Xist) long noncoding RNA is indispensable for XCI and initiates the process early during development by spreading in cis across the X chromosome from which it is transcribed. During XCI, Xist RNA triggers gene silencing, recruits a plethora of chromatin modifying factors, and drives a major structural reorganization of the X chromosome. Here, we review our knowledge of the multitude of epigenetic events orchestrated by Xist RNA to allow female mammals to survive through embryonic development by establishing and maintaining proper dosage compensation. In particular, we focus on recent studies characterizing the interaction partners of Xist RNA, and we discuss how they have affected the field by addressing long-standing controversies or by giving rise to new research perspectives that are currently being explored. This review is dedicated to the memory of Denise Barlow, pioneer of genomic imprinting and functional long noncoding RNAs (lncRNAs), whose work has revolutionized the epigenetics field and continues to inspire generations of scientists.In mammals, dosage compensation of sex chromosomal genes between females (XX) and males (XY) is achieved through X-chromosome inactivation (XCI). The X-linked X-inactive-specific transcript ( Xist) RNA was one of the first long noncoding RNAs (lncRNAs) to be discovered in the early 1990s [H19, at the Igf2 imprinted cluster [Airn, antisense to the imprinted Igf2r gene. Airn was uncovered by Denise Barlow as the first example of lncRNA playing a direct role in controlling the imprinted expression of neighboring genes [Airn, it took nearly 30 years of research to characterize the function of Xist, and several important questions still need to be answered.X-inactive–specific transcript , the epigenetic process that equalizes the dosage of X-linked genes between female (XX) and male (XY) mammals. At the onset of XCI, Xist is up-regulated from one of the two X chromosomes, and its RNA spreads in cis along the entire X and triggers the inactivation of most of its >1,000 genes. Ultimately, Xist spreading results in the conversion of one of the two active X chromosomes into a uniquely organized heterochromatic entity known as the “Barr body” [Xist alleles would lead to silencing of both X chromosomes and presumably consequent cell death, the spatio-temporal expression of Xist during development needs to be accurately regulated. Historically, the X-linked minimal genetic region that is necessary and sufficient to initiate XCI in female cells has been defined as the X-inactivation centre (Xic)  . The Xicnal unit . Tsix trromosome –32. Othe of Xist –40. Thus reviews –44. HereXist was initially proven to be indispensable for XCI by targeted mutagenesis and transgenesis in mouse embryonic stem cells (ESCs) and in mice. Deletion of the Xist gene results in skewed inactivation of the wild type X chromosome, indicating that this locus is essential for gene silencing [Xist transgenes integrated on autosomes demonstrated that Xist RNA alone is sufficient to trigger gene silencing [Xist’s function. First, the ability of Xist RNA to trigger gene silencing is strictly dependent on the developmental context. If Xist is ectopically induced outside of a critical temporal window during early ESCs differentiation, it will be no longer capable of initiating gene silencing [Xist has different tasks, such as cis-localization to the chromosome from which it is expressed and the ability to trigger gene silencing, and these tasks are mediated by genetically independent domains of the RNA [Xist have now been defined by genetic and biochemical approaches and overlap at least partially with blocks of tandem repeats named A–F repeats, which are conserved between human and mouse and were therefore suspected to play a role in the function of Xist [Xist-inducible transgenes, the highly conserved repeat A element, consisting of 7.5 copies of a 26 nt core sequence at the 5’ end of Xist RNA, was characterized as the necessary element to trigger gene silencing [Xist gene seems to affect Xist RNA’s capacity to be expressed from its endogenous promoter [Xist and for mediating its silencing function. On the other hand, Xist RNA localization to the X chromosome appeared to be mediated by a combination of different domains, including repeats C, E, and F, via redundant mechanisms [Xist might recruit to fulfil its multiple and genetically uncoupled functions. In this context, the transcription factor YY1 was reported to mediate Xist