evidence that the complex controlling Wnt/β-catenin is formed precisely by the protein LLPS driven by Axin.⁹⁰ The complex facilitates phosphorylation of β-catenin by GSK3β, which is essential for the regulation of β-catenin protein stability to maintain Wnt/β-catenin signaling.^{91, 92} The β-catenin destruction complex and its receptor signalosome can robustly and precisely regulate Wnt/β-catenin signaling. Assembly of Wnt signalosome is driven by Dishevelled and Axin copolymerization.^{93, 94} Dishvelled-2 (Dvl2) has an IDR at the N-terminus and can undergo LLPS in vitro and cells.^{95, 96} The formation of Dvl2 condensates depend on ubiquitylation of Dvl2 through K63 linkage by WWP2.⁹⁵ LLPS of Dvl2 has been found to be facilitated by signalosome components (e.g., Fzd5).⁹⁶ In addition, Dvl2 LLPS weakens LLPS of Axin that is organized into the signalosome and displays high mobility at low concentration.⁹⁶ Wnt signalosome and phase-separated β-catenin destruction complex emphasize the distinct mechanism LLPS provides in signal transduction.

3.4 RAS/MAPK signaling

The RAS/MAPK signaling pathway is involved in a variety of cellular processes, including differentiation, proliferation, and survival, and their mutations occur frequently in many tumors making it one of the most critical pathways associated with cancer.^97-99 The nonreceptor protein tyrosine phosphatase SHP2 is crucial in RAS–MAPK signaling during normal development.¹⁰⁰ The NH2-terminal end of SHP2 has two tandem SH2 structural domains, a central PTP-catalyzed structural domain, and a C-terminal tail.¹⁰¹ SHP2’ folded PTP structural domain mediates its LLPS by intermolecular electrostatic interactions.¹⁰² Wild-type SHP2 LLPS could be recruited and promoted by disease-related mutation of SHP2 to induce MAPK hyperactivation.¹⁰² In another study, ERK signal responsive repressor ERF has been determined to form condensates vis LLPS, and ERF condensates can be attenuated using ERK inhibitor U0126.¹⁰³ Generally, some components of RAS/MAPK signaling pathway display LLPS behaviors, and RAS/MAPK signaling could also participate in the modulation of LLPS to adjust cellular organization.

3.5 Hippo/YAP signaling

The Hippo–YAP signaling network integrates signals with its transcription factor YAP protein to control cell proliferation and differentiation.^{104, 105} Activation of this pathway promotes regeneration of damaged organs and is associated with tumorigenesis.^106-108 Several components of Hippo–YAP signaling include low-complexity domains and can undergo LLPS. Hippo signaling complexes undergoing LLPS to produce biomolecular condensates in the cytoplasm has been documented.¹⁰⁹ LLPS of the Hippo–YAP signaling pathway is closely related to super-enhancers. YAP LLPS occurs near chromatin compartmentalizes YAP from other relevant coactivators to induce transcription of YAP-specific proliferative genes.¹¹⁰ YAP coalescence occurs in the super-enhancer region, regulates cell proliferation and survival, and activates target genes.¹¹¹ It has been shown that hyperactivated YAP promotes super-enhancer condensates in embryonic stem cells through LLPS, leading to gene-specific and restrictive regulation to control lineage differentiation.¹¹² Glucose in tumor cells tends to be consumed in increased amounts to support tumor proliferation, and it has been found that accumulated glycogen can undergo LLPS.¹¹³ Glycogen droplets segregate Hippo kinase from YAP so that inhibition of the latter is deregulated, the Hippo pathway is inactivated, and the activity of the downstream proto-oncoprotein YAP is increased, ultimately promoting tumor growth.^{114, 115} Destroying YAP LLPS prevents tumor growth, increases immune response, and enhances the sensitivity of anti-PD-1 therapy,¹¹⁶ indicating that YAP LLPS can be applied as a therapeutic target for anti-PD-1 therapy. In addition, DDR1, a collagen-binding receptor tyrosine kinase, has been found to counteract the Hippo/YAP pathway in a LLPS-dependent manner.¹¹⁷ Therefore, LLPS can regulate the Hippo/YAP pathway and can also be controlled by the Hippo/YAP pathway to exert different functions.

4.1 TAR DNA-binding protein 43

As a significant nuclear RNA/DNA-binding protein with a prion-like structural domain, TDP-43 regulates various RNA processing steps. It accumulates in the cytoplasm of patients with a variety of neurodegenerative diseases.^{123, 124} TDP-43 consists mainly of two RNA recognition motifs, a structured N-terminal domain with a C-terminal glycine-rich low-complexity structural domain.^{125, 126} Recognition motifs of TDP-43 are primarily involved in RNA translocation, stability, and splicing,¹²⁷ whereas the disordered C-terminal structural domains^{126, 128, 129} with highly conserved N-terminal structural domains¹³⁰ are connected to LLPS. TDP-43 can undergo LLPS, and liquid phase-separated droplets containing TDP-43 can be converted into gel/solid structures under prolonged stress and even to amyloid fibril structures in vitro.¹³¹ In normal cells, phase separation of these RNA-binding proteins is ordered and controlled, with TDP-43 acting as a liquid shell and encapsulating the HSP70 family chaperone.¹³² As molecular chaperones, the HSP70 family maintains proteostasis to protect proteins from misfolding and aggregation^{133, 134} and is reported to be transcriptionally downregulated in AD.¹³⁵ Furthermore, under oxidative stress, HSPB1, along with HSP70 chaperone activity, is central to the maintenance of TDP-43 protein homeostasis through the interaction of the low-complexity structural domains with TDP-43.¹³⁶ Similar to the C-terminal domain, N-terminal domain interactions are also important for cellular function. In addition to ensuring that the expansion of the aggregation-prone region of the C-terminal structural domain drives endogenous TDP-43 aggregates and sequesters,¹³⁷ LLPS is also affected by the expansion of the aggregation-prone region of the structural domain. Wang et al.¹³⁰ reported that disrupted TDP-43 N-terminal domain polymer by phosphomimetic substitution at S48 can block LLPS and disrupt RNA splicing activity in vitro.

4.2 Fused in sarcoma

Similar to TP-43, FUS is a widely expressed RNA-binding protein involved in multiple RNA metabolic pathways.^{130, 138-140} The N-terminal domain of FUS is a highly conserved low-complexity structural domain that mediates protein–protein interactions and drives the aggregation of FUS into protein inclusion bodies. This region can undergo reversible LLPS.^{141, 142} Furthermore, it has been shown that cation–π interactions between tyrosine in the low-complexity structural domain and arginine in the structured C-terminal structural domain promote LLPS and gelation under the regulation of methylation.¹⁴³ ALS is the most common motor neuron disease in adults, and cytoplasmic mislocalization and aggregation of FUS in neurons and glial cells in affected individuals is the cause of ALS.^{144, 145} Transient SGs are formed when neurons encounter cellular stress, sequestering untranslated mRNAs and associated proteins to reduce energy requirements.¹⁴⁶ Overexpression of FUS induces the spontaneous formation of SGs and is recruited to this membraneless organelle.¹⁴⁷ The persistence of SGs provides an environment for mutations in RNA-binding proteins such as FUS,^{148, 149} and SGs may be a locus for disease biogenesis.⁴³ Also, there may be transmission factors in diseases such as ALS/FTD. It has been found that prion-like mechanisms in neurodegenerative diseases can transmit protein misfolding and aggregation.¹⁵⁰ TDP-43 and FUS in ALS have low-complexity structural domains similar in amino acid composition to yeast PrLDs (prion-like domains; PrLDs).¹⁵¹ Prion-like propagation mechanisms are active in ALS and FTD, and preformed recombinant TDP-43 fibers in neuronal cell lines can trigger overexpression and aggregation of endogenous TDP-43.^{152, 153}

