There is currently a lack of pharmaceuticals targeting neurodegenerative disorders via the regulation of phase separation. The potential of melatonin in the modulation of biomolecular condensate in the attenuation of aberrant condensate aggregation in neurodegenerative disorders is discussed in this review.Biomolecular condensates are membraneless organelles (MLOs) that form dynamic, chemically distinct subcellular compartments organizing macromolecules such as proteins, RNA, and DNA in unicellular prokaryotic bacteria and complex eukaryotic cells. Separated from surrounding environments, MLOs in the nucleoplasm, cytoplasm, and mitochondria assemble by liquid–liquid phase separation (LLPS) into transient, non-static, liquid-like droplets that regulate essential molecular functions. LLPS is primarily controlled by post-translational modifications (PTMs) that fine-tune the balance between attractive and repulsive charge states and/or binding motifs of proteins. Aberrant phase separation due to dysregulated membrane lipid rafts and/or PTMs, as well as the absence of adequate hydrotropic small molecules such as ATP, or the presence of specific RNA proteins can cause pathological protein aggregation in neurodegenerative disorders. Melatonin may exert a dominant influence over phase separation in biomolecular condensates by optimizing membrane and MLO interdependent reactions through stabilizing lipid raft domains, reducing line tension, and maintaining negative membrane curvature and fluidity. As a potent antioxidant, melatonin protects cardiolipin and other membrane lipids from peroxidation cascades, supporting protein trafficking, signaling, ion channel activities, and ATPase functionality during condensate coacervation or dissolution. Melatonin may even control condensate LLPS through PTM and balance mRNA- and RNA-binding protein composition by regulating N Present in all cells, biomolecular condensates are membraneless organelles (MLOs) containing proteins, ribonucleic acids (RNAs), and other nucleic acids . These mPhase separation at its core is a thermodynamic process driven by the reduction or a negative change in global free energy ,13. LLPSMutation, mis-regulated RNA processing, and the altered binding of RNA in MLOs that are enriched with RNA and RNA-binding proteins (RBPs)  often re16−22 and disrupted prefibril formations 1F0 ATP s [1F0, P —only F1Fform ATP ,130, wheform ATP , and V-tform ATP . Once bell lines ,133, endll lines , human ull lines , HepG2 [ll lines ,137 and ll lines , HaCaT kll lines , and evell lines .0 domain of both bacterial and eukaryotic ATP synthase is embedded within the plasma membrane, the hydrophilic, water-soluble catalytic F1 domain of eukaryotes is oriented towards extracellular space, whereas that of bacteria is directed inwards toward the cytoplasm [1F0 rotors during ATP synthesis [Although the Fytoplasm ,141. Botytoplasm ,145,146.ytoplasm ,148,149.ynthesis .1F0 ATP synthase on isolated rat hepatocytes plasma membrane was independently confirmed a few years later [2+-ATPases [2+ ATPase (PMCA) were found to be exclusively localized to cholesterol/sphingomyelin-rich lipid raft domains of caveolae in pig cerebellum synaptic plasma membranes [2+ ATPases (SERCA) were similarly identified in caveolae/lipid rafts in human uterine cells [For the first time, in 2004, ATP synthase alpha and beta were discovered to be expressed in lipid rafts isolated from rat HepG2 hepatocytes by immunofluorescence , and a frs later . In the rs later . P-type -ATPases  such as embranes , and sarne cells , rat hepne cells , and humne cells . Capablene cells , caveolane cells ,160,161.ne cells ,163,164,ne cells . In ordene cells ,167. Thene cells ,152 is r1F0 ATP synthases have been observed to translocate from mitochondria to lipid raft domains of various cell types, including plasma membranes of gonadotropes [The ATP synthases of mammalian mitochondria are usually arranged in rows of dimeric complexes of two identical monomers located at the highly curved apex of deep IMM invaginations known as cristae . Dimerizdotropes , and thedotropes .Biomolecular condensates adapt to changing endogenous or exogenous conditions  by conti1F0 ATP synthases in various human cancer cell lines translocated to cell surface lipid rafts or to lipid raft domains present in mitochondria [LLPS is further regulated by lipid raft membrane-anchored proteins that support the continuous restructuring and rearrangement of molecular components in condensates . Cell suchondria . The prechondria ,183 and chondria ,105 protchondria ,184. Faichondria  to functchondria , ATP synchondria  may contchondria  and cancchondria .Lipid bilayers in cell membranes are composed of hundreds of different lipid species with a propensity to segregate laterally into subcompartmentalized raft domains ,188. Fouo) and liquid-disordered (Ld) phases to induce elastic interactions by reducing line tension between Lo and Ld phases [o phase lipid raft domains [1 domains to cell surfaces while significantly doubling the degree of extracellular ATP production within 30 min of exposure [Line tension, or the energy required to create boundaries between lipid raft domains and the surrounding membranes, is one of the key drivers that can determine the size, form, and shape of physiological nanoscopic lipid compartments . The hydd phases ; in addi domains . Furtherexposure ,210.The ability of ATP synthase/ATPase to form dimerized rows on the IMM of mitochondria and other membrane surfaces may be highly dependent upon membrane lipid composition  and curvInability to neutralize excess reactive oxygen species (ROS) accumulated as products of normal cellular functions results in a state of imbalance often referred to as oxidative stress . Oxidati0 motor of the ATP synthase controls proton flux that powers the rotation of the F1 subunit protruding into the mitochondrial matrix, driving the synthesis of ATP [1F0 ATP synthase with higher affinity than all other mitochondrial phospholipids [0 section of ATP synthase [Cristae are dynamic, independent, bioenergetic IMM invaginations capable of remodeling in seconds to organize respiratory chain supercomplex assembly and ATP synthase for efficient ATP production ,222. Mits of ATP . CL is rs of ATP ,234. Thiholipids . ATP synsynthase , whereassynthase . Mitochosynthase ; therefosynthase , includisynthase , nonalcosynthase , thyroidsynthase , diabetesynthase ,244, cansynthase , as wellsynthase , Parkinssynthase ,248, amysynthase , Barth ssynthase ,251, andsynthase ,253. Thesynthase ,255. Altsynthase ,257.Cardiolipin (CL) is a mitochondria signature lipid distinctly attracted to membrane lipid domains with strong negative curvatures, such as the apex of IMM cristae ,228. CL α-syn demonstrates a high affinity for mitochondrial membranes, interacting in close proximity with mitochondrial OXPHOS proteins, including lipid raft-like domains at MAMs that are high in phospholipids ,283. NatPhysiological lipid rafts function optimally at nanoscopic sizes ,293,294.Saccharomyces cerevisiae under ROS attack [Melatonin is a potent antioxidant that has been shown to inhibit CL peroxidation in mitochondria, preventing mPTP opening and Cyt c release  by inhibS attack . The NLRS attack ,267,273.S attack ,305,306 ATP-dependent DEAD-box RNA helicases (DDXs) are ATPases that regulate RNA-containing phase-separated organelles in prokaryotes and eukaryotes ,308. DDXThe activation of the NLRP3 inflammasome is now associated with major neurodegenerative disorders such as AD, PD and ALS, where positive correlations have been found to exist between NLRP3 levels and abnormal protein aggregations such as Aβ and α-Syn, whereas the inhibition of the NLRP3 pathway attenuates pathological protein aggregations . MelatonNanoscopic transient lipid raft domains in biological membranes are formed by phase separation in response to external stimuli ,93,188. d phase to drive cholesterol into the ordered Lo phase [1–40 and Aβ1–42 peptides are known to interact strongly with negatively charged lipids by binding to anionic, negatively charged membranes [In 2005, melatonin was first observed to induce phase-separation in DPPC lipid bilayers ; recentlLo phase . These sembranes ,325,326.embranes . Althougembranes ,162, itsembranes ,330,331.25–35 peptides embedded in hydrocarbon cores of anionic lipid bilayers may further displace cholesterol molecules to increase oligomerization or fibrillation [In animal and in vitro studies, melatonin was able to prevent or ameliorate tau and Aβ pathology in AD ,334,335 illation , but theillation . Melatonillation . On the illation .Local variations in melatonin concentration also affected the re-ordering of lipids in membranes. At 0.5 mol% concentration, melatonin was documented to penetrate lipid bilayers to form fluid domains that