localization by bridging the repeat C region of the RNA with a “nucleation centre” at the repeat F of the DNA [Xist RNA by binding to regions previously reported to mediate Xist localization, including repeat C and a region downstream of repeat E within exon 7 [Xist locus known as Rep A and overlapping with Xist repeat A, was previously reported to recruit PRC2 to the Xi by directly binding its catalytic subunit: EZH2 [ilencing ,46. Subsilencing –49, althilencing . The earilencing . Second, the RNA . The var of Xist ,5,51,52 promoter , suggestchanisms –61. Finachanisms ,62–64. S the DNA . The hetn exon 7 ,61. Finait: EZH2 . EZH2 acit: EZH2 –71. Simiit: EZH2 .Xist was achieved when three independent proteomic approaches purified Xist RNA and its binding proteins in mouse ESCs and fully differentiated cells [Xist RNA, only hnRNP U was confirmed by all proteomic studies. On the contrary, neither EZH2 nor any other PRC2 components were identified, but PRC1 proteins were. YY1 was also not pulled down by Xist RNA, whereas ATRX was only identified as a weak interactor of Xist in one of the three studies [A major breakthrough in the characterization of the interactome of ed cells –75. AmonXist to trigger gene silencing [Xist and to identify its key protein partners. The research to understand their mechanisms of action in XCI is currently ongoing and will greatly enhance our general understanding of facultative heterochromatin formation, nuclear localization, and chromosome 3D spatial organization beyond the XCI field.In parallel, the functional relevance of several of the newly identified interactors including SPEN, RBM15, and WTAP was confirmed by two genetic screens aiming to define the key factors accounting for the ability of ilencing ,77. AltoXist RNA that was identified by all proteomic and genetic approaches is SPEN , a 400-kDa protein harbouring four N-terminal RNA recognition motifs (RRMs) and a highly conserved C-terminal Spen paralog and ortholog domain (SPOC). The functional role of SPEN in XCI was initially confirmed by knockdown and knockout experiments in which loss of SPEN resulted in defective silencing of the few X-linked genes that were tested [Xist allele leads to impaired gene silencing, resembling what is observed after SPEN knockdown, a model was put forward according to which SPEN might initiate transcriptional silencing by actively recruiting HDAC3 via SMRT binding [Xist RNA coating, and HDAC3 activity was specifically shown to promote silencing of most X-linked genes [Xist RNA, indicating that Xist RNA might initiate gene silencing through its interaction with SPEN by activating the prebound HDAC3 rather than by recruiting it de novo along the future Xi [The only binding factor of e tested ,74,76,77e tested . In othee tested –82. As ted genes . Neverthuture Xi .Xist RNA via the A repeat [Xist to localize to the Xi in cis [Xist RNA lacking the A repeat is still able to recruit both PRC1 and PRC2 and to create a repressive compartment from which most hallmarks of active transcription, including RNA Pol II and general transcription factors, are excluded [Xist RNA lacking the A repeat [Xist. Interestingly, none of the core PRC2 components were pulled down by the full length Xist RNA [Xist have been precisely addressed by following the X-chromosome–wide enrichment of these histone marks during XCI [Xist RNA lacking the A repeat is capable of recruiting PRC1 and PRC2 activity to regions of the Xi without active transcription, whereas spreading of both H3K27me3 and H2AK119Ub into domains of active genes is impaired [Xist-mediated silencing, the exact molecular mechanism by which it contributes to the establishment and/or maintenance of gene silencing remains elusive. For example, whether SPEN’s role in XCI is restricted to the SPOC domain or whether other domains may recruit additional factors to the Xi is not yet clear nor is the impact of SPEN on the function of Xist in vivo. Indeed, SPEN knockout mice do not survive through embryonic development but reach E12.5, a time point that is never reached by female mice carrying Xist deletions, which fail to undergo XCI [A key indication of SPEN’s function in XCI was the demonstration that it binds to A repeat , originaA repeat . Indeed,i in cis . Howeveri in cis , and losi in cis , suggesti in cis ,77. Howeexcluded –86. AccoA repeat , suggestXist RNA –75. The ring XCI . This stimpaired . Thus, aergo XCI ,87. HoweXist RNA interactor that was consistently identified