4.3 Tau

Tau consists of four distinct regions: the N-terminal projection domains, the proline-rich domains, the microtubule-binding domains, and the C-terminal domains.⁴¹ This structure facilitates binding to microtubules and allows for an intrinsically disordered and inhomogeneous charge distribution over the entire length of the tau.^{154, 155} Tau is a soluble neuron-specific microtubule-binding protein that is a significant component of inclusion bodies in various neurodegenerative diseases, including AD and FTD.^{22, 156, 157} Currently, one critical factor for the proteotoxicity of tau is the formation of neurofibrillary tangles, the abundance of which correlates with disease progression.^{22, 157} Proteins are susceptible to a large number of posttranslational modifications such as phosphorylation, acetylation, and ubiquitination, which are involved in the pathogenesis of neurodegenerative disorders.¹⁵⁸ neurons can release and take up hyperphosphorylated tau, triggering templated tau misfolding in neurons, leading to cytotoxicity.¹⁵⁹ It has been repeatedly observed that tau forms droplets via LLPS under physiological conditions, regulates microtubule assembly and function, and may be converted to amyloid aggregates upon aging.^{160, 161} It is currently believed that tau undergoes LLPS driven by attractive intermolecular electrostatic interactions between the negatively charged N-terminal and positively charged middle/C-terminal structural domains of the protein.^{160, 162} Also, aberrant LLPS may occur when tau binds to other molecules. For example, tau undergoes complex coalescence after binding to RNA.^{163, 164}

4.4 α-Synuclein

α-Synuclein is structurally and functionally highly distinct from TDP-43, FUS, and Tau and is intimately associated with Lewy body formation.^{165, 166} Lewy bodies are a characteristic hallmark of PD, leading to neuronal death.¹⁶⁷ α-Synuclein consists of a membrane-bound structural domain consisting of a positively charged N-terminal structural domain, a hydrophobic nonamyloid component, and an acidic structural domain containing the C-terminal structural domain, which is involved in its controversial nuclear localization and various interactions.^{168, 169} The C-terminal region of α-synuclein undergoes long-range interactions with the N-terminal region and can remain monomeric, highly flexible, and disordered after the occurrence of LLPS.¹⁷⁰ The α-synuclein LLPS is driven by electrostatic interactions in the amphiphilic N-terminal structural domain and hydrophobic interactions between nonamyloid components.¹⁷¹ High concentrations of α-synuclein trigger the process of LLPS and the formation of amyloid hydrogels containing oligomeric and fibrillar material.^{171, 172} Unstructured proteins of α-synuclein form partially folded intermediates, then oligomerize and protofibrillate, ultimately giving rise to amyloids.^{173, 174} Furthermore, it has been shown that abnormally aggregated α-synuclein molecules induce normal molecular misfolding to form polymers and are taken up by neurons, leading to neurodegenerative alternations.¹⁷⁵ α-Synuclein aggregates are also closely related to mitochondrial autophagy. Aggregates interfere with the normal function of mitochondrial autophagy-associated proteins, thereby affecting mitochondrial autophagy processes.¹⁷⁶

5.1 LLPS contributes to aberrant transformation via gene fusion and mutation

Disordered structures of various proteins form droplets through LLPS, repeated fusions among genes that bind proteins, and chromosomal translocations that produce multiple chimeras.²⁰⁰ SPOP–DAXX bodies, PML vesicles, and FET fusion proteins formed via multivalent interactions affect various cancers regarding gene fusion and mutation.

5.2 Involvement of LLPS in heterochromatin formation in tumor cells

LLPS affects the formation of chromatin structure, which might ultimately contribute to the development of cancer.²²⁵ The chromatin of eukaryotes consists of positively charged histones and negatively charged DNA. Heterochromosomes are chromosomes in a cohesive state with tight morphology, gene deletions, high duplication of genomic regions, and transcriptional inertia.²²⁶ Heterochromosomes are primarily in the perisynaptic and telomeric regions, allowing for flexible gene silencing at multiple levels. Methylation of Histone H3 lysine 9 (H3K9me), a vital disability for posttranslational modification of histones, is a marker of heterochromatin formation. The heterochromatin protein family HP1 is an essential component of the dynamic nuclear response that senses and corrects defects in epigenetic information encoded in chromatin through histone modifications and DNA methylation. Defects in this crucial “chromatin repair” response in transformed human cells can be ideal targets for killing cancer cells. IDR-containing HP1 and its homolog Swi6 can trigger LLPS on chromatin by recognizing H3K9me and forming heterochromatin.²²⁷ In contrast, the conformation of HP1 molecules altered by N-terminal chromatin phosphorylation exposes the binding site, disrupts the molecular network structure, and inhibits LLPS.²²⁸ LLPS occurs based on multivalent interactions. Therefore, the forces and associated networks between multivalent molecules affect the structure and function of the related chromatin on the basis of LLPS.^{229, 230} Inhibition of HP1 LLPS by phosphorylation provides preferential elimination of cancer cells.¹⁸⁶ In cancer cells, the nuclear morphology is frequently altered, a phenomenon known as atomic heterogeneity.²³¹ Alternation in nucleoli's composition, number, size, and activity can provide a diagnostic basis for cancer. Because LLPS controls the integrity of part of the nuclear structure and the integrity of the microenvironment, modulating LLPS may play a key role in cancer therapy.

In eukaryotes, heterochromatin maintains genomic integrity and stability and is organized into distinct nuclear domains. DNA damage proteins can access heterochromatin by specific mechanisms, and erosion of heterochromatin by abnormal factors is linked to carcinogenesis. Tethering LLPS condensates with heterochromatin adds complexity to regulating the nuclear microenvironment. Heterochromatin binding protein HP1 has three homologs termed HP1α, HP1β, and HP1γ that function in DNA repair, nuclear organization, chromosome segregation, telomere maintenance, and gene silencing. HP1 might participate in many types of cancers and can serve as a potential biomarker for cancer prognosis and therapeutic target. Growing evidences have supported that HP1 proteins act as key modulators in micro-LLPS and the segregation of heterochromatin-like domains/complexes.²³² HP1α and HP1β have been reported to form phase-separated droplets to contribute to forming heterochromatin compartments.^{233, 234} Besides HP1, 53BP1 is another important player in regulating heterochromatin formation. It has been shown that 53BP1, together with HP1α undergoes LLPS at heterochromatin in a mutually dependent manner.²³⁵ Modulating the LLPS of factors, such as HP1 and 53BP1, to maintain heterochromatin integrity and genome stability can aid in preventing tumorigenesis and improve cancer therapy outcomes.

5.3 LLPS regulates gene expression to induce carcinogenesis and malignant behavior

Phase-separated regulation of gene expression is involved in both transcription and translation processes. LLPS plays a critical role in both regulating DNA damage response pathway and RNA transcription process. LLPS regulates DNA-responsive injury pathways to maintain genomic stability. DNA damage response and repair occur when signaling factors are activated and repair proteins are concentrated. 53BP1 is a scaffold for LLPS to bind DNA damage recognition and repair factor assembly to cell fate decisions.¹⁸⁴ p53-binding protein1 (53BP1) is a significant player in the DNA damage responding and repairing pathway and accumulates at DNA lesions, creating a nuclear environment called “53BP1 nuclear bodies” to scaffold factors for downstream signal cascades.²³⁶ p53 (a transcription factor) and USP28 (a deubiquitinase stabilizing p53) are assembled in 53BP1 by dynamic fusion and fission, symbolizing LLPS to encode an oncogenic factor.²³⁷ Generally, the 53BP1 nuclear body coordinates the DNA damage recognition with gene expression. Besides 53BP1, RAP80 LLPS has also been found to enhance the recruitment of BRCA1, a well-known tumor suppressor gene, to DNA double-strand breaks.²³⁸

Three different types of enzymes mainly regulate transcription, Pol I, Pol II, and Pol III.²³⁹ Pol I synthesizes preribosomal RNAs (rRNAs), Pol II produces messenger RNAs (mRNAs) and a variety of precursors for ncRNAs, and Pol III is responsible for transfer RNAs (tRNAs). LLPS differentially phosphorylates 239 RNA polymerase II at transcription initiation, elongation and termination to generate transcription factory or condensates.¹⁹⁴ Upon entering the extension phase, RNA polymerase II dissociates from the transcriptional condensate in response to differential phosphorylation and relocates to another condensate used for splicing factors.²⁴⁰ These findings imply that LLPS is critical for transcription processes, including initiation and the switch to elongation, by concentrating factors and conferring dynamics with phosphorylation.²⁴¹

In the first step of gene transcription, Pol II is a scaffold for the preinitiation complex, recruiting other factors to interact with itself and coordinate the transcription initiation. The CTD is a critical element of the scaffolding of Pol II and is highly structurally disordered, which is essential for the development of LLPS.²⁴² CTD can promote LLPS through interactions with other regulators, and these binding sites enrich multiple transcriptional regulators in the condensate of CTD to achieve efficient activation of transcription initiation.²⁴³ CTDs can also be recruited to transcription centers formed by specific transcription factors to enhance transcription initiation.^{192, 244, 245} After escaping the promoter, Pol II is in a suspended state near the proximal region of the promoter.^{246, 247} To release from this proximal paused state, the cell cycle protein-dependent kinase CDK9 phosphorylates residues of the Pol II CTD thereby removing the inhibitory effect of paused Pol II.²⁴⁸ CycT1 possesses an IDR and can fold into a structural domain through its N-terminal region that binds tightly to CDK.²⁴⁹ CycT1 concentrates and compartmentalizes transcription factors into phase-separated environments by LLPS through histidine-rich structural domains within its IDR, leading to hyperphosphorylation of Pol II CTD and continued transcription elongation.^{250, 251} At the end of the regulatory transcription cycle, the Arabidopsis RNA-binding protein FCA undergoes LLPS, in which the curly helix protein facilitates to reduce transcriptional read-through.^252-254 It can be concluded that LLPS participates in the whole process of gene transcription.