enriched lipid membranes where melatonin molecules aligned parallel to phospholipid tails with the electron-dense regions slightly below hydrophilic headgroups; however, at 30 mol% concentration, melatonin molecules aligned parallel to the lipid bilayer, close to the headgroup regions where one melatonin molecule was associated with two lipid molecules to form an ordered, uniform, lateral membrane structure distributed evenly throughout the membrane model . VariatiMembrane fluidity reveals the degree of molecular disorder and motion within lipids in membrane bilayers . There ad phase [o–Ld phase separation over a range of temperatures and domain sizes, effectively preventing the formation of a non-raft Ld phase, possibly by reducing line tension or acting as a surfactant at Lo–Ld interfaces [It is believed that lipid rafts function optimally as nanodomains ,293, whed phase . Interesterfaces . ATP is terfaces ,31 capabterfaces . Whetherterfaces  to inducterfaces . NonetheDuring lipid peroxidation events, oxidized moieties were found to mainly reside close to the lipid headgroups forming hydrogen bonds with water. These oxidized lipids can perturb membrane bilayer structures and modify membrane properties, including decreasing the membrane fluidity ,354,355.Lipid rafts are phase-separated regions in lipid bilayers responsible for important biological functions including signal transduction ,93 as we+ as a result [Present in all eukaryotes , CYB5R3 a result ,371. Thea result , whereasa result ,373,374.a result ,376.Originally known as mitochondrial porin after its identification in yeast (1985)  and humaCYB5R3 enzymes form large redox centers in lipid rafts that enhance mitochondrial respiration rate and ATP production, albeit resulting in increased production of ROS ,373,374.VDAC is the gatekeeper which controls the export of ATP out of mitochondria into cytosol and the import of essential respiratory substrates such as ADP and Pi into mitochondria ,399; theThe mitochondrial electron transport chain is a major ROS-generating site where complex III and mitochondrial glycerol 3-phosphate dehydrogenase can produce large amounts of redox signaling molecules such as superoxide and hydrogen peroxide to the external side of the IMM as well as the matrix ,404. BisMelatonin is an ancient, potent antioxidant that protects lipid nanodomains from peroxidation caused by excess oxidative stress. The addition of micromolar concentrations of melatonin to rat heart mitochondria dramatically inhibited CL oxidation by tert-Butylhydroperoxide (t-BuOOH), a peroxidation promoting peroxide, reversing cytochrome c release, matrix swelling, and proton motive force (ΔΨ) collapse in treated cells . The melN-acetyl-5-methoxytryptamine) is a mitochondria-targeted molecule found in cells of all tested eukarya and bacteria [N-acetyltransferase (AANAT), the enzyme responsible for the rhythmic production and release of melatonin in bacteria, fungi, unicellular green algae, and chordates [Rhodospirillum rubrum and cyanobacteria, precursors to mitochondria and chloroplasts, respectively [Melatonin  in prokaryotic bacteria ,428, whehibitors .0CF1) control transmembrane electrochemical proton gradients for the production of ATP [The gene sequences of cyanobacteria ATP synthase subunits are extremely similar to those in chloroplasts . Embedden of ATP ,438,439.n of ATP , PG is tn of ATP . Both CLn of ATP ,445,446.•OH) through the sequestration of metal ions and deactivating •OH during Fenton-like reactions in Type II antioxidant reactions [Melatonin and its secondary, tertiary, and quaternary metabolites actively scavenge potent free radicals ,426,447 eactions . In addieactions .•OH employing density functional theory found that one molecule of melatonin effectively scavenged two •OH radicals to produce the stable footprint metabolite, cyclic 3-hydroxymelatonin (3-OHM) [•OOH) at rates 98.4 times faster than Trolox in aqueous solution [•OOH and other free radicals that penetrate deep into lipid molecules.A recent study that analyzed the mechanistic interactions between melatonin and  (3-OHM) , in perf (3-OHM) ,459,460.solution . Melatonsolution ; therefo•OH [•OOH, the two most prevalent ROS responsible for the chain oxidation of unsaturated phospholipids [All biological cell membranes comprise amphipathic lipid molecules with hydrophilic heads and hydrophobic tails that naturally form bilayers with headgroups oriented towards an aqueous environment and tails facing each other . The mel•OH  and •OOHholipids ,466 in tholipids ,468.•OOH and •OH and terminates with reactive aldehyde end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) [•OOH and •OH are derived from ubiquitous superoxide radicals (O2•−) generated from the one-electron reduction of oxygen (O2) that may be catalyzed by nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) during respiratory bursts [−1·s−1 [2•− behaves more similarly to an unimpressive reductant (E°′(O2/O2•−) = −0.33 V) than an oxidant (E°′(O2•−/H2O2) = 0.93 V) [•OOH [•OOH or HO2•), also known as a perhydroxyl radical, is a chemically active, protonated form of superoxide radicals (O2•−) [2•− into hydrogen peroxide (H2O2) which then can further be transformed via Fenton’s/Haber–Weiss reactions [•OH, possibly the most reactive and mobile species of oxygen that interacts with almost all molecules in cells [•OOH exists primarily as the less reactive O2•−, where the ratio of protonated •OOH to anionic O2•− is ~130:1 (less than 1%), •OOH can be a potent initiator of lipid peroxidation [Lipid peroxidation, a physiological process in all aerobic cells , is a caal (HNE) ,472,473.y bursts  and/or ey bursts . Due to  [−1·s−1 , O2•− be 0.93 V) ,477,478 V) [•OOH ,479. Hyds (O2•−) , engagedeactions  into •OHin cells ,481. Evexidation ,466.•OOH to accumulate at lipid headgroup membrane–water interface at concentration enhancement of over one order of magnitude [•OH, •OOH, and H2O2 with polar headgroups of phospholipid bilayer revealed that all three species traveled deep into the water layer to reach phospholipid biomolecules, oxidizing hydrophilic headgroups before hydrophobic tails [•OOH staying adsorbed for the longest duration at headgroup regions [When reacting with phospholipids, the advantageous free energy profile of −8.5 kJ/mol free energy minimum relative to the aqueous phase allowed agnitude . Multi-lic tails , with •O regions . The hea regions , support regions . +) concentration (~700 to ~800) [+ concentration at membrane surfaces may cause accumulation of •OOH, the protonated form of O2•− [The strong negative curvature of cristae in the IMM is primarily sustained by the distinct molecular geometry of CL with its smaller, elongated, conical-shaped, double-phosphate dianonic headgroups that increase lateral pressure within the acyl chain regions and stabilize cylindrically curved, tubular cristae structures ,485,486.to ~800) , is the to ~800) . At the  of O2•− .•OOH remains adsorbed at polar headgroups longer than other ROS tested [S tested ; therefoS tested ,497,498 S tested  and/or uS tested . In the S tested ,501 becaS tested . The abiS tested .As early as 2002, melatonin was found to increase mitochondria OXPHOS activity and elevate the production of ATP . Recent Saccharomyces cerevisiae and human hepatocarcinoma cells challenged with hypoxic stress, the non-canonical RNA-binding proteins in glycolytic enzymes have been observed to promote phase separation [Interestingly, in paration  that facparation . Melatonparation ,504,505;paration . These rparation . Alteratparation . Increasparation ,517,518.Biomolecular condensates containing protein, RNA, and other nucleic acids  are formCells also employ post-translational modifications (PTMs) to induce non-equilibrium thermodynamic chemical reactions in order to tune the molecular interactions of key condensate components where external energy input drives reactions out of equilibrium to control the size and number of MLOs . PTMs, iCellular stress in eukaryotes activates defense mechanisms such as stress granules (SGs) that can promote either survival or apoptosis . IntegraUnder cellular stress conditions, phosphorylation can initiate the formation of SGs  and alsoMissense mutations associated with diseases are found mostly within IDRs . These mIntriguingly, ATP has recently been reported to exhibit a unique biphasic relationship with TDP-43 PLD. At a molar ratio of only 1:25 (PLD:ATP), TDP-43 PLD was induced to undergo LLPS to start forming liquid droplets in a dose-dependent manner where many droplets could be produced at a 1:100 molar ratio. Further increases in ATP, in contrast, led to a reduction in droplet formation. At 1:750, only a few droplets could be detected, and at 1:1000, all droplets were disassembled by ATP. Importantly, in the absence of ATP, TDP-43 PLD was