is RNA-binding motif protein 15 (RBM15), a component of the N6-adenosine (m6A) RNA methylation machinery [6A methyltransferase complex, which was found to interact with Xist in an A-repeat–dependent manner [6A is the most abundant mRNA modification, and its impact on post-transcriptional gene regulation is being increasingly recognized [6A-readers”, such as the YT521-B homology (YTH) domain-containing proteins, which specifically binds to m6A and trigger downstream alterations affecting several aspects of mRNA metabolism, including alternative pre-mRNA splicing, 3’-end processing, nuclear export, translation, and decay [Xist RNA interactors pointed to a potential role of the m6A RNA methylation pathways in mediating the function of Xist. Indeed, human XIST RNA is methylated at 78 m6A residues, some of which encompass the repeat A element, and these modifications appeared to be required for proper gene silencing, although only a few loci along the Xi were tested [XIST RNA methylation was reported to rely not only on the activity of RBM15 but also on its paralog RBM15B [Xist interactor [6A) RNA methylation machinery METTL3 to Xist RNA in a WTAP-dependent manner, and their functional redundancy is likely to explain previously contradictory results obtained upon RBM15 depletion [Xist-mediated silencing of the Gpc4 gene as assessed by single molecule RNA FISH [6A sites in the 5’ region of Xist RNA results in a limited silencing defect, similarly to RBM15 and WTAP knockouts, indicating that the m6A pathway might not play a central role in Xist-mediated silencing, and highlighting the need to further assess the exact impact of m6A methylation on XCI [6A residues along Xist by the YTHDC1 reader. Depletion of YTHDC1 was shown to result in defective XCI, whereas tethering YTHDC1 to Xist rescues the phenotype in the absence of a functional m6A methylation complex [6A methylation recruited via Xist RNA to the X chromosome might affect the stability of X-linked mRNAs. In fact, although the exact impact of m6A on gene regulation is not yet fully understood, specific functions have been reported for at least some readers, resulting in opposite effects on gene expression. For example, binding of YTHDF1 improves the efficiency of translation of m6A-methylated mRNAs [Another achinery –75. Inteachinery , anothert manner . m6A is cognized –91. Oncend decay . The idee tested . Furtherg RBM15B , also idepletion ,78,92. IRNA FISH , and chred mRNAs , whereased mRNAs . This diXist by two proteomic approaches, although not in an A-repeat dependent manner [Xist-mediated silencing [Xist RNA independently of the A-repeat domain, and more recent studies have shown this interaction to be dependent on Xist RNA repeats B and C [Xist-mediated silencing by knockdown experiments and were able to show that its depletion significantly reduces the accumulation of H3K27me3 and H2AK119ub along Xi [Xist repeat B and a small part of repeat C  was identified as the required element to mediate hnRNP K/Xist interaction, and deletions of XR-PID abolished Polycomb recruitment and resulted in reduced gene silencing [Xist transgene itself, as this study employed a short form of Xist RNA encompassing repeats A, F, B, and part of C, but lacking the elements downstream the first 3.9 kb of Xist exon 1. Indeed, the lack of these sequences might somehow enhance the role of Polycomb recruitment in silencing establishment. More recently, three independent studies further explored the contribution of Polycomb recruitment to the establishment and maintenance of XCI by generating a series of Xist mutants in which repeats B and C have been deleted from the Xist endogenous locus [Xist RNA that lacks repeats B and C and is unable to recruit PRC1/PRC2, although slightly less efficiently, Polycomb recruitment and transcriptional silencing appear to be largely uncoupled [Xist RNA spreading [Xist-dependent recruitment of PRC2 to the Xi [Xist-dependent deposition of H2AK119ub and H3K27me3 during XCI [Xist interactors [The heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as an interactor of t manner –75. Howeilencing ,77. Chu  B and C ,63. Funcalong Xi . Subsequus locus ,62,78. Tpreading . Regardlo the Xi . Accordiring XCI . The receractors , and theeractors .Xist RNA is the Lamin B receptor (LBR). LBR is a transmembrane protein anchored to the inner nuclear membrane where it binds Lamin B and works as a structural scaffold for proteins involved in chromatin silencing at the nuclear