As a flexible and variable molecule, RNA is a potentially powerful provider of polyvalency in LLPS.²⁵⁵ lncRNA SLERT acts as an RNA modulator to regulate LLPS of fibrillar center/dense fibrillar component to facilitate Pol I transcription.²⁵⁶ Complex network structures formed by RNA–RNA interactions may directly trigger LLPS or provide a platform for protein coalescence and undergoing LLPS.²⁵⁷ Posttranscriptional modification of RNA regulates the properties and metabolism of RNA.^{258, 259} Numerous membraneless chambers promote gene regulation through various mechanisms, such as concentrating the activity of chelating factors to facilitate biochemical reactions or constructing interchromosomal hubs to promote the occurrence of macromolecules associated with gene expression.^{31, 260} Recently, several groups have reported that multivalent m⁶A-modified RNAs act as scaffolds to gather YTHDF proteins and thus lead to LLPS both in vitro and in vivo.²⁶¹ In addition, a membraneless organelle, the P-body is involved in the regulation of translational repression and mRNA decay mechanisms. Changes in P-body mediators affect mRNA metabolism and may lead to alternations in expression profiles and epigenetic regulation. P-body mediators, such as miRNA and m⁶A, are associated with cancer.¹⁸ LLPS in RNA modification regulation would be a new research perspective because RNA biology is widely associated with various biological processes.

Abnormal transcriptional machinery is a widely accepted cancer hallmark. Therefore, based on the above description, it is easy to reach a consensus that LLPS holds a dominant role in cancer development. LLPS of RNA-binding protein triggers the phosphorylation and release of Pol II to enhance active transcription.²⁶² Many RNA-binding proteins, such as TIA1, LIN28, ZEB1/2, RBM38, and TRBP, have been shown to be aberrantly expressed in cancers.^{263, 264} The transcriptional regulators’ YAP/TAZ can mediate cancer cells reprogramming into cancer stem cells, inciting carcinogenesis and cancer metastasis.²⁶⁵ LLPS of YAP fusions, YAP-MAMLD1 and C11ORF95-YAP have been considered the leading mechanism in initiating ependymoma from neural progenitor cells.²⁶⁶ Furthermore, inhibiting transcriptional coactivator activity mediated by condensates can prevent tumorigenesis,²⁶⁶ confirming the important role of YAP LLPS in oncogenic activity. SNHG9, a cancer-promoting lncRNA, drives the formation of a liquid-like droplet of LATS1 and downregulates the Hippo signaling.²⁶⁷ SNHG9 together with phosphatidic acids binds to LATS1 C-terminal domain to trigger LAST1 LLPS, thus blocking LATS1-mediated YAP phosphorylation.²⁶⁷ These results indicate a novel regulatory strategy to modulate cancer-promoting transcription apparatus by facilitating LLPS.

In addition, the nuclear microenvironment of cancer is altered in response to LLPS.²⁶⁸ Super-enhancers, which act as enhancer clusters to mediate dysregulation of transcriptional programs, are essential oncogenic drivers that sustain cancer cells.^{269, 270} Super-enhancer-rich transcriptional coactivators BRD4 and MED1 can form LLPS droplets at the super-enhancer IDR, concentrating the factors in specific genomic regions.²⁷⁰ LSD1, also named KDM1A, is involved in the progression of multiple cancers. It has been demonstrated that LSD1 interacts with BRD4 and FOXA1 and is enriched at super-enhancer regions, showing LLPS behavior.²⁷¹ NUP98–HOXA9, a homeodomain-containing transcription factor chimera in leukemias, contains IDR and can establish LLPS puncta of chimera.²⁰⁰ Phase-separated NUP98–HOXA9 generates a “super-enhancer”-like binding pattern to potentiate transcriptional activation and induce leukemic transformation.²⁰⁰ NUP98–HOXA9 LLPS leads to the formation of proto-oncogenes enriched CTCF-independent chromatin loops.²⁰⁰ HOXB8 and FOSL1 are core regulatory circuitry components that control chromatin accessibility in super-enhancers. HOXB8 and FOSL1 form dynamic phase-separated droplets to incite the release of RNA Pol II from the promoter of super-enhancer-driven genes and the condensates can be specifically destroyed by GSK-J4, a H3K27 demethylase inhibitor, to suppress metastasis and re-establish sensitivity to chemotherapy treatment.²⁷² LLPS remodels nuclear microenvironment provide mechanistic and therapeutic insights for targeting epigenetic factors to regulate chromatin accessibility in cancer therapy.

5.4 LLPS maintains cellular homeostasis to regulate cancer progression

The cell is separated from its surroundings by a cell membrane, and the internal conditions of the cell are very different from the fluid surrounding the cell. The cell constantly exchanges substances with the surrounding fluid and maintains its internal constancy, which is called cellular homeostasis. Cellular homeostasis includes proteostasis, redox homeostasis, calcium homeostasis, and so on. Cellular homeostasis has many essential roles in cancer therapy. For example, cellular metabolism can modulate the effector T cells to influence the adaptation of cellular functions in cancer immunity.²⁷³

Degradation of misfolded proteins is essential for cellular homeostasis. Selective autophagy mediates the degradation of harmful substances by separating these proteins into larger cohesions in stages through multivalent interactions and tethering them to the autophagosomal membrane.²⁷⁴ Membraneless particles(such as postsynaptic densities, signaling granules at DNA damage repair sites, and p62 granules involved in selective autophagy) have been found to assemble in membraneless compartments in several biochemical pathways and cellular pathways.^{49, 275, 276} During proliferation and spread, the intracellular environment of cancer cells, including the homeostasis of proteins, is altered. Gene activation or genomic instability impedes protein folding in cancer cells. Inadequate amino acid supply, glucose deprivation, and so on challenge protein processing in ER.²⁷⁷ p62 is a scaffolding and stress-inducible protein. By regulating autophagy and apoptosis, this multifunctional protein controls cell viability in response to cytotoxic stress (Figure 3).^278-280 Selective autophagy mediates the degradation of harmful substances by segregating these protein stages into larger endosomes through multivalent interactions and binding them to the autophagosome membrane. P62-dependent hepatic differentiation is activated by autophagy inhibition. When the mTOR pathway is activated, p62 autophagy would be inhibited to cause protein accumulation. p62 is also closely associated with causing resistance to cancer therapy. Reduced p62 levels through autophagy upregulation in cisplatin-resistant ovarian epithelial cancer have been observed to increase tumor sensitivity to the drug.^{278, 281}

The oxidation and reduction reactions within the cell change the properties of the cellular components and provide energy to the organism. Redox imbalances in cells can lead to oxidative stress that impairs intracellular functions, for example, the susceptibility of the purine nucleotide guanine to oxidation, leading to DNA mutations that may initiate and propagate cancer.²⁸² LLPS enables efficient energy production, promotes cell survival under acute hypoxia, and protects cells from apoptosis during cellular translational remodeling.^{282, 283} Nuclear factors erythroid 2-related factor 2 (NRF2)-mediated redox-regulated pathways can sense cellular damage through LLPS and activate autophagy to degrade these damaged organelles. NRF2 has been shown to be a key activator of cancer-supporting anabolism, and NRF2 activation has significant and protumor solid effects, particularly in the reprogramming of cancer cell metabolism.²⁸⁴

Calcium homeostasis controls autophagy in cancer cells. Ca²⁺ is a multifunctional second messenger, and as one of the most important factors regulating processes such as cell death, Ca²⁺ activates various regulatory proteins, including enzymes and transcription factors.²⁸⁵ Alterations in Ca²⁺ concentration in the cytoplasm and endoplasmic reticulum are integrated into the core through different regulatory mechanisms of autophagic activity.²⁸⁶ Ca²⁺ triggers the FIP200 complex to drive assembly via LLPS, triggering the formation of FIP200 sites, after which the autophagosome complex assembles in the endoplasmic reticulum.²⁸⁷ As a survival-promoting and death-inducing factor, autophagy boasts an excellent scope for tumorigenesis and prevention regulation.

