Search Results (29)

Phase transition or phase separation occurs in cells and tissues in many physiological events, such as protein granules (P granules) in early C. elegans development. Inappropriate phase transition is often associated with pathologic processes such as RNA–protein complex (RNP) and FUS-mutation-associated diseases. Given its ubiquity, phase transition has been considered a new frontier for comprehending physiological processes and pathological diseases. However, molecular and cellular mechanisms of phase transition in situ remain poorly described. Combining histochemistry with polarization analysis and biochemical thin-layer chromatography, we identified a massive phase transition change during the development of Taihe fowl (Gallus gallus domesticus Brisson). During embryonic day 12 (E12), the livers of these Silkie chicken demonstrated a sudden massive transition from hepatic lipid droplets (HLD) into hepatic liquid crystal droplets (HLCDs). We identified these changes by characterizing the sudden appearance of birefringent Maltese crosses (MCs) typical to liquid crystals (LC) where non-birefringent lipid droplets used to reside within hepatic cells. LC status was confirmed by fluidity with shape-changing and in vitro thermal phase transition tests. These HLCDs were present consistently until the early postnatal days after hatching. Using thin-layer chromatography combined with X-ray diffraction analysis, we determined that these HLCDs were composed of cholesterol, cholesterol ester and lecithin, which are the same as the components of cytoplasmic membrane. There was no change in the quantity of lipid components during liver development to suggest a critical mass of components triggering these changes. However, expressions of membrane-associated autophagy markers LC3A and Beclin 1 increased dramatically during this HLD to HLCD transition. Increases in membrane-associated LC3A and Beclin 1 are localized with massive increases in membrane lipid components of HLCDs. Areas with enhanced LC3A and Beclin 1 signaling have been associated with liquid crystal MCs to the thickness of 69 Å (Bragg d value). These associations indicate the possible regulatory role autophagy plays during liquid crystal phase transition in embryonic liver development. Reactivation of this autophagy pathway may be a possible mechanism behind the development of non-alcoholic fatty liver disease in adulthood. Full article

Mutations in an RNA-binding protein FUS are known to cause familial amyotrophic lateral sclerosis (ALS). Since this discovery, mutations in several other RNA-binding proteins (RBPs) have also been linked to ALS. Some of these ALS-associated RBPs have been shown to colocalize with ribonucleoprotein (RNP) granules such as stress granules and processing bodies (p-bodies). Increasing evidence has emerged supporting a hypothesis that the impaired clearance, inappropriate assembly, and dysregulation of RNP granules play a role in ALS. Through the genome-scale overexpression screening of a yeast model of FUS toxicity, we found that TAF15, a human RBP with a similar protein domain structure and belonging to the same FET protein family as FUS, suppresses FUS toxicity in yeast. The suppression by TAF15 is specific to FUS and not found in other yeast models of neurodegenerative disease-associated proteins. We showed that the RNA recognition motif (RRM) of TAF15 is required for its suppression of FUS toxicity. Furthermore, FUS and TAF15 physically interact, and the C-terminus of TAF15 is required for both the physical protein–protein interaction and its protection against FUS toxicity. Finally, while FUS induces and colocalizes with both stress granules and p-bodies, TAF15 only induces and colocalizes with p-bodies. Importantly, the co-expression of FUS and TAF15 induces more p-bodies than individually expressing each gene alone, and FUS toxicity is exacerbated in yeast that is deficient in p-body formation. Overall, our findings suggest a role of increased p-body formation in the suppression of FUS toxicity by TAF15. Full article

The central role of YB-1 in messenger ribonucleoprotein particle (mRNP) metabolism and stress-granule biology highlights the importance of defining the determinants of its self-assembly. YB-1 fibrillogenesis has been attributed primarily to the cold shock domain (CSD). Here, we show that the YB-1 fragment spanning residues 1–129 (AP–CSD) form amyloid fibrils under near-physiological ionic strength (0.12–0.15 M KCl). Fibrillization proceeds without a pronounced exponential growth phase and increases approximately linearly over 45–50 h. Far-UV circular dichroism (CD) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) indicate no substantial change in overall secondary-structure content during aggregation. In parallel, ¹H nuclear magnetic resonance (NMR) spectroscopy reveals the depletion of soluble species, and oriented fiber X-ray diffraction displays the hallmark cross-β reflections at approximately 4.7 Å and 10 Å. The prolonged formation time implies an activation barrier that is unlikely to require global refolding. Instead, it may reflect early association events such as dimerization or other local rearrangements required for primary nucleation, followed by consolidation into stable intermolecular contacts. Aggregation that preserves a largely native-like fold while establishing cross-β order may reduce recognition by cellular quality-control systems that preferentially target globally unfolded or strongly destabilized states. This provides a plausible framework for how YB-1 derived assemblies could persist under stress and during age-associated proteostasis decline. Full article