lamina [Xist RNA and also showed that losing this interaction results in defective gene silencing [Xist-coated chromosome can still be recruited to the nuclear lamina in case of a major silencing defect, for example, upon SPEN depletion [Xist-mediated silencing upon differentiation of LBR knockout ESCs and were able to show that lack of LBR leads to a minor silencing defect [Xist RNA mediating the interaction with LBR resulted in slightly more affected gene silencing, but the overall effect on XCI remained weak. Thus, although the LBR–Xist interaction might be necessary to maintain and/or stabilize gene repression, the exact role of LBR during XCI still remains unclear.Another factor that has been proposed to associate with r lamina . McHugh r lamina . Subsequilencing . The samilencing . Howeverepletion , and theepletion , indicatepletion ,99. Moreg defect . DeletinXist concerns how it remains associated only with the chromosome in cis rather than diffusing to neighboring chromosomes in trans. Based on RNA fluorescent in situ hybridization analysis (FISH), the Xist RNA domain appears to be confined to the same nuclear territory of Xi throughout the entire cell cycle [XIST was reported to dissociate from the Xi during mitosis [Xist RNA might be temporarily displaced from the Xi through cell division, its localization does not seem to rely on a sequence-specific mechanism. For example, neither RNase H nor DNase treatment affects the Xist RNA domain within the nuclear space, indicating that Xist does not bind the Xi simply via formation of RNA/DNA hybrids [Xist RNA remains with the nuclear matrix fraction after removal of chromosomal DNA, its localization to the Xi chromatin might be directed by proteins of the nuclear matrix [One of the most fascinating and yet open questions about the function of ll cycle , althoug mitosis ,101, and mitosis . RegardlXist RNA localization to chromatin. hnRNP U is enriched along the Xi [Xist was confirmed in proteomic studies [XIST/Xist RNA [Xist localization by reporting diffusion of Xist RNA from the Xi territory upon hnRNP U depletion [XIST [Xist RNA seems to be at least partially cell type specific, indicating that Xist localization is likely to rely on a combination of anchoring factors rather than on hnRNP U alone [The matrix attachment protein hnRNP U has been proposed to play a key role in g the Xi ,104, and studies –75. In pXist RNA ,105. Sevepletion ,73,74. Non [XIST , and its U alone ,107. Fur U alone , but givXist interactor [Xist localization [Xist RNA during the earliest stages of XCI [Xist RNA via the C-terminal nuclear matrix–anchoring domain. Indeed, the C-terminal domain is required to recruit CIZ1 to Xi, and deleting the E repeat within exon 7 of Xist abolishes this recruitment [Xist RNA or rather anchors the Xi via binding to other Xist RNA interactors is not yet clear. It should be noted that in hnRNP U knockout cells, CIZ1 remains localized with Xist RNA, although Xist RNA is dispersed throughout the nucleoplasm. Thus, CIZ1 and hnRNP U interact with Xist independently of each other, highlighting the complexity of the mechanisms directing Xist RNA coating and the formation of the Xi territory [Xist localization in mouse embryonic fibroblast (MEFs) and activated B and T lymphocytes but is certainly dispensable for XCI initiation as knockout mice survive embryonic development [Xist localization in lymphocytes and impaired XCI might be the cause of the sex-specific phenotype. Consistent with this, deletion of Xist RNA in the mouse blood compartment was previously shown to lead to hematologic cancer [Xist RNA delocalization, X-linked gene dosage, and cancer development is yet to be clearly defined.Cip1-interacting zinc finger protein 1 (CIZ1) is another nuclear matrix protein identified as a teractor  and recelization ,58. CIZ1s of XCI ,58. Funcs of XCI  and a C-s of XCI . Thus, oerritory . Finallyc cancer . HoweverXist RNA is its ability to direct a major reorganization of the 3D spatial architecture of the Xi. Evidence of such structural reshaping was originally provided by assessing the local organization of X-linked genes during XCI [Xist RNA domain to a more internal compartment, which is depleted of RNA Pol II and general transcription factors, whereas genes that escape XCI remain external of the silent compartment [Xist RNA domain early upon XCI and only start to intermingle with it at a later stage, when silencing has occurred [Xist RNA coating results in partitioning