5.5 LLPS remodels microenvironment to control cancer progression

The tumor microenvironment consists of tumor cells and their surrounding immune and inflammatory cells, tumor-associated fibroblasts, nearby mesenchymal tissues, microvasculature, part of the neuronal regulatory circuits, as well as a variety of cytokines and chemokines, and is a complex integrated system.^{288, 289} In recent years, there is increasing evidence for the diversity of LLPS in the regulation of immune regulation, including the maturation and activation of immune cells, immune signaling, and so on.⁴⁷ LLPS is also involved in the regulation of intracellular immune signaling, such as the cGAS–STING pathway (Figure 4).²⁹⁰ Cancer-associated fibroblasts are essential components of the tumor microenvironment, and are activated to promote tumor growth, angiogenesis, invasion and metastasis. In addition, cancer-associated fibroblasts can interact with tumor-infiltrating immune cells and other immune components within the tumor microenvironment by secreting various factors and other effector molecules, resulting in immune effector cell dysfunction and suppression of antitumor immunity.²⁹¹ Then, whether modulation of immune cells by LLPS and attenuation of their action with tumor-promoting factors by immune components can inhibit cancer growth and invasion in the tumor microenvironment.

6.1 N⁶-methyladenosine

6.2 N¹-methyladenosine

m¹A, another type of RNA methylation, is a crucial internal RNA modification that controls gene expression, appearing on the first nitrogen atom of adenosine in RNA.³⁵² Similar to m⁶A, m¹A can be installed by “writer” methyltransferases, removed by “eraser” demethylases and recognized by m¹A-binding proteins called “reader.”³⁵³ m¹A is very abundant in tRNAs and is most conserved and common in bacteria, archaea and eukaryotes.³⁵⁴ m¹A at position 58 is the first m¹A modification of the initial transcripts of tRNA in the nucleus, whose methyl methyltransferase complex has been first discovered in Saccharomyces cerevisiae.³⁵⁵ m¹A plays an important role in the regulation of pre-RNA processing, RNA structure, translation and stability.³⁵⁶ It should be noted that the biological function and modification sites of m1A modification vary in different species.

m¹A is involved in regulating disease initiation and progression. m¹A methylation upregulates microfibril-associated protein 2 (MFAP2) expression and promotes colorectal cancer metastasis by blocking autophagic degradation of CDC Like Kinase 3 (CLK3).³⁵⁶ In gastric cancer cells, m¹A regulates the PI3K/AKT/mTOR pathway and ErbB pathway. Knockdown of ALKBH3, the “writer” protein of m¹A, has been observed to result in poor prognosis and metastasis in gastric cancer.³⁵⁷ In addition, m¹A is also involved in the prognosis of gliomas.³⁵⁸ m¹A-related proteins, including ALKBH1, TRMT6, TRMT10C, and YTHDF1, are significantly overexpressed in gliomas. TRMT6 regulates cell cycle distribution and increases glioma cell proliferation and death.^{358, 359} m¹A methylation in mitochondria and cytoplasm has been reported to be dysregulated in AD.^{360, 361} For example, aberrant m¹A deposits lead to complex I dysfunction in AD.³⁶² In sum, there has been increasing evidence of alterations in m¹A occurring in various diseases, and these m¹A modifications may be potential drug targets.

6.3 m⁵C and hm⁵C

In eukaryotes’ RNA, m⁵C and hm⁵C are the prominent modifications and are thought to contribute to epigenetic regulation through various mechanisms.³⁶³ NOL1/NOP2/NSUN family and DNMT2 catalyze m⁵C, which covalently transfers methyl from S' adenosine methionine to the cytosine pyrimidine ring.^{363, 364} m⁵C methylation is a reversible epigenetic modification with a constant turnover of cytosine modifications.³⁶⁵ This dynamic regulation of reversible chemotaxis is important in cell fate determination and can act as an epigenetic barrier by limiting the developmental potential of cells and restricting their differentiation.³⁶⁶

The methyltransferase of m⁵C has been observed in a variety of cancers, including glioma, gastric cancer and leukemia.³⁶⁴ In hepatocellular carcinoma cells, the methyltransferase NSUN2 of m⁵C can target the tumor-associated gene lncRNA H19 to promote tumorigenesis and progression. G3BP1 regulates a variety of tumorigenesis-related pathways, including Ras, Wnt/β-catenin, PI3K/AKT, and NF-κB/Her2 signaling pathways. RNA purification and mass spectrometry analyses indicate that m5C modifications regulate the binding of G3BP1 protein to H19 RNA.³⁶⁷ In addition, NSUN2 is upregulated in gastric cancer and promotes cancer cell proliferation, migration and invasion. SUMOylation is a significant regulatory posttranslational modification, and its covalent bond interaction with NSUN2 to form the SUMOylation–NSUN2–m⁵C axis is a novel mechanism and therapeutic target for gastric cancer cells.³⁶⁸ The methyltransferase DNMT2, in turn, can interact with RNA-binding proteins to mediate the 5-azacytidine response in leukemia.³⁶⁹ Azetidine acrylamides, stereoselective covalent inhibitors of human NSUN2, can cause a global reduction in tRNA m⁵C content in cancer cells.³⁷⁰ Growing studies are currently undergoing to unveil the role of m⁵C modification in cancer development.

NSUN2 are all enriched in the developing brain³⁷¹ and are knocked down to delay or block late differentiation in specific tissues including skin and testis.³⁷² Several mutations in NSUN2 may cause growth and mental retardation, unusual facial and skin abnormalities.³⁷³ And mutations in NSUN3 have been reported to be detected in patients with mitochondrial-deficiency disorders.^{374, 375} NSUN5 is located in the deletion genome of a neurodevelopmental disorder called Williams-Beuren syndrome,³⁷⁶ and the deletion of NSUN5 may be one of the pathogenic causes of the disorder.³⁷⁷ NSUN7 is widely expressed in testicular cells and may lead to reduced sperm viability under induced mutations.³⁷⁸ In summary, m⁵C is commonly involved in a variety of diseases.

As the hydroxylated form of m⁵C, hm⁵C is now widely recognized as the “sixth base” of the mammalian genome.^{379, 380} The hm⁵C modification has also been identified in RNA, and ten-eleven translocation (TET) enzymes are responsible for the oxidation of m⁵C into hm⁵C in RNA.^{381, 382} It has been found that the deposition of hm⁵C on tRNA depends on TET2, and TET2 knockout impairs the abundance of several ncRNAs.³⁸³ In a recent study, it has been confirmed that ALKBH1 is the primary enzyme for hm⁵C modification in RNA other than TET2.³⁸⁴ It is well known that both TET2 and ALKBH1 are essential for cancer initiation and progression. Besides hm⁵C, there are also different types of oxidative metabolite of m⁵C in RNA, including 5-Formylcytidine (f⁵C)³⁸⁵ and 2′-O-methyl-5-hydroxymethylcytidine (hm⁵Cm).^{384, 386} However, the biological function of these modifications in RNA remains largely unknown.