The localization and remodeling of mRNPs is inextricably linked to translational control. In recent years there has been great progress in the field of mRNA translational control due to the characterization of the proteins and small RNAs that compose mRNPs. But our initial assumptions about the physical nature and participation of germ cell granules/condensates in mRNA regulation may have been misguided. These “granules” were found to be non-membrane-bound liquid–liquid phase-separated (LLPS) condensates that form around proteins with intrinsically disordered regions (IDRs) and RNA. Their macrostructures are dynamic as germ cells differentiate into gametes and subsequently join to form embryos. In addition, they segregate translation-repressing RNA-binding proteins (RBPs), selected eIF4 initiation factors, Vasa/GLH-1 and other helicases, several Argonautes and their associated small RNAs, and frequently components of P bodies and stress granules (SGs). Condensate movement, separation, fusion, and dissolution were long conjectured to mediate the translational control of mRNAs residing in contained mRNPs. New high-resolution microscopy and tagging techniques identified order in their organization, showing the segregation of similar mRNAs and the stratification of proteins into distinct mRNPs. Functional transitions from repression to activation seem to corelate with the overt granule dynamics. Yet increasing evidence suggests that the resident mRNPs, and not the macroscopic condensates, exert the bulk of the regulation. Full article

Circadian rhythms are endogenous ~24 h oscillations that regulate diverse biochemical processes. Although stress responses can exhibit circadian modulation, evidence for rhythmic regulation of stress granules (SGs)—cytoplasmic RNA–protein condensates formed under stress—remains limited. We investigated sodium arsenite-induced SG dynamics in NIH/3T3 cultures. SG number, eIF3 signal intensity—an established SG marker—and area oscillated with a period of ~24 h. These rhythms persisted in Bmal1^−/− mouse embryonic fibroblasts (MEFs), despite lacking a transcription–translation feedback loop (TTFL) that constitutes the canonical circadian clock, but with altered amplitude and phase, indicating partial dependence on the molecular clock. Several SG-associated RNA-binding proteins (TIA-1, BRF1, hnRNP Q, and LARK) exhibited time-dependent changes at the mRNA and/or protein level, suggesting potential mechanisms for rhythmic SG modulation. Unlike previous in vivo reports linking SG variation to eIF2α phosphorylation, no temporal changes in phosphorylated eIF2α were observed, highlighting differences between isolated cells and tissues. Our results show that SG rhythmicity can persist without BMAL1, supporting alternative oscillatory mechanisms that contribute to the temporal organization of stress responses. Given their role in cell survival and the association of SG dysfunction with disease, these rhythms provide insight into how cellular stress responses are temporally regulated. Full article

Stress granule formation is a type of liquid–liquid phase separation in the cytoplasm, leading to RNA–protein condensates that are associated with various cellular stress responses and implicated in numerous pathologies, including cancer, neurodegeneration, inflammation, and cellular senescence. One of the key components of mammalian stress granules is the DEAD-box RNA helicase DDX3, which unwinds RNA in an ATP-dependent manner. DDX3 is involved in multiple steps of RNA metabolism, facilitating gene transcription, splicing, and nuclear export and regulating cytoplasmic translation. In this study, we investigate the role of the RNA helicase DDX3’s enzymatic activity in shaping the RNA content of ribonucleoprotein (RNP) condensates formed during arsenite-induced stress by inhibiting DDX3 activity with RK-33, a small molecule previously shown to be effective in cancer clinical studies. Using the human osteosarcoma U2OS cell line, we purified the RNP granule fraction and performed RNA sequencing to assess changes in the RNA pool. Our results reveal that RK-33 treatment alters the composition of non-coding RNAs within the RNP granule fraction. We observed a DDX3-dependent increase in circular RNA (circRNA) content and alterations in the granule-associated intronic RNAs, suggesting a novel role for DDX3 in regulating the cytoplasmic redistribution of non-coding RNAs. Full article