of the Xi into megadomains via DXZ4 is not yet clear. Similarly, the exact causality and temporal dynamics between the collapse of TADs along the Xi and the establishment of gene silencing also remains to be defined. For example, Xist RNA was proposed to direct the 3D organization of Xi by repelling architectural factors, thus possibly avoiding the establishment of a transcription-favorable chromatin status (Minajigi 2015). However, how Xist RNA can repulse a subset of factors involved in chromatin 3D organization remains elusive, as do the sequence elements within Xist RNA that would account for this function.Another remarkable feature of ring XCI . In partpartment . Similaroccurred . More reoccurred ,112. Comoccurred ,113–116.Xist RNA [XIST depletion [Recently, the structural maintenance of chromosomes flexible hinge domain containing 1 (SmcHD1) protein was hypothesized to participate in reshaping the 3D conformation of Xi –119. SmcXist RNA  and was Xist RNA ,121. Mecepletion , and strepletion –119, altepletion ,119.Xist spreading [Xist locus and correspond to the regions that become first targeted by Xist RNA at the beginning of XCI [Another unexplored possibility is that the distribution of chromatin features along the X chromosome before XCI initiation might direct its structural reorganization. For example, the involvement of the Polycomb complexes in directing the 3D folding of chromosomes is increasingly recognized –125. In preading , indicatg of XCI ,126.Xist RNA. For example, FUS, hnRNPA2B1, and RBM14 all carry low-complexity domains that are prone to mediate phase separation and were previously reported to be implicated in the formation of paraspeckles [Xist RNA might direct the reorganization of Xi by forcing a local high concentration of specific proteins via RNA–protein interactions, thus creating a phase-separated silent compartment in which factors important for the establishment of facultative heterochromatin are sequestered. However, whether or not such a structure is assembled and its potential impact on the initiation and/or maintenance of XCI remains to be proven.Finally, Polycomb complexes might contribute to the 3D reorganization of the Xi by supporting the formation of membrane-less organelles similar to paraspeckles, defined as protein-rich nuclear condensates built around a specific lncRNA scaffold and able to influence gene regulation by sequestrating a subset of specific proteins by RNA-protein interactions (for review ). Indeedspeckles ,129–131.speckles ,133, an Xist RNA continues to provide a powerful model system for exploring a multitude of epigenetic mechanisms, including the developmentally regulated formation of facultative heterochromatin and the 3D organization of the genome in nuclear space. Future studies are likely to unveil the molecular mechanisms through which noncoding RNAs induce or change the 3D structure of the genome, as well as whether these changes in structure are a cause or a consequence of changes in gene activity and how they play a role in maintaining differences between active and inactive compartments of the mammalian genomes.Almost thirty years after its discovery,


Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as β-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood.The filamentous fungus bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular β-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei.Deletion of the bgl3i offers a new strategy to improve T. reesei strain performance.BGL3I may take part in the complex regulating system of cellulase induction. The deletion of The online version of this article (10.1186/s13068-018-1314-6) contains supplementary material, which is available to authorized users. Trichoderma reesei (teleomorph Hypocrea jecorina) is one of the most well-known industrial strains producing cellulase and hemicellulase, which releases fermentable soluble sugars from plant biomass. The high production levels of lignocellulose-degrading enzymes in T. reesei are dependent on induction by cellulose- and hemicellulose-containing plant polysaccharide mixtures. Disaccharide lactose  also serves as an inducer of cellulase formation in T. reesei and is presently the only soluble substrate that can be applied economically for this purpose  was used to collect the GST-tagged BGL3I. SDS-PAGE was used to verify the purified protein according to standard protocols  [The BGL3I coding sequence was expressed by the pGEX system according to