6.4 Adenosine-to-inosine editing

Irreversible deamination reaction A-to-I converts adenosine to inosine in the dsRNA region, thus functionally acting as an A-to-G mutation.^{387, 388} Adenosine deaminase acting on the RNA (ADAR) family is the most prominent A-to-I modifying enzyme and is dysregulated in many human diseases.³⁸⁹

ADAR1, which is much more enriched than ADAR2, plays the most important role in the family and is closely associated with cancer development.³⁹⁰ ADAR1 recognizes the hairpin structure of dsDNA and edits it, leading to inactivation or activation of the final translation product and disrupting miRNA binding sites to alter mRNA stability.³⁹¹ Knockdown of ADAR1 results in mice exhibiting massive apoptosis, defective erythropoiesis, and abnormal innate immune responses.³⁹² ADAR2 enzyme targets the Q/R locus in the mammalian central nervous system in the mouse brain, and defects at this locus may result in cell death of excitotoxic neurons, leading to excitotoxicity.^{393, 394} As an important target of ADAR1, Antizyme inhibitor 1 (AZIN1) is involved in forming various cancers, including colorectal and lung cancer. Takeda et al.³⁹⁵ proposed a new mechanism by which AZIN1 promotes the accumulation of proteins necessary for polyamine synthesis, activating and increasing fibroblast invasion into the tumor microenvironment. AZIN1 promotes tumor angiogenesis in colorectal cancer by upregulating IL-8, and the IL-8 receptor profoundly affects the tumor microenvironment, increasing the proliferation, survival, and migration of vascular endothelial cells and the permeability of endothelial cells.³⁹⁶

6.5 N⁷-methylguanosine

m⁷G is the RNA methylation of guanine at the N7 position, which primarily occurs in the cap region of mRNAs with RNAs and tRNA internals.³⁹⁷ The METTL1/WDR4 complex appears to be the “writer” in m⁷G modification.³⁹⁸ Although METTL1 is mainly responsible for mediating m⁷G methylation, WDR4 has more favorable binding sites on RNA for stabilization.³⁹⁹ In cancer, METTL1 is frequently overexpressed in inducing oncogenic cell transformation. Arg-TCT-4-1, the m⁷G-modified tRNAs, can increase translation of AGA codon-rich mRNAs and control tumorigenesis and growth, and the upregulated phenotype of tRNA–Arg–TCT-4-1 has been shown to be a METTL1/WDR4 overexpression phenotype.^{400, 401} A missense mutation in WDR4 may be the causative variant in microcephalic primordial dwarfism, a neurological disorder with facial deformities and brain malformations.³⁹⁴ Furthermore, WDR4 expression levels were significantly reduced in hippocampal tissues of Down syndrome mice,⁴⁰² and overexpression of WDR4 promoted cognitive function and hippocampal plasticity.⁴⁰³ Mutations in METTL1/WDR4 cause neurodevelopmental disorders through multiple mechanisms.⁴⁰⁴ Furthermore, in head and neck squamous cell carcinoma, METTL1 accelerates cancer progression by promoting the translation of cell cycle proteins and related oncogenes through the PI3K/AKT/mTOR signaling pathway.⁴⁰⁵ The METTL1/WDR4 complex continues to promote oncogene translation and regulates the progression of cell cycle-related proteins in lung cancer through a codon-dependent manner.⁴⁰⁶ Hence, targeting m⁷G to inhibit oncogene translation would be a potential antitumor strategy. WBSCR22/TRMT112 is another significant m⁷G methyltransferase complex.^{407, 408} As an m⁷G writer, WBSCR22 has been reported to be involved in organ regeneration and enhancing glucocorticoid receptor function in lung cancer.^{404, 409, 410} In addition, WBSCR22 has been shown to affect tumor cell survival and drug resistance in a variety of cancers, including breast cancer, melanoma, and colorectal cancer.^411-414 TRMT112 is a cofactor of WBSCR22, maintains its stability and aids in m⁷G modification installation.^{407, 415} The information concludes that m⁷G modifications and related methyltransferases function in regulating disease development and drug resistance.

6.6 Pseudouridine

The C5-glycoside isoform of uridine, Ψ, deposits in both coding and noncoding regions of RNA and is highly conserved among species.⁴¹⁶ Ψ in tRNA contributes to its structural stabilization with inhibition of protein synthesis.^{417, 418} Possibly, since C-C is more inert than C-N, Ψ’s “reader” and “writer” proteins are less detectable than other modifications.∖Mutations in the DKC1 enzyme in the Ψ-mediated catalytic mode by RNA-dependent mechanisms are associated with aberrant protein translation and have been reported to be upregulated in a variety of cancers.^{407, 419} Impaired telomerase activity and gene expression in the absence of DKC1 activity leads to decreased proliferation and survival of cancer cells.⁴²⁰ DKC1 is significantly upregulated in colorectal cancer tissues, acting as a critical regulator for cancer cell proliferation. Three hundred eighty-three thousand three hundred eighty-four rRNA mutants are very common in colorectal cancer,⁴²¹ so ψ modification of reduced ribosomes may affect translation in cancer cells. Ψ can regulate the misreading of mRNA, and might have a unique application in the development of RNA drugs.

6.7 N⁴-acetylcysteine

ac⁴C is currently the only type of acetylation modification in eukaryotic RNAs, mainly occurring on tRNAs and rRNAs and, to a lesser extent, mRNAs.⁴²² ac⁴C in tRNA helps to maintain translational stability and plays a role in thermophilic bacteria for their heat resistance.^{423, 424} ac⁴C is vital in the diagnosis and treatment of cancer. Studies have found that cancer patients have a more pronounced increase in ac⁴C than standard nucleosides and that ac⁴C levels are significantly lower after tumor resection.⁴²⁵ In addition to cancer diagnostics, ac⁴C has a regulatory role in bladder, gastric, and colorectal cancers.^{426, 427} N-acetyltransferase 10 (NAT10) exhibits acetyltransferase and RNA binding activity and can catalyze ac⁴C modification. NAT10-mediated acetylation improves mRNA stability and translational efficiency.⁴²⁸ High expression of NAT10 is necessary for the tumorigenic properties of bladder cancer. Regulation of ac⁴C via NAT10 involves proliferation, migration, and stem cell properties of bladder cancer cells.^{429, 430} In gastric cancer, ac⁴C acts as the inducer of hypoxia tolerance through glycolytic mechanisms. NAT10 receives regulation of a critical transcription factor HIF-1α in the hypoxia response, which triggers the activation of the HIF-1 pathway and regulates ac⁴C to enhance hypoxia tolerance in gastric cancer cells.⁴³¹ NAT10 mediates an increase in ac⁴C modification and promotes the binding of polysaccharides, which enhances translational efficiency and induces central sensitization for neuropathic pain.⁴³² Knockdown of NAT10 significantly reduced macrophage inflammatory responses, whereas overexpression resulted in the opposite result. It has been demonstrated that NAT10 regulates macrophage inflammatory responses through the NOX2–ROS–NF-κB pathway.⁴³³ The abundance of ac⁴C modifications in lncRNA significantly differed in AD as obtained by highly repetitive sequence analysis.⁴³⁴ These studies highlight the critical role of ac⁴C in cancer development by controlling various pathological events, such as glucose metabolism reprogramming and cancer hypoxia.

6.8 2ʹ-O-methylation

2ʹ-O-methylation is the main methylation of the 2′-OH portion of the ribose and is widely present in tRNA, rRNA and mRNA in mammalian cells.⁴³⁵ Absence of 2′-O-methylation in vertebrate development leads to severe morphological defects and embryonic lethality.^{422, 436} Fibrillarin is a 2′-O-RNA methyltransferase located in the dense proto-fiber component of the nucleolus accumbens.⁴³⁷ Fibrillarin is an essential nucleolin protein involved in pre-rRNA processing and regulates rRNA transcription, and it is overexpressed in different cancers.⁴³⁸ 2ʹ-O-methylated modification in ncRNAs is involved in colorectal carcinogenesis.⁴³⁹ Among these ncRNAs, SNORD11B functions in cancer proliferation and invasion and increases the processing and maturation of 18S rRNA by introducing 2ʹ-O-methylation on 18S rRNA G509 site. lncRNA ZFAS1 is significantly overexpressed in several human malignant tumors, including colorectal, hepatocellular, and gastric cancers, and knockdown of ZFAS1 inhibits cancer cell proliferation and migration and increases apoptosis.⁴⁴⁰ Overexpression of NOP58 reverses this inhibition of ZFAS1 and promotes 2ʹ-O-methylation.⁴⁴¹ Another small nucleolar RNA, SNORD104, can be knocked down by antisense oligonucleotide in Ishikawa cells to reduce proliferation and migration in endometrial cancer.⁴⁴² In this study, SNORD104 is found to bind to the 2′-O-methyltransferase fibrillarin and increases PARP1 2′-O-methylation.⁴⁴² In malignant melanoma and acute myeloid leukemia, 2ʹ-O-methylation has also been recognized as a new target for treatment.^{443, 444} Loss of function of FTSJ1, a characteristic 2′-O-methyltransferase targeting tRNA, has been identified as the cause of nonsyndromic X-linked intellectual disability.^{445, 446} Another 2′-O-methyluridine methyltransferase, TRMT44, is associated with idiopathic epilepsy.⁴⁴⁷ It still demands more studies to understand the role of 2ʹ-O-methylation in preventing and treating diseases.