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid–liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated. Full article

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4–8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood–testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, β-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, β-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies. Full article

Cellular biomolecular condensates, termed ribonucleoprotein (RNP) granules, are often enriched in messenger RNA (mRNA) molecules relative to the surrounding cytoplasm. Yet, the spatial localization and diffusion of mRNAs in close proximity to phase separated RNP granules are not well understood. In this study, we performed single-molecule fluorescence imaging experiments of mRNAs in live cells in the presence of two types of RNP granules, stress granules (SGs) and processing bodies (PBs), which are distinct in their molecular composition and function. We developed a photobleaching- and noise-corrected colocalization imaging algorithm that was employed to determine the accurate positions of individual mRNAs relative to the granule’s boundaries. We found that mRNAs are often localized at granule boundaries, an observation consistent with recently published data. We suggest that mRNA molecules become spontaneously confined at the RNP granule boundary similar to the adsorption of polymer molecules at liquid–liquid interfaces, which is observed in various technological and biological processes. We also suggest that this confinement could be due to a combination of intermolecular interactions associated with, first, the screening of a portion of the RNP granule interface by the polymer and, second, electrostatic interactions due to a strong electric field induced by a Donnan potential generated across the thin interface. Full article

Transposable elements (TEs) are DNA sequences that can transpose and replicate within the genome, leading to genetic changes that affect various aspects of host biology. Evolutionarily, hosts have also developed molecular mechanisms to suppress TEs at the transcriptional and post-transcriptional levels. Recent studies suggest that stress-induced formation of ribonucleoprotein (RNP) granules, including stress granule (SG) and processing body (P-body), can play a role in the sequestration of TEs to prevent transposition, suggesting an additional layer of the regulatory mechanism for TEs. RNP granules have been shown to contain factors involved in RNA regulation, including mRNA decay enzymes, RNA-binding proteins, and noncoding RNAs, which could potentially contribute to the regulation of TEs. Therefore, understanding the interplay between TEs and RNP granules is crucial for elucidating the mechanisms for maintaining genomic stability and controlling gene expression. In this review, we provide a brief overview of the current knowledge regarding the interplay between TEs and RNP granules, proposing RNP granules as a novel layer of the regulatory mechanism for TEs during stress. Full article

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of β-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal. Full article

Cells possess membraneless ribonucleoprotein (RNP) granules, including stress granules, processing bodies, Cajal bodies, or paraspeckles, that play physiological or pathological roles. RNP granules contain RNA and numerous RNA-binding proteins, transiently formed through the liquid–liquid phase separation. The assembly or disassembly of numerous RNP granules is strongly controlled to maintain their homeostasis and perform their cellular functions properly. Normal RNA granules are reversibly assembled, whereas abnormal RNP granules accumulate and associate with various neurodegenerative diseases. This review summarizes current studies on the physiological or pathological roles of post-translational modifications of various cellular RNP granules and discusses the therapeutic methods in curing diseases related to abnormal RNP granules by autophagy. Full article

Membrane-less biomolecular compartmentalization is a core phenomenon involved in many physiological activities that occur ubiquitously in cells. Condensates, such as promyelocytic leukemia (PML) bodies, stress granules, and P-bodies (PBs), have been investigated to understand the process of membrane-less cellular compartmentalization. In budding yeast, PBs dispersed in the cytoplasm of exponentially growing cells rapidly accumulate in response to various stresses such as osmotic stress, glucose deficiency, and heat stress. In addition, cells start to accumulate PBs chronically in post-exponential phases. Specific protein–protein interactions are involved in accelerating PB accumulation in each circumstance, and discovering the regulatory mechanism for each is the key to understanding cellular condensation. Here, we demonstrate that Nst1 of budding yeast Saccharomyces cerevisiae is far more densely associated with PBs in post-exponentially growing phases from the diauxic shift to the stationary phase than during glucose deprivation of exponentially growing cells, while the PB marker Dcp2 exhibits a similar degree of condensation under these conditions. Similar to Edc3, ectopic Nst1 overexpression induces self-condensation and the condensation of other PB components, such as Dcp2 and Dhh1, which exhibit liquid-like properties. Altogether, these results suggest that Nst1 has the intrinsic potential for self-condensation and the condensation of other PB components, specifically in post-exponential phases. Full article

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3′-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs. Full article

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules. Full article