unable to phase-separate into droplets . NeuronsMutations in fused in sarcoma (FUS) are associated with ALS pathology, and are believed to be a major cause behind familial ALS ,599. UndPhysiological FUS is a transcription factor  which haC. elegans, phosphorylation also promoted IDR granule disassembly, whereas dephosphorylation promoted granule assembly [Phosphorylation is one of the most important PTMs that can control the assembly/disassembly of MLOs  as well assembly . Under dassembly ,351,536,assembly ,538.The ATP-dependent DEAD-box helicase  DDX3X reAD is associated with the aggregation of Aβ as well as the intracellular deposition of neurofibrillary tangles (NFTs) of tau, a major neuronal protein with important physiological functions of stabilizing and promoting the assembly of microtubules (MTs) in the central nervous system (CNS) ,635. TheLLPS of tau has been demonstrated to promote amyloid aggregation . Tau canTau phosphorylation has been proposed as a neuroprotective mechanism  where ph1–42-induced memory impairment, synaptic disorder, and tau hyperphosphorylation-associated neurotoxicity in C57BL/6N mice [25–35 in pheochromocytoma (PC12) cells by activating melatonin-PTEN/Akt/GSK3β signaling [The use of melatonin in neurodegenerative disorders has been extensively studied and reviewed ,692,693./6N mice ; restori/6N mice ; and dec/6N mice . Melaton/6N mice . Even thignaling . The majignaling  adds an Even though melatonin is able to ameliorate tau-pathology-related symptoms such as oxidative stress, neuroinflammation, cognitive impairment, cell death, and tau hyperphosphorylation in vivo , the fac1–40 and Aβ1–42 to inhibit the formation of β-sheet and/or amyloid fibrils [1–40 and Aβ1–42 with only 100 μM of melatonin could not be replicated in control experiments using a potent free radical scavenger N-t-butyl-a-phenylnitrone (PBN), or a melatonin analog 5-hydroxy-N-acetyl-tryptamine (NAT) [An in vitro study on Neuro2A cells reported that melatonin at 10 μM concentration reduced intracellular ROS levels induced by tau aggregate treatment, and at 50 μM, melatonin reduced phospho-tau as well as GSK3β mRNA and subsequent protein levels. Melatonin increased cell viability in tau-exposed neurons in a dose-dependent manner, with 80% viability observed at 20 μM melatonin and a complete reversal at 200 μM, compared to only a 60% viability in controls without melatonin . In an e fibrils , and thene (NAT) . Even thne (NAT)  by disrune (NAT) ,711 critne (NAT) , the conne (NAT)  or 5000 ne (NAT)  requiredne (NAT) ,708, or ne (NAT) . More imMLOs are found abundantly in the nucleus, cytoplasm, and on the membranes of almost all eukaryotic cells , where t−/−) mouse model demonstrated the formation of tau filaments elevated intraneuronal unesterified cholesterol, which may result in a vicious circle where tau fibrils alter cholesterol homeostasis and disturb cholesterol metabolism which continues to promote tau pathology [c domains [c domains [c domains to become more fluid, mechanistically deforming membrane bilayer structure [c domains that have become more fluid will transition into Lo lipid raft domains [Mutations in the Niemann–Pick type C (NPC) gene cause disturbances in cholesterol metabolism in lipid rafts , where aathology . Physiolathology . A recenathology . The binathology  includinathology . As a re domains  in anion domains . Increas domains  may caustructure . Melatontructure , bindingtructure . Lc doma domains ,724 that domains ,198.o–Ld phase separation over a range of temperatures [Melatonin may regulate lipid dynamics and composition, modifying the lipid hydrocarbon chain order to promote phase separation in ternary membrane models ,319, as eratures . The abieratures ; therefoeratures  can poteeratures ,722,725.The tumor suppressor p53 has been found to interact with tau and Aβ, forming pathological aggregates that result in the mislocation and impairment of its essential physiological DNA repair functions ,727,728.Often called the “guardian of the genome” , p53 is N-termini that flank the DBD [N-termini of p53 [More than 50% of all human cancers have been associated with missense  and/or s the DBD ,767,768. the DBD ; therefo the DBD . In addi the DBD ,771,772  the DBD . More imi of p53 .N- and C-termini disordered domains [Recent in vitro studies have demonstrated that formation of highly fluid p53 droplets at neutral and slightly acidic pH, and a low-salt environment is mediated by its  domains . The DNA domains . Molecul domains ,609. It A recent study demonstrated that wild-type p53 expressed in living yeast not only had the capacity to form liquid-like, dynamic, unstable droplets that appeared and disappeared in response to the presence or absence of stress, respectively, but when overexpressed, was able to propagate into true amyloid-like prions that could seed other molecules, and at the same time suppressed p53 transcription activities to precipitate tumorigenesis , confirmIn AD human brain tissues and animal models, the interaction between p53, pathological tau oligomers, and Aβ form aggregates resulting in the mislocalization and impairment of its important physiological functions in DNA repair ,727,728.The main function of UPS is to degrade and eliminate abnormal proteins damaged by oxidative stress and/or mutations after they are covalently bound to ubiquitin in an ATP-dependent pathway . Many MLIn eukaryotes, the UPS may be the most complex, extensive, cytosolic proteolytic enzyme system that performs essential functions  includin+, CoA into acetyl-coA, NADH and CO2. The phosphorylation of the E1α subunit of pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 2 (PDK2) blocks the entrance of acetyl-coA into the tricarboxylic acid (TCA) cycle, inhibiting the OXPHOS production of higher ATP [The theoretical maximum of ATP calculated from simultaneous measurements of extracellular acidification and oxygen consumption indicated that OXPHOS ATP production was close to or more than 16 times above glycolysis, at 31.45 ATP/glucose  and 2 ATP/glucose, respectively . Whethergher ATP ,825,826.gher ATP ; thus, agher ATP . Decreasgher ATP  as well gher ATP .VDAC is the gatekeeper that controls the export of ATP out of mitochondria into cytosol and the import of essential respiratory substrates such as ADP and Pi into mitochondria ,399; the+, CoA into acetyl-coA, NADH and CO2, supporting the TCA cycle in OXPHOS to generate higher levels of ATP [p53 is a transcription factor  that ress of ATP ,825. AD s of ATP ,830; thes of ATP ,846,847 s of ATP ,851,852,s of ATP .Breast cancer (MCF-7) and human colorectal carcinoma cells (HCT116) treated with 1 μM melatonin exhibited p53 activation and accumulation which inhibited proliferation and protected against DNA damage through the ATP-dependent phosphorylation of p53 at serine 15 (Ser15) . PhosphoAs part of the complex PTM/UPS modification system, SUMO modification, similarly to ubiquitination, is an ATP-dependent enzymatic cascade involving activating, conjugating, and ligating E1, E2, and E3 enzymes, respectively ,873. SmaNucleoli PMLs are phase-separated quality control MLOs that compartmentalize misfolded proteins for clearance . In mammIn the cytoplasm, exposure to heat ,909, oxiFrontal cortex tissues of double-transgenic APP/PS1 AD mice that were given daily intraperitoneal injections of melatonin at pharmacological concentration of ~10 mg/kg  for 3 weeks exhibited a significant degradation of Aβ as a result of the SUMOylation of the amyloid precursor protein (APP) intracellular domain (AICD) at lysine 43 by the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) . AICD SU2O2 increased SUMO conjugation, but exposure to low levels of H2O2 (1 mM) induced a severe, rapid deSUMOylation within 5 min, resulting in the disappearance of SUMO conjugates including transcription factors [Stress granules are formed in response to external stress factors; therefore, hypoxia, heat, oxidative, osmotic, and genotoxic stress can also significantly increase SUMO conjugate levels as a protective response . Under s factors . Even th factors , a dysre factors  due to t factors  but also factors .Ribonucleic acid (RNA) is a single-stranded molecule with alternating ribose and phosphate groups attached to adenine, uracil, cytosine or guanine bases. The evolution of RNA is believed to precede that of DNA; nonetheless, the origin of the “RNA World” has not been resolved to date ,927,928.Phase-separated MLOs are enriched in RNA and RNA-binding proteins with IDRs ,630,941.Transcription factors (TFs) can undergo LLPS to form dynamic regions that compartmentalize and concentrate other TFs, enriching transcription-related proteins to activate the transcription of target genes . RNA proAberrant RNA–RNA interactions leading to the sequestration and/or dysregulation of RNA-binding