the manufacturer’s guidelines. The first-strand cDNA was used as a template to amplify the fragment encompassing the ORF using the primers indicated in Additional file oduction . The rec 90 min) . The GSTt test was performed with WPS Office software , employing a two-tailed test. Significance was at P < 0.05.Student’s Additional file 1. Deletion of bgl3i gene from QmU2–3 derived strain. (A) Schematic map of replacing the bgl3i coding region with marker ura5 gene by the homologous integration, generating bgl3i deletion strain; (B) Identification of positive deletion strain by PCR: M, marker 1 kb ladder; +, positive control; -, negative control.Additional file 2. Protein concentration and enzyme activities of QmU2–3 derived strain. QmU2–3: parent strain; Δbgl3i: bgl3i deletion strain; rebgl3i: complementation strain. Samples were supernatants of each strain after 7-day incubation with 1% lactose.Additional file 3. Cellulase activity of T. reesei on other carbon source. (A) CMCase activity, (B) pNPCase activity, (C) pNPGase activity, (D) PFU activity, (E) extracellular protein concentration of WT and Δbgl3i mutant on 1% cellobiose, Avicel or glucose. Vertical bars indicate SD and each reaction was done in triplicate.Additional file 4. Transcriptional levels of the major cellulase genes response to Avicel. (A) cbh1, (B) egl1, (C) bgl1. Cellulase genes were detected 4 h, 16 h and 24 h after the beginning of the induction on 1% Avicel in WT and Δbgl3i mutant. Transcripts were normalized to the housekeeping gene actin. Vertical bars indicate SD and each reaction was done in triplicate.Additional file 5. Analysis of transcription levels of transcription factors. (A) transcription factor xyr1, (B) transcription factor cre1, (C) transcription factor ace1, (D) transcription factor ace2. They were detected 4 h, 16 h and 24 h after the beginning of the cultivation on 1% Avicel in strains WT and Δbgl3i mutant. Transcripts were normalized to the housekeeping gene actin. Vertical bars indicate SD and each reaction was done in triplicate.Additional file 6. Western blot analysis of BGL3I-his overexpressed in T. reesei QmU2–3 using promoter pdc, xyn2 and cbh1. Western blot analysis of BGL3I-his in the intracellular protein extract (A) and in the culture supernatant (B) of BGL3I-his overexpressed transformants. Lane 1: transformant of bgl3i overexpressed under control of promoter pdc; lane 2: transformant of bgl3i overexpressed under control of promoter xyn2; lane 2: transformant of bgl3i overexpressed under control of promoter cbh1; M: marker.Additional file 7. Western blot analysis of BGL3I-his overexpressed in T. reesei QmU2–3 using constitutive promoter tef1. Western blot analysis of BGL3I-his in the intracellular protein extract (A) and in the culture supernatant (B) of BGL3I-his overexpressed transformant. Lane 1: sample cultured for 5 days; lane 2: sample cultured for 7 days; M: marker.Additional file 8. Oligonucleotides used in this study.


Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model system comprised of an arginine-rich peptide and RNA, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a general mechanism for modulating the biochemical environment of RNA coacervates in a cellular context. In particular, liquid-liquid phase transitions can drive dynamic intracellular compartmentalization to form membrane-less organelles (MLOs) such as the nucleolus and stress granules5. Unlike their membrane-bound counterparts such as the nucleus, the unique physicochemical features of MLOs can enable liquid-like behavior such as facile formation, fusion, and dissolution2. These dynamic qualities provide a means for cells to sense and respond rapidly to changing environments, such as in the cytoplasm during stress2. In this context, stimulus-dependent liquid-liquid phase separation (LLPS) of biopolymers has recently emerged with numerous implications in biology7.Phase separation aids in regulating essential functions of cells and organisms. Within the cellular context, phase separation of biomolecules can selectively compartmentalize essential proteins, nucleic acids, and biochemical processes8. A quantitative understanding of this underlying interaction network and the elucidation of the molecular driving forces that alter them are therefore key topics of research in this context. The interchain associations of proteins and nucleic acids are largely determined by their primary sequences8. Intrinsically-disordered proteins (IDPs) with