6.9 LLPS in RNA modifying enzymes

Many RNA modifications have their own “reader,” “writer,” and “eraser” proteins that play an important role in the relationship between previously mentioned RNA modifications and diseases. For example, YTHDC1 undergoes LLPS requiring m⁶A, and the nuclear YTHDC1–m⁶A condensates maintain cell survival and the undifferentiated state of acute myeloid leukemia cells.³²⁴ Possibly, m⁶A acts as a bridge between LLPS and RNA to fine-tune the subcellular localization of RNA to implement or even modify its functions in controlling cancer cells' occurrence, proliferation, apoptosis, and metastasis.¹⁷⁸ Proteins with disordered structure are likely to undergo LLPS or have some involvement in forming membraneless organelles. As the m¹A “writer” protein TRMT6/61A is significantly enriched in SG-chelated mRNAs, it was shown that TRMT6/61A methyltransferase localizes to SGs under stress and protects mRNAs during heat shock.⁴⁴⁸ ALKBH5 specifically binds to proteins in the paraspeckles and forms phase-separated droplets that bind to the paraspeckles through its C-terminal IDR.³¹⁷ In a hypoxic environment, proteins in the paraspeckles before the upregulation of ALKBH5 expression are induced with its rapid condensation to promote the formation of paraspeckles, reflecting an essential role in hypoxia induction.⁴⁴⁸ ADAR1 is localized to SGs formed in response to arsenite-induced oxidative stress or long dsRNA transfection.⁴⁴⁹ METTL3 has a large number of disordered structures that are not only capable of LLPs independently but also of stabilizing METTL14 to bind constitutively to each other and form heterodimers. In addition, certain LLPS behaviors of METTL3 cancer mutants have now been observed.³⁰⁶ DNMT2 can relocate from the nucleus to SGs and participate in the protein processing of RNA.⁴⁵⁰ In sum, many RNA-modifying enzymes harbor IDRs that must be explored in their association with LLPS.

6.10 Relationship between RNA modifications and LLPS

The phase boundaries of condensates can be controlled by changing RNA length, sequence, structure, modifications, and RNA–RNA and RNA–protein interactions.²⁵⁵ The use of RNA–RNA and RNA–protein interactions to influence RNA-driven LLPS realizes that it is possible to use RNA to control condensates, such as the LLPS regulation of m⁶A.⁴⁵¹ The RNA modifications can alter LLPS behaviors by refining RNA–RNA and RNA–protein interactions.

As mentioned earlier, membrane-free fluidic regions play a role in many cellular functions; they are often enriched in RNA and influence the transcriptome. For example, nuclear speckles, which are significant regulators of gene expression and contain proteins and RNAs involved in gene expression, play a role in some cancers, and speckles, working together with p53, can directly enhance gene activity. Speckle-associated p53 target genes are more likely to be involved in tumor suppressor functions, such as stopping cell growth and triggering cell suicide, than other p53 target genes.^{452, 453} Among them, nuclear speckles or splicing speckles, also called interchromatin granule clusters, are meaningful RNA-containing membraneless chambers that function as sites for RNA splicing and RNA m⁶A methylation. LLPS of these compartments selects biomolecules to become concentrated, presumably to implement their functions.^{454, 455} Phase-separated multivalent interactions are mediated by scaffold proteins with tandem repeat binding sites with other partners or proteins with IDRs (or low-complexity structural domains), often in conjunction with nucleic acids.²⁵³ The m⁶A can be recognized by the YTH structural domain for LLPS in the IDR, and the LLPS potential can be further facilitated by multiple m⁶A modifications to the RNA.²⁶¹

The YTH family of binding proteins of m⁶A consists of a m⁶A-binding YTH domain of approximately 15 kD and a low complexity domain of approximately 40 kD.²⁹⁸ Under the action of LLPS, some low-complexity structures (e.g., YTHDF family) form protofibrils, hydrogels or droplets.¹⁴⁹ The heated sample of YTHDF proteins becomes turbid when warmed to 37°C, indicating that the warming causes lower critical solution temperature LLPS. Adding as low as 10% glycerol and reducing the salt concentration decreases the YTHDF2 concentration required for the LLPS transition to 1−8 µM.²⁶¹ These results demonstrate that YTHDF2 LLPS is enhanced by increasing protein concentration but weakened by salt. Furthermore, studies reveal that polymethylated m⁶A-RNA provides a scaffold to juxtapose multiple YTHDF proteins, causing these proteins to undergo LLPS through interactions between their low-complexity domains.²⁶¹ It possibly demonstrates that m⁶A-RNA binding to the YTH domain regulates LLPS.²⁶¹

YTHDF proteins localize to neuronal RNA granules, P-bodies, and SGs, each of which is considered to be a phase-separated compartment in the cytosol raising the possibility that LLPS may govern the localization of these proteins and potentially m⁶A-mRNA. It is well known that nearly all m⁶A formation in mRNA is catalyzed by the METTL3–METTL14 heterodimeric methyltransferase.⁴⁵⁶ METTL14 knockout has been conducted to examine the effect of m⁶A-mRNA on YTHDF2 protein localization to SGs and P-bodies. Results reflected that METTL14 knockout did not alter the formation of SGs, but there was less YTHDF2 relocalization in SGs.²⁶¹ In addition, the YTHDF2 mutant that lacks m⁶A binding exhibited less relocalization to SGs in wild-type cells, indicating that binding to m⁶A mRNA is necessary for YTHDF2 to partition into SGs efficiently. YTHDF2 does not enrich the P-body in the cells with METTL14 knockdown, suggesting that YTHDF2 being included in P-bodies to form complexes is accompanied by m⁶A-mRNA. In SGs, m⁶A-mRNA occupies a higher percentage than in total cellular mRNA, further confirming that polymethylated m⁶A RNAs play an important role in LLPS.

Additionally, it is found that, unlike other forms of RNA-scaffolded LLPS, m⁶A regulates the fate of cytoplasmic m⁶A through the scaffold YTHDF protein, leading to the formation of phase-separated YTHDF-m⁶A-mRNA complexes that then partition into phase-separated structures in cells.²⁶¹ This series of experiments by Rise et al. demonstrated that m⁶A regulates the fate of mRNA by scaffolding YTHDF proteins to form phase-separated YTHDF–m⁶A–mRNA complexes. The presence of m⁶A enhances RNA–RNA and RNA–protein interactions, targeting different intracellular condensates. Furthermore, O-GlcNAcylation modification can occur on YTHDF1 and YTHDF3 to modulate the stability, assembly and disassembly of SGs.⁴⁵⁷ The regulatory mechanism of O-GlcNAcylation in RNA m⁶A methylation highlights additional complexity to the posttranscriptional regulation function of LLPS.

Investigation shows that the LLPS of YTHDC1 and 3′ end processing factor FIP1L1 can be enhanced by binding to the m⁶A sites, probably playing a crucial role in their interaction and APA regulation.⁴⁵⁸ LLPS of YTHDC1 also undergoes liquid-like condensates in highly active enhancers marked by m⁶A-enhancer RNAs.⁴⁵¹ YTHDC1/m⁶A-enhancer RNAs condensates can further facilitate BRD4 coactivator condensates formation to implement enhancer activation and gene transcriptional control.⁴⁵¹ Considering these essential findings, m⁶A might provide a regulatory LLPS mechanism based on m⁶A multivalency, potentially a new strategy for cancer treatment.

RNA is a crucial component of phase-separated cellular compartments, and large amounts of protein-free mRNA can be released from multimers into the cytoplasm under stress conditions.⁴⁵⁹ Free RNA is susceptible to aberrant interactions with misfolded proteins and undergoes protein accumulation, inducing the formation of irreversible RNA–protein coaggregation. Participation of m¹A in mRNA granulation and protection during stress via TRMT6/61A methyltransferase.¹⁴ Strong binding of TDP-43 to m1A stimulates cytoplasmic mislocalization and formation of TDP-43 gel-like aggregates.⁴⁶⁰ TDP43 promotes stemness in breast cancer stem cells, and deletion of TDP43 inhibits the progression of triple-negative breast cancer.^{461, 462} m⁷G is predominantly present in mRNA processing and protein synthesis. Within mRNAs, m⁷G can be selectively recognized by quiver proteins (QKIs). m⁷G regulates mRNA stability and translation under stress conditions, and the bound complex QKI7 interacts with SGs core proteins (via the C-terminus) and shuttles internally. Through extensive experimental validation, Zhao et al.¹⁵ found that QKIs can regulate target mRNA metabolism and cellular drug resistance by binding m⁷G. Also, A-to-I editing has been shown to be associated with alterations in the interaction sites of proteins involved in LLPS.¹⁶ However, there are still plenty of unknowns in investigating the relationship between RNA modifications and LLPS. The phenomenon and underlying mechanism of many RNA modifications in LLPS have not yet been discovered.