proteins in MLOs may be one of the major driving forces behind neurodegenerative diseases ,948,949.6A) in the 5′ UTRs of mRNA [6A mRNA modification is dynamic, reversible, and has been observed to be oxidative-stress-dependent. Stress-induced methylation is recognized by the m6A cytoplasmic “reader” protein, YTH domain family 3 (YTHDF3) [6A to affect heterogeneous RNA and protein contents of SGs resulting from stress-specific differentiation in composition, dynamics of assembly and disassembly may ultimately determine the viability or pathology of cells in neurodegenerative diseases [The mechanism of selection for mRNA inclusion in SGs is determined by mRNA modification mediated by a prevalent methylation at position 6 of adenosine (m of mRNA . m6A mRN(YTHDF3) , which t(YTHDF3) . The abidiseases .6-methyladenosine (m6A), which involves the transfer of a methyl group to the sixth position of the purine ring in RNA adenosine [6A is installed by m6A methyltransferases and removed by m6A demethylases. RNA splicing, transcription, stability, and metabolism are all regulated by RNA m6A modifications [6A mediates structural switches that affect RNA stability and activity, regulating the access of RNA-binding proteins to their RNA binding motifs [6A is possibly the most prevalent internal, dynamic, reversible chemical modification identified to date, and plays critical roles in the growth, differentiation, and metabolism of cells [6A RNA methylation is involved in most aspects of RNA processing that may affect the regulation of cellular processes [Modification of eukaryotic messenger RNAs (mRNAs) occurs mostly via Ndenosine . m6A is ications ,955,956.g motifs . Among hg motifs , m6A is of cells ,959. An rocesses  such as rocesses ,962,963,rocesses , circadirocesses , fertilirocesses ,968,969,rocesses .6A RNA modification of eukaryotic RNAs is dynamic and reversible, where the methylation of mRNAs, tRNAs, rRNAs, and long non-coding RNAs by “writers” (RNA methyltransferases) such as METTL3 [6A-binding proteins) such as YTH domain proteins [6A “writers”, “readers”, and “erasers” are increasingly associated with degenerative and metabolic diseases. Fat mass and obesity-associated (FTO) protein, the m6A “eraser” associated with human obesity and energy homeostasis [6A “eraser”, was found to be overexpressed in the tumorigenesis of glioblastoma stem-like cells [6A “writer” was also identified as a critical regulator of a chromatin-based pathway that maintained cells in a leukemic state, where the inhibition of METTL3 removed the myeloid differentiation block in human and mouse AML cells [6A “reader”, was able to enhance antitumor immune responses in the dendritic cells of tumor-bearing mice [6A is increasingly associated with tumorigenesis [The ms METTL3  is removs METTL3 ,973, andproteins ,975. Dyseostasis ,978,979,eostasis , hepatoceostasis , melanomeostasis , and acueostasis ; howevereostasis . ALKBH5,ke cells . The METML cells . Depletiing mice . The dysigenesis ,988,989 igenesis ,992,993.6A methylation may be highest in the brain, regulating embryonic stem cell differentiation and brain development [6A methylation potentially drives neurodevelopmental disorders [6A RNA methylation in the brains of double-transgenic APP/PS1 with those of control mice revealed statistically significant elevations of m6A methyltransferase METTL3 and downregulations of m6A demethylase FTO in the cortex and hippocampus of AD mice [6A methyltransferases where METTL3 and the RNA-binding motif protein 15B (RBM15B) were downregulated and upregulated in the hippocampus, respectively. METTL3 was observed to be accumulated in the insoluble fractions of tau proteins, possibly implying an epitranscriptomic mechanism in altered gene expression in neurodegenerative disorders [melopment ,998,999;isorders . Investi AD mice . Post-moisorders . RNA epiisorders  may provisorders  to facilisorders .6A modifiers can lead to changes in the regulation of gene expression, affecting cancer [6A methylations, such as METTL3, METTL14, YTHDF1, YTHDF2, FTO, and ALKBH5, are significantly correlated with p53 mutations in AML patients [6A regulatory genes. In addition, their overall and event-free survival is worse than patients without m6A genetic alterations [6A methyltransferase METTL3 in hepatocellular carcinoma cells (HepG2) caused alterations in gene expression and alternative splicing in more than 20 genes, including MDM2, MDM4, and p21, involved in the signaling of p53 [6A methylation by METTL3 at the point-mutated codon 273 (G > A) of p53 pre-mRNA promoted a preferential pre-mRNA splicing that produced p53 R273H mutant genes that were resistant to multiple anticancer drugs in colon cancer cells [Dysregulation of mg cancer ,1004, neg cancer ,991,1005g cancer , blood pg cancer , and carg cancer . More thg cancer  and/or sg cancer ,760,761.patients . Most  inhibited SG formation and the recruitment of mRNAs into SGs [6A-RNA before they can be complexed into stress granules, implying that polymethylated m6A-RNA may act as a scaffold for YTH proteins, causing them to undergo LLPS through interactions within their own low-complexity domains [6A can be regarded as a beacon that attracts YTH proteins into stress granules [6A modifications.Aberrant RNA–RNA interactions leading to the sequestration and/or dysregulation of RNA-binding proteins in MLOs may be one of the major driving forces behind neurodegenerative diseases ,948,949.” YTHDF3 ,951. The” YTHDF3  enrichedinto SGs . YTH pro domains ,1018. Thgranules . PTMs sugranules  and SUMOgranules ,916. Mel6A “writers” and “readers” is also associated with the progression of cancer.Even though AICD SUMOylation has been shown to exert beneficial effects in transgenic AD mice models, the study of SUMOylation in neurodegenerative disorders and other diseases has led to controversial and often contradictory observations, because pathways can undergo SUMOylation at different sites, yielding conflicting consequences. For example, the SUMOylation of many important proteins in AD, including APP and tau, have been associated with the pathogenesis of AD ,1021 and6A levels in vitro and in vivo—a process that can be reduced by deSUMOylation enzyme sentrin/SUMO-specific protease 1 (SENP1) [6A “reader” YTHDF2 by SUMO1 at lysine residue K571 in vitro and in vivo increased the binding affinity of YTHDF2 to m6A-modified mRNAs, altering gene expression profiles, resulting in the increased proliferation, migration, colony formation and tumor growth of lung cancer H1299 cells [The SUMOylation of METTL3 was found to promote tumor growth and colony formation in human non-small cell lung carcinoma (NSCLC) cells (H1299) , where t (SENP1) ,1040. In (SENP1) . However99 cells .6A “reader” targets and destabilizes m6A-modified mRNAs, facilitating the localization and degradation of m6A mRNA in MLOs such as P-bodies [5C, a ubiquitous post-translational RNA modification found in mRNAs [5C in RNA, significantly regulating 208 out of 1350 identified m5C sites that may affect pre-rRNA processing through the modification of m5C levels in rRNA [The YTHDF2 mP-bodies ,953. UltP-bodies . The proin mRNAs . YTHDF2  in rRNA . More imN-acetyl-l-cysteine (NAC), an antioxidant, also augments YTHDF2 expression in oxidative-stress-induced senescent cells; therefore, it is believed that oxidative pathways may negatively regulate YTHDF2 expression [Stimulation of oncogene Ras led to the suppression of YTHDF2 that stabilized the transcription of MAP2K4 and MAP4K4, upregulating the senescence-associated secretory phenotype (SASP) in human ovarian surface epithelial cells (HOSEpiC). Treating HOSEpiC cells with 1 μM of melatonin enhanced the expression of YTHDF2, reversed telomere shortening, and blocked Ras-induced growth arrest . The actpression . On the Adult male C57BL/6J mice pretreated with melatonin intraperitoneal injections (25 mg/kg b.w./day × 14 days) exhibited attenuated cell viability loss, ROS generation, mitochondrial dynamics imbalance, and mitophagy in spermatogonial stem cells (SSCs) induced by daily intraperitoneal injections with chromium (VI) (16.2 mg/kg b.w./day × 14 days), an environmental toxin and carcinogen that can cause male infertility by damaging SSCs ,1048. In6A regulates the pluripotency of embryonic stem cells (ESCs) [6A modification and significant reduction in m6A “writer” METTL3 [6A mRNA methylation and altered the subcellular location of METTL3, preventing m6A-dependent mRNA decay to stabilize key pluripotency factors Nanog, Sox2, Klf4, and c-Myc [6A methylation [6A modification compared to wild-type ESCs treated with melatonin [6A mRNA methylation.On the other hand, melatonin was found to decrease METTL3 expression and modification in another report. ms (ESCs) . Treatme” METTL3 . Melatonnd c-Myc , known thylation . It has hylation . Using