low-complexity repetitive sequences have been identified as drivers of MLO biogenesis, with charged sequences being one of the most common motifs found in intracellular MLOs9. Phase separation of charged low-complexity motifs is largely driven by electrostatic interactions11. This phenomenon, commonly known as complex coacervation, can be recapitulated in an in vitro model consisting of an arginine-rich peptide and RNA mixtures13. Our recent work demonstrated that a peptide-RNA mixture can display a reentrant phase behavior, where droplets can form and dissolve due to monotonic variation of ionic peptide-RNA ratios alone12. This type of behavior can be modeled simply using early polymer chemistry theories15, using the assumption that electrostatic interactions are the only interaction parameter. In addition to complex coacervation of RNA and IDPs, recent reports have described observations of liquid-liquid phase transitions via homotypic interactions of RNA chains in the absence of protein17.One of the driving forces of MLO formation, stability, and dynamics is the formation of weak multivalent interactions among proteins and nucleic acids18. Hence, IDP-RNA mixtures can be sensitive to small changes in solution conditions such as temperature, ionic environment, pH, and IDP-RNA stoichiometry8. These alterations in conditions can prompt dynamic responses in phase behavior by influencing the relative strengths and interplay of homotypic and heterotypic interactions. Along these lines, in this work we reasoned that the interplay of different interactions raises the possibility of an alternative stimulus-dependent LLPS modulation via tuning of the relative magnitudes of competing interactions by solvent components, in particular divalent cations.The global phase behavior of IDP-RNA systems is determined by a complex interplay between homotypic (IDP-IDP and RNA-RNA) and heterotypic (IDP-RNA) interactions24. Under rapidly changing cellular conditions, such as during stress, the flux of signaling cations coincides with MLO formation and dissolution dynamics24. Several recent studies exemplify the critical nature of divalent ions and their relationship to phase separation. One study shows that the phase separation of a stress granule-related IDP, TIA-1, can be regulated by Zn2+ 25. Another study revealed heat-induced phase separation of single stranded DNA in the presence of divalent cations26. Given the importance of divalent cations in MLO dynamics, we set out to determine how fluctuations in divalent ion concentrations could control the phase behavior of proteins and RNA. Our hypothesis is founded on the potential for divalent cations, such as Mg2+ and Ca2+, to have a destabilizing effect on heterotypic IDP-RNA interactions, while stabilizing RNA structure and interactions28. We anticipate that these opposing effects could give rise to a switch-like behavior with the capacity to regulate between complex coacervates (heterotypic) and RNA coacervates (homotypic) by modulating cation concentration in solution. Additionally, the same underlying effects on molecular interactions could also result in continuous tuning of droplet physical properties and composition.The cytoplasmic concentrations of divalent ions are among the most regulated conditions in the cellin vitro model IDP-RNA system, consisting of an arginine-rich peptide {RP3 (RRxxxRRxxxRRxxx)} and a long homopolymeric single-stranded RNA {polyU (800–1000 kD)}12. This peptide-RNA system was chosen because it has been previously used to understand the electrostatically-mediated phase behavior of intracellular ribonucleoprotein (RNP) granules13, which often contain IDPs featuring arginine-rich motifs9. Our previous studies showed that monovalent (Na+) cations reduce the propensity for phase separation in this system, which is consistent with the notion that heterotypic electrostatic interactions drive LLPS in such RNP systems12.We previously studied important biophysical aspects  of LLPS using an 2+) cations reduced LLPS, as demonstrated by a reduced turbidity  cations  and a molecular crowder  can lead to phase separation of polyU17. In our system, we tested if variation of divalent cation concentration was sufficient to induce polyU coacervation in the absence of PEG.We then tested the influence of divalent cations on homotypic phase separation of RNA. It is well known that the structure and assembly of both RNA and DNA are heavily influenced by the presence of divalent