6.11 RNA modifications regulate LLPS to affect disease development

As mentioned above, m⁶A enhances the LLPS potential of mRNA, which in turn regulates various metabolic processes in organisms.²⁶¹ Considering that both LLPS and m⁶A might participate in multiple pathological progression, it would be reasonable to hypothesize that LLPS can be regulated by m⁶A to control disease development.

Human papillomavirus (HPV), a diverse family of small double-stranded DNA viruses, is the major risk factor for various types of cancers, including cervical cancer, head and neck cancer, and nonmelanoma skin cancer. The E7 protein encoded by high-risk HPVs contributes to HPV-associated carcinogenesis through multiple signaling pathways. Viral E7 mRNA contains modified m⁶A that IGF2BP1 stabilizes in HPV-infected cells.⁴⁶³ Heat treatment has been reported to reverse HPV-associated tumorigenesis both in vitro and in vivo, which enhances IGF2BP1 aggregation depending on the presence of m⁶A-modified E7 mRNA to generate obvious m⁶A E7 mRNA–IGF2BP1 granules,⁴⁶³ indicating that controlling LLPS is a possible cancer therapeutic strategy in the m⁶A context. RNA driving IGF2BP1 LLPS has also been documented in several research studies.^{464, 465} IGF2BP1 is an important m⁶A modulatory gene that has always increased in many malignant tissues. RBM15 contains IDR and potentially forms “liquid-like” condensates in vivo and in vitro.⁴⁶⁶ RBM15 condensates are prone to contact or partially overlap with nuclear speckles rather than other membraneless chambers.⁴⁶⁶ Most importantly, RBM15 condensates promote the m⁶A modification of the transcripts of highly expressing cancer-related genes and are partially colocalized with m⁶A-modified transcripts in the nucleus.⁴⁶⁶ In myeloid leukemia, m⁶A is found to be required for YTHDC1 to undergo LLPS, generating nuclear YTHDC1–m⁶A condensates to maintain cell survival and myeloid leukemic undifferentiated state.³²⁴ METTL3 can also undergo LLPS, and its self-interacting ability provides the necessary multivalency for LLPS of mRNA m⁶A methyltransferase complex.³⁰⁶ Researchers also investigated the LLPS behavior of METTL3 cancer mutants, including R508H, R415C, and E516K, finding that R415C and E516K mutants fail to undergo LLPS separation.³⁰⁶ Because the METTL3 has been documented to function in many types of cancers,^{329, 334} modulating its LLPS behavior may be a potentially critical facet for inhibiting cancer progression.

Many cancer-related genes can undergo LLPS and be regulated by m⁶A modification. Low-complex domain RNA-binding protein FUS can mediate LLPS.⁴⁶⁷ FUS is overexpressed in prostate adenocarcinoma, and patients with FUS overexpression show a low survival rate. Increased abundance of m⁶A modification promotes FUS expression to enhance P53 and Gpx4 expression to promote the proliferation of prostate adenocarcinoma.⁴⁶⁸ m⁶A-modified RNAs have been shown to prevent cytoplasmic TLS/FUS aggregation induced by hyperosmotic stress.⁴⁶⁹ SGs in U2OS cells induced by NaAsO₂ treatment are enriched of m⁶A modified mRNA, and m⁶A binding of YTHDF proteins enhances SG formation.¹⁷ YTH domain family proteins, including YTHDF and YTHDC subtypes, have been reported to function in several types of cancers.^{470, 471} YTHDF2 has an essential regulatory role in various cancers (Figure 6),^472-474 and acts as either an oncogene or tumor suppressor gene in different cancers depending on context.⁴⁷⁵ Generally, YTH domain family proteins play an essential role in tumor proliferation, invasion, migration, metabolism, and apoptosis.

In addition, it has been noted that SG formation can be beneficial for the survival of stressed cells, and persistent induction of SGs can cause cell death. SG assembly can be attenuated by circRNA–CREIT to activate the RACK1/MTK1 apoptosis signaling pathway in doxorubicin-resistant triple-negative breast cancer and combinatorial treatment of SG inhibitor ISRIB and doxorubicin would synergistically suppress the growth of doxorubicin-resistant triple-negative breast cancer.⁴⁷⁶ In another report, chemotherapy-driven SGs have been shown to disassemble by glucocorticoids,⁴⁷⁷ suggesting that controlling SGs assembly can increase cancer cell chemosensitivity. However, a study has argued that m⁶A modification in mRNAs plays a minimal role, if any, in mRNA recruitment to SGs.⁴⁷⁸ Generally, m⁶A modification might modulate LLPS to regulate the development of cancer-representative diseases under certain circumstances. However, more LLPS phenomena and their precise mechanism should be further exploited to benefit disease therapy.

7.1 Phase-separated polymers can be used for diagnostics

Errors at any step of cell division might lead to aberrance in chromosome replication. Meanwhile, rearrangements and aneuploidy of the genome might occur in steps, such as chromosome condensation, breakdown of sister chromatids, and individualization of chromosomes as spatial segregates. Therefore, it is reasonable to apply phase-separated polymers for disease diagnostics.

The expression of human Ki-67 protein strictly correlates with cell proliferation and can be used as a value-added marker of tumor cells.⁴⁷⁹ Only antigens can be localized in the nucleus during inter-LLPS cell division, and proteins would be precisely localized on chromosomes during mitosis. The Ki-67 protein presents during all active cell cycle phases (G1, S, G2, and mitosis). It is absent from resting cells (G0), making it an excellent marker for determining the so-called growth fraction of a given cell population.⁴⁸⁰ Ki-67 acts as a space, electrostatic charge barrier, or surfactant by phase-separating individual mitotic chromosome arms.⁴⁸¹ Generally, modulation by LLPS has the potential to enhance cancer diagnosis. To utilize LLPS behaviors for tumor diagnostics, real-time tracking and mapping of the numerous endogenous proteins at once in living cells is critical. Recently, TransitID has been applied to unbiasedly map endogenous proteome trafficking among cytosol and different organelles in living cells, including nucleolus and SGs.⁴⁸² Such a powerful approach for distinguishing protein populations even under the occurrence of LLPS can be conducive to developing precision medicine.

7.2 SGs can be targets for preventing disease progression and therapeutic resistance

Untranslated mRNPs bind over weak interactions and form cores or droplets that aggregate into SGs.^{483, 484} Normally, SGs are in dynamic equilibrium with polyribosomes, whereas pharmacological intervention or stress disrupts this equilibrium and blocks translation by inhibiting translation initiation.^{484, 485} It has been demonstrated that SGs regulate the body's behavior in various cancers, including lung, breast, pancreatic, and gastric cancer (Figure 7).^486-493 Particularly, SGs show strong antiapoptotic effects in pancreatic cancer.⁴⁹⁴ Breast cancer cell lines containing SGs exhibit lower sensitivity and less apoptosis when exposed to drugs.⁴⁹⁵ Generally, the formation of SGs would help the cancer cell to cope with the encountered stress to survive. Several studies have shown that some mRNA molecules contained in SGs can undergo a complete translation cycle and participate in and regulate various cellular biological processes.⁴⁹⁶ SGs-related components are upregulated in multiple tumor cells, including G3BP1 and G3BP2. In lung cancer, G3BP1 has been shown to negatively regulate the p53 tumor suppressor gene by interacting with lncRNA to suppress apoptosis.^{497, 498} In non-small cell lung cancer, a member of the TRIM protein family named MG53 has been revealed to modulate G3BP2 activity to inhibit SGs formation and suppress cancer progression.⁴⁹⁹ In addition, SGs may also be triggered in the high-pressure tumor microenvironment, where SGs coordinate the cellular response to the harsh cellular environment. Targeting SGs by interfering with SGs formation or assembly and altering stress conditions can affect tumor progression and sensitize cancer cells to chemotherapeutic agents.⁵⁰⁰ Sodium arsenite induces production-dependent SGs, participates in the regulation of apoptosis, protects cells in response to stress, inhibits apoptosis, and promotes cell survival.^{499, 501} Silvestrol-induced production of nondependent SGs impairs cellular resistance to stress, reduces cell survival, and promotes apoptosis.⁵⁰² Formation of SGs can contribute to the establishment of doxorubicin resistance, and circRNA–CREIT attenuates doxorubicin-induced SGs formation via the PKR/eIF2α axis in triple-negative breast cancer.⁴⁷⁶ In fact, many widely used chemotherapeutics have been found to induce SGs formation to drive cancer cells to obtain chemoresistance. SGs regulate tumor proliferation, apoptosis, invasion and drug resistance by participating in various tumor-related signaling pathways.^{503, 504} mTOR is a critical signaling pathway that regulates cellular biological behavior, and studies have found that upregulation of mTOR promotes SGs formation in cancer cells.^{501, 505} Mutations in the RAS genes (K-RAS, N-RAS, and H-RAS), the most frequently mutated genes in tumor cells, regulate the biosynthesis and catabolism of lipid signaling molecules, and contribute to the formation of SGs.^{499, 506} Therefore, engineering these pathways to destroy the aberrant formation of SGs and impaired SGs disassembly can contribute to eliminate the pathological phenomena in cancer.