delatonin . Howeverelatonin ) and/or elatonin ) loweredelatonin , then it6A mRNA methylation can be oxidatively reversed or “erased” by m6A demethylases such as FTO [mh as FTO ,1055 andh as FTO ,1056. Juh as FTO , the defh as FTO . In facth as FTO . DDX3 ish as FTO . DDX3 wah as FTO .NLRP3 inflammasome, a widely documented target of melatonin associated with pathological protein aggregates in neurodegenerative disorders ,313,314,When life originated, LLPS driven by multivalent macromolecular interactions might have been the organizing principle behind the subcellular compartmentalization of MLOS in eukaryotes and prokaryotes ,277,428.Adenosine triphosphate (ATP) is one of the four nucleotide monomers used during RNA synthesis . RNA hasCells rely upon RNA to regulate condensates because RNA molecules contain powerful electrostatic forces due to the high negative charge densities buried in their phosphate backbones ,531,532.Saccharomyces cerevisiae in 1999, DX19 (human)/Dbp5 (yeast) [Since its discovery in  (yeast)  has been (yeast) ,1081, mR (yeast) , and tra (yeast) , as well (yeast) ,1084. On (yeast) . NPCs no (yeast) .It has been proposed that the ATP-dependent catalytic cycle of DDX19 involves the cycling between open and closed conformations to bind RNA for export into cytoplasm . Even thNPCs can be visualized as thousands of toroid-shaped “ultradonut”-like pores with extremely high curvatures generated by nanoscale buckling instabilities triggered by membrane stresses during nuclei growth . These “Saccharomyces cerevisiae with defects in regulating membrane fluidity assembled NPCs that were defective, whereas the restoration of membrane fluidity via the addition of membrane-fluidizing agents attenuated defects in NPC biogenesis and normalized mRNA export [•OH [•OOH, the two most prevalent ROS responsible for the chain oxidation of unsaturated phospholipids [As early as 1979, the regulation of nuclear RNA release was found to be directly correlated with nuclear membrane fluidity where a reduction in membrane fluidity caused a linear decrease in RNA release . It has A export . The perA export ,1101 mayA export ,354,355.A export ,341,356.A export , especiaort [•OH  and •OOHholipids ,466 suchholipids  , preserving nanoscopic lipid domain structure and composition, possibly by reducing membrane line tension [o-phase lipid rafts in cells and nuclei (During protein membrane adsorption, the complex interactions between lateral membrane organization and proteins often enhance the propensity of membrane lipids to form domains or to phase-separate . These d binding . Cells m tension . Lipid p tension ,218,296.d nuclei .Rhodospirillum rubrum and cyanobacteria, precursors to mitochondria and chloroplasts, respectively [The relationship between melatonin and RNA is likely an ancient one that might date as far back as ~4 billion years ago, possibly after the height of the “RNA world” ,1117, whectively ,424,425,ectively ,426,427.The physiological and pathological functions of biomolecular condensates in neurodegenerative disorders are shaped by powerful, complex, interdependent relationships between membraneless organelles, membranes/lipid rafts, ATP, RNA, and most of all, stress and its timely resolution. Melatonin’s intimate association with each of these decisive influencers may position the potent, ancient antioxidant as an important mediator of the phase separation of condensates in health and disease via principal ATP-dependent mechanisms including post-translational modifications and RNA m6A modifications . This no


One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a chemically-accurate sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1, and TDP-43 proteins along with that of their corresponding prion-like domains and RNA recognition motifs from absence to moderately high RNA concentration. By characterizing the phase diagram, key molecular interactions, surface tension, and transport properties of the condensates, we report a dual RNA-induced behavior: on the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whereas at high concentration, it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity even at high concentration, but barely modify protein self-diffusion which mainly depends on RNA concentration and on the effect RNA has on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases. Biomolecular condensates are liquid-like membraneless compartments that contribute to the spatiotemporal organization of the cellular material. Remarkably, the assembly of these condensates can be critically regulated by RNA. Here, by means of a residue-resolution coarse-grained model, we investigate the role of RNA concentration and length in the stability and kinetics of FUS, hnRNPA1, and TDP-43 protein condensates. Besides observing the “so-called” RNA-induced reentrant behavior with concentration, we find that RNA length plays a major role in condensate dynamics. We identify a minimal critical RNA length at which phase separation is enhanced at low RNA concentration while condensate viscosity can be still reduced at moderate RNA concentration. Overall, our simulations provide a detailed characterization of the underlying molecular driving forces behind RNA-binding-protein aggregation.Liquid-liquid phase separation (LLPS) is one of the key processes employed by cells to control the spatiotemporal organization of their many components , 4, 5, 6π-π interactions) have been found to be critical in regulating LLPS  or proteins with globular domains connected by flexible linkers that can establish multiple homotypic or heterotypic interactions with cognate biomolecules  over their interactions with the solvent ,11. Seveing LLPS , 53, 54.In vitro experimental evidence shows how protein aggregation can be enhanced upon addition of RNA at low concentration but inhibited at high concentration ,55,56. SRecent experimental advances in single-molecule Förster resonance energy transfer have enabled the direct observation of the structural and dynamic protein behavior in diluted conditions , 70, 71;In that respect, computer simulations emerge as a great tool to enlighten such a blind spot , 79, 80.This work aims to narrow down this problem by shedding light on the RNA modulation of transport properties and stability of RBP condensates. By employing a high-resolution CG model for RNA and intrinsically disordered proteins (IDPs) ,119,126,A detailed explanation of our model and methods can be found in the π interactions by means of short-range pairwise potentials and for electrostatic interactions through Yukawa long-range potentials (see π and electrostatic interactions are described by Lennard-Jones (Eq. S5) and Yukawa/Debye-Hückel potential terms (Eq. S3), respectively. The low salt physiological concentration regime (∼150 mM) of the implicit solvent is controlled by the screening length of the Yukawa/Debye-Hückel potential. Given that the original HPS model  of the encoded protein amino acids ,111. HerPS model  has beenractions , we emplractions . Additioractions ,127. AllA). We evaluate the phase diagram for the full protein sequences as well as for some of their specific protein domains such as the RRMs or the prion-like domains (PLDs). More precisely, we focus on the following sequences: FUS (full sequence), FUS-PLD, hnRNPA1 , hnRNPA1-PLD, hnRNPA1-RRM, TDP-43 (full sequence), and TDP-43-PLD  and another in which the whole PLD region remains fully disordered (wild-type wt-TDP-43). Despite h-TDP-43 and wt-TDP-43 only differing by less than 10% of their sequence structural conformation  of all the aforementioned proteins  against the experimental saturation concentration of the proteins to undergo LLPS for FUS  demonstrates that the cation-π reparameterization proposed by Das et al.  simulations , 138 in  for FUS ,85, FUS- for FUS , hnRNPA1 for FUS , hnRNPA1 for FUS , wt-TDP- for FUS ,85, h-TD for FUS ,85, and  for FUS  are plotx et al.  correspos et al.  on top os et al.  is able s et al.  Fig. S2B of all orted in . Notablyorted in . On the orted in . To ratiB). To further characterize the molecular, structural, and interfacial properties of the previous protein condensates, we now perform a comprehensive full analysis of their surface tension, LLPS-stabilizing most frequent contacts, protein conformational ensembles in and out of the droplet, and condensate structure.The specific composition and patterning of the amino acids along the sequence has a huge impact on the protein macroscopic phase behavior ,103,134.A, we plot the surface tension (γ) between the condensate (protein-rich) and protein-poor liquid phases as a function of temperature . An advantage of computer simulations is that γ between two coexisting fluid phases (or between a fluid and a vapor one) can be easily computed, as explained in the γ than h-TDP-43. At the same temperature, the presence of the helical structure in h-TDP-43 promotes a more compact assembly of proteins in the condensed phase, increasing the surface tension. Additionally, TDP-43-PLD droplets present much smaller γ than those of any of its two full-sequence variants at moderate temperatures, explaining why TDP-43-PLD domains are markedly exposed toward the interface in wt-TDP-43 condensates (B). Similarly, the surface tension of FUS-PLD droplets is lower than that of FUS (full sequence). However, interestingly, γ for hnRNPA1 and hnRNPA1-A-LCD droplets are remarkably similar . Proteins with higher γ can typically phase separate until higher temperatures or at lower protein concentration.In densates B. Simil B and γ B. ProteB), evidencing that their PLD interactions barely favor aggregation (B (center). Conversely, the α-helix structure of h-TDP-43 notably favors the interaction between helical domains and hence between the rest of the intrinsically disordered neighbor regions, significantly enhancing the PLD connectivity and thus reducing droplet heterogeneity as experimentally suggested .Next, we focus on the structural organization of the different protein condensates. A significant contrasting behavior between both FUS and hnRNPA1 droplets and those of TDP-43 (both variants) is observed. Although both FUS and hnRNPA1 exhibit homogeneous density droplet distribution with their PLDs indistinctly located along the condensate , TDP-43 shows a highly marked heterogeneous distribution exposing its prion-like domains toward the droplet boundaries B, evideregation ,135. Thiregation . In the uggested . Moreovendensate B.C and To rationalize the molecular driving forces behind these structural differences, we compute 1) the amino acid contact map frequency of the proteins within the condensates C and S8A), highlighting how hydrophobic, cation-π, and more modestly electrostatic interactions contribute to stabilize the droplets. Because glycine (G) represents nearly 30% of the residues along FUS sequence, the frequency of G-G is the highest despite not being one of the strongest pair of amino acid interactions (π interaction R-Y becomes the most relevant one inducing LLPS (B). Furthermore, when analyzing the FUS contact map (C), we observe that its prion-like domain, despite showing a much lower ability to phase separate on its own than the full protein, markedly interacts with the three RGG domains. The top contacts of the PLD alone are very different from those of the full-sequence FUS (A), resulting in much worse phase-separation capabilities for the PLD than for the full-FUS sequence (B) as experimentally observed (C).In FUS condensates, the most repeated contacts are G-G, R-Y, and G-Y A, highlractions . Howevering LLPS ,77 accoring LLPS B. Furthtact map C, we obence FUS A, resulsequence B as expobserved ,61. We aobserved C.C). Nonetheless, the wt-TDP-43 PLD region is still the second protein domain establishing more contacts in total after the RRM1 segment, but mostly because of its length. The three most predominant contacts in wt-TDP-43 (according to our model (A), clearly denoting the key role of cation-π and electrostatic interactions in driving condensation. However, when the structured helical region is present (h-TDP-43), R-F contacts sensibly increase, becoming the third most dominant interaction. Interestingly, the renormalization of contacts by amino acid abundance in TDP-43 barely modifies the list of the most frequent interactions, probably because of the very homogeneous distribution of amino acids along its sequence (C) when compared with that of FUS. However, similarly to FUS, TDP-43-PLD shows a completely different list of the most repeated interactions compared with the full protein (A), which is likely contributing to reducing its critical temperature (B) and surface tension (A).Although in FUS condensates, the PLD plays a vital role in LLPS ,103, theur model ) are K-Fur model A, clearsequence C when c protein A, whichperature B and su tension A.A), but because glycine is the most abundant amino acid (∼25%), followed by serine (∼15%), the normalized contacts by amino acid abundance show that R-Y, R-F, and K-Y are dominant interactions, again highlighting the importance of cation-π interactions in hnRNPA1 LLPS. The list of top interactions of hnRNPA1-PLD, even after normalization, is very similar to that of hnRNPA1 (A and B), which explains why the phase diagrams of both sequences are hardly distinguishable (A). Surprisingly, the list of the most frequent interactions of hnRNPA1-A-LCD is also remarkably similar to that of the hnRNPA1 full sequence (A). In fact, the detailed contact map of hnRNPA1-A-LCD corresponds to the region of hnRNPA1 that contains more LLPS-stabilizing interactions (dashed lines in A).In hnRNPA1, the most frequent contacts are G-G, G-S, and G-R A, but b hnRNPA1 , A and Buishable A. Surprsequence A. In faP(Rg). Our simulations reveal that in all cases, when proteins transition from the diluted to the condensed phase, their conformations adopt larger radii of gyration (D). Also, the width of P(Rg) considerably increases, indicating the more versatile conformations that proteins can exhibit within the condensate. This structural behavior allows proteins to maximize their number of intermolecular contacts and thus the droplet connectivity, as recently shown in (T (E), in contrast to those measured in the diluted phase as a function of T (P(Rg) distributions, we find almost identical protein ensembles, exhibiting the wild-type variant slightly more open conformations. Such a small surplus of extended conformations shown by wt-TDP-43, which can enable a higher number of intermolecular contacts . Moreovcontacts , is not h-TDP-43 .RNA has been recently shown to critically regulate both the phase behavior of different RNA-binding proteins ,57,145 aFirst, we mix poly-U RNA strands of 250 nucleotides (nt) with the proteins studied above. Remarkably, not all proteins were able to favorably interact with poly-U in our simulations. We find that FUS-PLD and TDP-43 (including both variants) do not associate with poly-U even at very low RNA concentration . We further test the affinity of wt-TDP-43 with poly-U strands by performing a separate analysis of each of its major protein sequence domains . None of these domains exhibited a conclusive interaction with poly-U at temperatures moderately below the critical one. That is not entirely surprising because 1) several experimental studies have shown that TDP-43-RRM1 only presents a strong affinity for RNA strands enriched in UG nucleotides , 148 andA and B for FUS (note that poly-U/hnRNPA1-PLD condensates show a very mild LLPS enhancement at low poly-U concentration (E). This reentrant behavior has been experimentally observed for synthetic peptides such as RP3 and SR8 in poly-U mixtures (C). At the pure FUS critical temperature, we observe a closed-loop diagram (green curve), and for slightly lower temperatures, reentrant phase behavior is also recovered in agreement with experimental findings (red) exhibits the highest variation in critical temperature at either low or high RNA concentration (D). Interestingly, hnRNPA1 (blue) shows an intermediate behavior between that of its A-LCD (cyan) and RRM (purple) domains. The maximal critical temperature in hnRNPA1-RRM is reached at the lowest RNA concentration of the set, and it sharply decays after the maximum. Contrarily, hnRNPA1-A-LCD suffers only a moderate increment of the critical temperature, but its reentrant behavior is smoother and appears at much greater concentration (two times higher) than that of hnRNPA1-RRM. Overall, hnRNPA1 condensates present higher RNA-induced stabilization in the low RNA regime than that of their PLD and RRMs separately. Moreover, it is worth mentioning that in all sequences, the larger enhancement of LLPS is reached at a poly-U concentration close to the electroneutrality point (depicted by crosses in D), which emphasizes the major importance of electrostatic nucleotide-amino acid interactions in RNA-RBPs phase separation  that favorably interact with poly-U; these are FUS, hnRNPA1, hnRNPA1-PLD, hnRNPA1-A-LCD, and hnRNPA1-RRMs. In all these systems except for hnRNPA1-PLD, the resulting phase behavior is similar to that shown in ntration , so herentration E. This mixtures  and for mixtures , 56, 57,mixtures , G3BP1 . We strikingly find that despite varying the stability and density of the droplets with RNA concentration , the structural conformation of the proteins and RNA does not significantly change . Regarding poly-U/hnRNPA1 droplets, our simulations reveal that G-G contacts remain as the dominant amino acid pair interaction  and R-U and G-U become the next two most frequent contacts (further details in purple curve in E), even at the lowest