As one of the most representative membraneless chambers, SGs also have an essential role in the pathophysiology of neurodegenerative diseases.⁵⁰⁷ SGs in neurons appear to be different from SGs that are generated rapidly after stress, possibly due to the presence of cytoplasmic factors that inhibit LLPS, such as posttranslational modifications, protein chaperones, specific cytoskeletal proteins, and so on.⁴⁸⁵ Neurodegenerative diseases with their slower pressure changes may also make the body's regulatory mechanisms more adaptable.¹⁴⁷ Mutations in genes encoding pathologic aggregation proteins such as TDP43 and FUS lead to excessive accumulation of SGs in cells exposed to stress.^{508, 509} The adaptive response and accumulation of SGs to transient stress is fundamentally susceptible to aggregation-based disease, and this high local concentration of increased amyloidogenic interactions would lead to the formation of persistent pathological oligomers. Targeted SGs could be used as a new option for treating neurodegenerative diseases. Studies have demonstrated the therapeutic benefit of inhibiting SGs-associated RNA-binding proteins. Knockdown of Atxn2 in an animal model of ALS overexpressing TDP43 increased the median lifespan of the animals by 80%,⁵¹⁰ and inhibition of T1A1 prolonged survival of tau mice by 26%.⁵¹¹ In addition, targeting the neural pathways involved in SGs, including the mTOR pathway and the eIF2α pathway, is also considered as a potential therapeutic means.

7.3 RNA enzymes act as potential anticancer drug targets

RNA methylation is catalyzed by nucleoside methyltransferases, which act as critical proteins in a variety of RNA modifications (writers, erasers, and readers). Recently, developing anticancer epigenetic drugs has become a hot topic in cancer therapy research.^{512, 513} METTL3 is a well-known m⁶A methyltransferase and is upregulated in multiple tumors and metastatic tissues. Elvitegravir, an anti-HIV drug, directly targets METTL3 to prevent metastasis of esophageal squamous cell carcinoma.⁵¹⁴ Actually, small-molecule STM2457 has been designed to inhibit METTL3 and show antitumor ability against acute myeloid leukemia.⁵¹⁵ Etrazepam, a thrombopoietin receptor agonist for the treatment of chronic immune thrombocytopenia and aplastic anemia, has been shown to act as a potential METTL3 inhibitor.⁵¹⁶ In addition, renal inflammation and programmed cell death associated with the administration of cisplatin were attenuated in the presence of METTL3 silencing.⁵¹⁷ METTL14 displays extremely high methyltransferase activity and is a crucial RNA-binding scaffold that is essential in recognizing substrate RNAs. The SOCS family of proteins is involved in the negative feedback regulation of cytokine signaling, which disrupts cytokine downstream signaling and leads to the development of inflammatory and autoimmune diseases and malignant tumors.⁵¹⁸ METTL14 can inhibit the proliferation and metastasis of colorectal cancer by targeting oncogenic lncRNA XIST.⁵¹⁹ METTL14 is both a promoter and a tumor suppressor in cancer, and activators and inhibitors targeting METTL14 have recently been reported. METTL14 affects many biological processes and plays multiple roles in cancer,⁵²⁰ making it a potential drug target. ALKBH5, a demethylase of m⁶A, is involved in the induction of cancer immune escape and adaptive immune responses.⁵²¹ Since ALKBH5 has both cancer-inhibitory and cancer-promoting roles, methods to induce and inhibit ALKBH5 are a viable therapeutic option.⁵²² In addition, ALKBH5 demonstrated a regulatory role in renal impairment. ALKBH5 knockout mice exhibit less pathologic injury and better renal function, and the imposition of the ALKBH5 inhibitor IOX1 promotes Ccl28 modification to inhibit inflammatory cells.⁵²³ ALKBH5 has a high potential targeting capacity, and compounds, including small molecule modulators and protein hydrolysis-targeted chimeras function at targeting the ALKBH5 regulatory mechanism.^{524, 525} Fibrillarin in 2ʹ-O-methylation has been reported to be a favorable target against tumors because it is an autoantibody for various cancers.⁴⁴⁶ Fibrillarin as a crucial constitutive protein affecting ribosome abundance and composition, offers significant possibilities for targeting key ribosome biogenesis components to reduce genotoxic activity in cancer cells.⁵²⁶ Investigating inhibitors targeting RNA modifications for the application of diseases is promising, but it is necessary to consider the role of LLPS during treatment as discussed above.

7.4 Targeting LLPS in drug design and delivery

Transcriptional control affects nearly all biological processes and events, including cancer initiation, proliferation, metastasis, recurrence, and drug resistance. LLPS has been observed to contribute to the maintenance of cellular homeostasis. Therefore, regulating the LLPS in forming SQSTM1/p62, NRF2, or FIP200 condensates would be conducive to recovering cellular homeostasis and preventing cancer progression. Furthermore, LLPS is also connected to cancer chemotherapeutic resistance, especially for involving with the formation of SGs. For example, cisplatin has been shown to suppress the formation of SGs.⁵²⁷ Meanwhile, future drug delivery system designs should focus on utilizing LLPS to enhance drug concentration and precision on targets. Based on some of the current applications of LLPS in drug delivery, precise delivery of drugs by RNA phase change to form polymers is possible.

Traditional drug development typically involves drugs that bind to and inhibit pathogenic targets, targeting proteins, and RNA via small molecule or RNA Phase modulators based on their propensity to phase separate.^{255, 528} Phase modulators can directly target RNA or proteins in IDR.⁵²⁹ In general, IDR are generally considered nondruggable therapeutic targets because they do not display stable, well-defined binding sites. Depending on the screening of inhibitors of protein–protein interactions and phenotypic screening, a small molecule targeting the inherently disordered region of p53 interaction with MDM-2 has been developed.⁵³⁰ YTHDF1 can bind to ribosomal proteins to facilitate translation of the mRNAs it targets,⁵³¹ providing a direction for this protein to be used in targeted drug development.

Numerous anomalous phase-separated drug screening and development platforms have been established to screen phase-separated targeted drugs. In 2023, Wang et al.⁵³² established DropScan, which could perform extensive screening and multidimensional analysis, identifying LY2835219, a condensate that solubilizes Ewing sarcoma fusions. Through this recognition of phase separation and the application of deep learning techniques, this platform could, in the future, screen suitable drugs in neurodegenerative, cardiovascular diseases, and so on.

Currently, there are also several applications of LLPS in drug delivery.^{533, 534} Regarding drug protection and degradability, Feng et al.⁵³⁵ used LLPS coupled with sodium alginate beads to enable drug release downstream in the gastrointestinal tract. The use of LLPS to deliver drugs, forming LLPS droplets to insulate their internal drug from physicochemical properties such as temperature, can lead to the design of LLPS systems capable of transporting active protein cascades to specific locations in human patients.⁵³⁶ In 2020, research validates the drug delivery potential of LLPS condensates by successfully delivering myoglobin to human mesenchymal stem cells using characteristics such as the noncytotoxic nature of the condensates and their spontaneous interaction with cell membranes.⁵³⁷ Nevertheless, LLPS that might impair or even reverse the destination is one of the prospects for designing and delivering cancer drugs.