RNA/protein ratio at which the droplet stability attains its maximal value, R-U and K-U emerge as some of the most frequent contacts despite the very modest poly-U concentration  of the condensates with the dilute phase in presence of poly-U as a function of temperature (F). Both for FUS (F) and hnRNPA1-A-LCD  within the bulk condensate to maximize the molecular connectivity with poly-U, rather than remaining more exposed to the interface as in the pure component, and therefore, contributing to minimize the droplet surface tension because of their higher hydrophilicity. On the contrary, at moderately high poly-U ratios, the surface tension seems to decrease, although the scattering of our data does not allow us to conclude whether a non-monotonic behavior in γ may also exist . Our simulations reveal that very short poly-U strands (∼10 nt) do not enhance phase separation in FUS and hnRNPA1-A-LCD droplets (A and B). In fact, 10 nt poly-U strands in hnRNPA1-A-LCD droplets inhibit LLPS even at low concentration. On the other hand, we observe that RNA strands longer than ∼100 uridines  promote a similar droplet stabilization independently of their length (B). To further investigate the molecular insights behind these observations, we analyze the FUS-RNA conformational ensemble within phase-separated droplets with distinct RNA lengths by computing their radius of gyration histograms. As can be seen in C, RNA strands with radii of gyration comparable or longer than those of the proteins  promote maximal condensate stabilization, whereas RNA poly-U strands with shorter Rg than those of FUS proteins  cannot achieve the same degree of droplet stabilization (and density) for the same RNA/FUS concentration. We note that the observed minimal critical RNA length in FUS and hnRNPA1-A-LCD droplets may be also modulated by some protein- or RNA-specific features and modifications such as RNA sequence, secondary structure interactions, protein charge distribution, post-translational modifications, and RRM patterning effects (B), we find that RNA strands longer than the minimal critical length promote a higher number of protein intermolecular interactions, whereas short RNA chains  considerably hinder the liquid-network connectivity of the proteins within the droplets  is usually obtained by bead tracking within droplets using microrheology techniques (G(t) of the system (G(t) provides useful information about the underlying relaxation mechanisms of the proteins (see A for FUS condensates with and without poly-U), either at short times (white region) at which the relaxation modes mostly depend on short-range and intramolecular interactions  or at long timescales (beige region) at which G(t) is dominated by intermolecular forces, long-range conformational relaxation, and protein diffusion within crowded liquid-like environments. Moreover, in A, the fact that G(t) presents a faster decay when condensates contain RNA (purple circles) suggests that their viscosity will be lower than those of pure FUS droplets (red circles).In vitro, viscosity  and at the corresponding bulk droplet equilibrium density corresponding to each poly-U concentration at that temperature. First, we introduce poly-U strands of 125 nucleotides. As shown in B, the phase diagram for a given concentration is not expected to change either by using strands of 125 or 250 nucleotides. For both FUS and hnRNPA1-A-LCD droplets, we observe a mild nonmonotonic behavior with a maximum in viscosity at low poly-U ratios (solid circles in B), which might be directly related to the maximum in droplet stability shown in D or due to a coincidental scattering of our measurements. Nevertheless, at moderate poly-U mass ratios , the viscosity of the condensates (using 125 nt RNA strands) is about 30% lower than that without poly-U. On the other hand, a monotonic decreasing trend in viscosity was detected for hnRNPA1 condensates, for which almost a ∼50% drop in η is found at high poly-U mass fractions (0.24 mg poly-U/mg hnRNPA1). Even though the observed maximum in viscosity could be easily related to the reentrant behavior depicted in E, further work needs to be devoted to clarifying whether this is a real feature of the model and ultimately of these RBP-RNA condensates. Furthermore, we investigate how poly-U strands of 250 nucleotides can regulate droplet viscosity at the same concentrations. Although poly-U 125 nt strands significantly reduce viscosity at high mass ratios, poly-U 250 nt strands barely varies the condensate viscosity at the same concentrations except for FUS, for which a moderate viscosity increase was detected (open symbols in B). These observations are in full agreement with those reported for LAF-1 condensates in presence of short (red cross symbol), the viscosity of the phase-separated droplets is almost two times lower than that of condensates containing 250 nt at the same poly-U/FUS ratio (∼0.06 mg poly-U/mg FUS). In the η for the different RBP condensates as a function of poly-U concentration and length, as well as details on the statistical analysis for estimating the uncertainty of these calculations. We note that our G(t)-values for FUS do not quantitatively match with those from experiments of  within the condensates for all previous poly-U concentrations and strand lengths of 125 and 250 nt. In all cases, we find a coherent correlation between viscosity and protein mobility, the latter being considerably higher at moderate poly-U/protein ratios than for pure protein condensates (C). Strikingly, protein diffusion hardly depends on poly-U strand length (open symbols) as viscosity does (B). Only when extremely short RNA chains of 10 nt are added, as those tested in FUS condensates , protein diffusion noticeably increases. Although the shear stress relaxation modulus of the condensates crucially depends on the RNA strand length (longer RNAs imply longer G(t) relaxation decay), the protein diffusion coefficient does not. The latter mainly depends on droplet density (and temperature), and, as shown in A, condensate densities remain similar when using strands of either 125 or 250 nucleotides. However, when 10 nt strands are added at the same poly-U concentration, the droplet density critically decreases from ∼0.28 g/cm3 (for 125 and 250 nt chains) to ∼0.20 g/cm3. Therefore, our simulations suggest that the condensate dynamics dependence on RNA concentration is intimately related to the droplet density decrease as a function of poly-U concentration and length shown in A. To better comprehend the underlying mechanism behind such behavior, we also measure D for poly-U strands of different lengths  within the condensates. In D, we observe the severe impact of RNA chain length on its own mobility, as expected. Whereas FUS D  barely depends on the poly-U length (at least between 125 and 250 nucleotides), a twofold decrease in the RNA diffusion coefficient when adding 250 nt chains instead of 125 nt is behind the augment of droplet viscosity at high RNA concentration shown in B. Moreover, when adding 10 nt chains, FUS (and RNA) diffusion considerably increases with respect to those with 125 or 250 nt (at the same RNA concentration) because of the droplet density reduction (D). Interestingly, we also note that FUS, despite having the lower critical temperature to phase separate and thus weaker LLPS-stabilizing interactions than wt-TDP-43 and hnRNPA1 (B), displays the lowest protein diffusion of the set in the absence of poly-U. Such an intriguing fact, which might be related to patterning sequence effects  has beeOur simulations with poly-U RNA also reveal how the formation of protein condensates is clearly enhanced at low poly-U concentration , whereasD). However, the addition of moderately short RNA strands  could help in promoting condensate dynamics without significantly destabilizing phase separation (B). Our results suggest that the enhanced droplet dynamics at high RNA concentrations is mediated by a density reduction upon poly-U addition due to electrostatic repulsion. Taken together, our observations shed light on the crucial role of RNA (concentration and length) on the formation and phase behavior of RNA-protein complexes (Finally, we focus on the transport properties of the RBP condensates as a function of RNA concentration and length. Our simulations demonstrate that although viscosity severely depends on the length of the added RNA chains—i.e., poly-U strands of 10 and 125 nt reduce droplet viscosity , whereasparation B. Our romplexes ,58,125. J.R.E. and J.R. designed the research. A.R.T. and A.G. built the protein models, A.R.T. performed the simulations. A.R.T., J.R.E., A.G., and J.R. analyzed the data. A.R.T. and J.R.E. wrote the initial version of the manuscript. All authors contributed and edited the manuscript. J.R.E. and J.R. supervised the research.


RNA:amyloid protein interactions have been observed in the past few years. Nevertheless, the molecular basis and physiological implications of these interactions are still poorly understood. Here we focus on a bacterial amyloid protein, Hfq. This protein is a pleiotropic bacterial regulator that mediates many aspects of RNA metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small noncoding regulatory RNAs. This regulation contributes to bacterial adaptation to stresses. Our results show that the amyloid region of Hfq significantly influences the efficiency of annealing between DsrA small noncoding RNA to its target mRNA. This unexpected result opens perspectives for a novel physiological role of amyloids, including those associated with neurodegenerative diseases.Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of  Any change in environmental conditions such as temperature, pH, nutrients starvation, salts, and oxidation inflict stresses on bacteria ,2. Many bacteria ,7,8,9. Hhia coli ,11. Apprhia coli . As a gll length ,14.E. coli, it is composed of an Sm N-terminal region  which forms a homohexameric ring-shaped structure, and an intrinsically disordered C-terminal region  [Hfq is a member of the Sm and Sm-like family of RNA-binding proteins ,16,17,18 66–102) ,20. The  66–102) ,22,23. T 66–102) . The dis 66–102) . Recent  66–102) ,26. Ther 66–102) ,28,29 an 66–102) ,31.Although the functional importance of the Sm domain is well established, the function of the presumably disordered CTR is poorly understood . Indeed,rpoS [hns [mreB [rbsD [hns, mreB, or rbsD mRNAs with different conformational states [rpoS mRNA, which encodes for the stress sigma factor σS, by acting as an anti-antisense RNA [rpoS mRNA forms a large stem-loop structure that shields the ribosome binding site  and therefore inhibits ribosome binding [rpoS, it causes this stem-loop to open, releases the rbs and activates the translation of rpoS mRNA. Opening of DsrA SL1 is required for efficient DsrA:rpoS annealing [rpoS annealing, Hfq recruits DsrA for base-pairing with rpoS mRNA [rpoS mRNA without Hfq at room temperature [Among Hfq-dependent sRNAs, DsrA is one of the first sRNA regulators found to regulate translation of multiple mRNAs, such as rpoS , hns [55poS [hns , mreB [5ns [mreB , and rbseB [rbsD . DsrA fol states . The SL1ense RNA ,60. The  binding ,61. Whennnealing . HoweverpoS mRNA . The prepoS mRNA . Meanwhiperature . These rrpoS mRNA. Following RNA annealing using ElectroMobility Shift Assay (EMSA) is usually challenging for polymerizing proteins, as in the case of amyloids [Here, we used a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to study the effect of the Hfq-CTR on DsrA SL1 annealing to amyloids . SRCD thamyloids . UnexpecAll chemicals were purchased from Sigma-Aldrich  or Thermofisher scientific .E. coli bacteria as it is unstable when translated independently of the NTR region. The sequence of Hfq-CTR is SRPVSHHSNNAGGGTSSNYHHGSSAQNTSAQQDSEETE [Full-length Hfq and NTR were purified as described previously . Hfq-CTRQQDSEETE . Before QQDSEETE . When thQQDSEETE . We checrpoS fragments described in Hwang et al. [core WT and mutant) and two fragments of rpoS (rpoSrbs and rpoSreg) were used  5′-AACACAUCAGAUUUCCUGGUGUAACGAAUUUUUUAAG-3′, DsrAcoremut  5′-AACACAUCAGGGAACUGGUGUAACGAAUUUUUUAAG-3′. Sequences of rpoS fragments were rpoSreg: 5′-AUUUUGAAAUUCGUUACAAGGGGAAAUCCGUAAACCC-3′; rpoSrbs: 5′-CAAGGGAUCACGGGUAGGAGCCACCUUAUGAGUCAGAAU-3′.Sequences of DsrA fragments were: DsrAcore:rpoSreg, rpoSrbs:rpoSreg…) were formed using the same protocol after stoichiometric addition of the two oligonucleotides in the same tube.Before use, the oligonucleotides (Eurogentec) were heated at 90 °C for 3 min and then slowly cooled down at 20 °C to allow proper folding. Duplexes of oligonucleotides -camphor-10-sulfonic acid (CSA) was used to calibrate amplitudes and wavelength positions of the experiment. Data analyses  were carried out with CDtoolX [SRCD measurements were carried out on the DISCO beamline at SOLEIL Synchrotron as described previously   concentration were 0.2 or 0.4 mM, respectively (full-length Hfq concentration was lower due to its lower solubility). RNAs were in excess relative to proteins. The effect of Hfq or CTR on RNAs was analyzed at 15 °C. For kinetic measurements, an apparent catalytic kinetic constant kcatapp was determined during initial rate conditions. This constant was expressed in mdeg·M−1·min−1 and depends on protein concentration .Two types of experiments have been performed: the effect of Hfq or CTR were tested either on individual RNA, or on duplexes such as DsrAm). Note that a shift of the peak may occur in some cases and that the maximum is not always precisely at 180 nm [m − x)/slope)).For melting curves, triplet SRCD spectra were acquired every 3 °C between 15 °C and 81 °C. Averaged SRCD values of the maximum of the peak around 180 nm are presented as a function of temperature to measure the melting point (Tt 180 nm  (Supplemcore and Cy5-rpoSreg) or unlabeled RNA . After incubation with CTR for one hour, samples were loaded on a polyacrylamide gel (10% or 4–20%) and subjected to electrophoresis in Tris-Acetate-EDTA (TAE) buffer. The gels were scanned using a fluorescence imager.The Electromobility Shift Assays were performed with labelled RNAs   are used permitting very small loading volumes (3–4 µL). Therefore, high concentrations are chosen following Beer–Lambert law. This has the advantage of reducing overall precious sample consumption as well as extending the spectral-band down to 170 nm. With Hfq-NTR we could not reach a concentration of ~0.2 mM and increasing its concentration clearly promoted precipitation of the protein which is incompatible with SRCD measurements because it results in diffusion of the UV light. We tried to measure the annealing activity of Hfq-NTR using lower concentrations but, unfortunately, were not able to detect a significant signal change for SRCD, because the signal to noise ratios were dropping considerably below 10 in contrast to above 200 for high concentration and short pathlengths. We thus presume NTR activity is significantly lower than that of the full-length protein, even if this region is able to promote annealing in vivo [SRCD kinetics of ntaloop) . DsrAcorentaloop . Many otAcoremut . In compDsrAcore ; neverth in vivo . This recore:rpoSreg complex is more stable in the presence of both Hfq and CTR. This confirms that the full protein stabilizes the sRNA:mRNA complex, but this result was unexpected for the CTR.The effect of Hfq and Hfq-CTR on the stability of the complex formed was evaluated using meting curves. As seen in coremut is significantly more stable than DsrAcore in the absence or in the presence of CTR , we suspect this loop is important to form the complex. This is confirmed using EMSA where we show that Hfq-CTR binds DsrAcoremut but less than DsrAcore , which is extremely widespread, comprising one-third of the tetraloops in ribosomal RNA [m measurement  and more important stabilization of DsrA  and ribonucleic (RNA) acid conformational changes has been observed previously . In part


Antimicrobial stewardship programs are needed to improve antimicrobial use among not only physicians but also dentists. This study aimed to investigate the factors influencing the decision of prescribing penicillin as first choice among dentists at clinics in Japan. We conducted a nationwide cross-sectional study of dental clinics in Japan between July and September 2020. Data on the following were collected using questionnaires: basic information, types of antimicrobials stocked, first-choice antimicrobials, and knowledge and practice of antimicrobial resistance and infectious endocarditis. Using logistic regression, odds ratios (ORs) and 95% confidence intervals (CIs) were estimated to assess the factors influencing penicillin prescription.Among the 1700 participating dental clinics, 342 dental clinics responded. The median age of the study cohort was 57 (49–65) years, and there were 298 (87.1%) men. The first choice of antimicrobials was third-generation cephalosporin (169 [49.4%]), followed by penicillin (103 [30.1%]) and macrolide (19 [5.6%]). In multivariate analysis, clinics with stocked penicillin (OR = 27.30 [95% CI: 12.04–63.00]) and with more than two dentists (OR = 0.48 [95% CI: 0.24–0.92]) were associated with penicillin use as first choice.This is the first study investigating the factors influencing the decision of prescribing penicillin as first choice among dentists in Japan. Further studies evaluating the relationships between penicillin use as first choice and stocked penicillin in the clinic and the number of working dentists are needed.All Authors: No reported disclosures