cations2+ and Ca2+ , Ca2+ (≥50 mM), and Sr2+ (≥75 mM) in a concentration dependent manner, with Ca2+ and Sr2+ showing a lower concentration threshold required for phase separation. One potential explanation for this difference between the phase separation thresholds of Mg2+ and Ca2+/Sr2+ is the variance in ion charge density, which is known to have a profound influence on RNA stability35. Ca2+ and Sr2+ are more effective at screening the negative charge of the RNA phosphate backbone and have weaker nonspecific interactions with RNA than Mg2+. This difference is also consistent with previous data that exhibited a lower temperature threshold for single-stranded DNA phase separation with Ca2+ than with Mg2+, however, phase separation of single-stranded DNA was not observed in the presence of Sr2+ in that study26. In our work, the droplets displayed liquid-like characteristics such as fusion, circular appearance, and recovery of fluorescence after photobleaching , consistent with previous data26, but it showed liquid-liquid phase separation at lower concentrations (75 mM–150 mM)  Figs  and S5. 2+ from 300 mM Mg2+ (0% PEG) to 75 mM Mg2+ (5% PEG) to 50 mM Mg2+ (10% PEG)  Fig. . This isPEG Fig. . This su2+ should be able to act as a stimulus to create a switch-like phase behavior in the mixed system. In other words, starting with RP3-polyU droplets, a titration of Mg2+ should first lead to dissolution of these heterotypic droplets and subsequent formation of homotypic RNA droplets. Sequential turbidity measurements of RP3-polyU mixtures over increasing [Mg2+] corroborated this prediction, displaying a window of miscibility between the two-phase regions of the phase diagram , we added 150 mM Mg2+ into the solution, which resulted in the dissolution of the droplets. Subsequent addition of Mg2+ to a final concentration of 500 mM produced new homotypic polyU droplets  becomes smaller. Therefore, the overall effective electrostatic stabilization decreases with increasing ionic strength of the solution. In contrast, the homotypic RNA self-assembly is stabilized at higher salt due to (a) a decrease in the repulsive potential of the negatively charged RNA phosphate backbone, and (b) the ability of Mg2+ ions to mediate 42. Numerical simulation of the free energy curvature, which determines the thermodynamic stability of a mixture with respect to phase separation, demonstrated that the system has two critical points, one at a low salt concentration and one at a higher salt concentration, with a homogeneous mixture being populated at intermediate salt concentrations   to ~80 kDa (large globular proteins), along with diversity in charge and structure  Fig. 44. Thesere Table .2+ we observed a continuous decrease in RP3 partitioning into homotypic polyU droplets, eventually leading to preferential exclusion at 1500 mM Mg2+ 20 (containing 20 arginines) with significantly higher positive charge than RP3 (6 arginines) showed favorable partitioning even under these conditions 20 at 1500 mM Mg2+  experiments, where the scaled FRAP time  substantially from 0 mM Mgg2+ Fig. . Additio2+ Table . The risery Fig. . Togethe2+ and in the presence of 10% PEG, the droplet fluidity decreases to a point where the FRAP recovery becomes extremely slow  and RP3C: ({RRASL}3C) were ordered and custom synthesized from GenScript  at ≥95% purity. (GR)20: (C{GR}20) was ordered and custom synthesized from PEPSCAN  at ≥95% purity. RP3 was dissolved in DEPC-treated water (Santa Cruz Biotechnology) and used without further purification. RP3C and (GR)20 were subsequently fluorescently labeled and purified (See Fluorescent Labeling).RP3: ({RRASL}54. E. coli (BL21(DE3)) cells were transformed with the eGFP-MBP plasmid. The transformed cells were grown at 37 °C to an OD600 of ~0.6. Protein expression was induced with IPTG to a final concentration of 0.3 mM and allowed to grow for another 4 hours at 37 °C. The cultures were harvested by centrifugation and lysed by sonication in lysis buffer  with a protease inhibitor cocktail. The cell debris was removed by centrifugation and the protein was collected from the supernatant by incubation in Ni-NTA resin. The supernatant was then run through a gravity column, and the protein eluted with an elution buffer containing 250 mM imidazole and 150 mM NaCl. Dialysis was used to remove imidazole from the buffer. Presence and purity of the protein was checked using SDS-PAGE, A280/A260 measurements, and mass spectrometry.α-synuclein and Hsp27 were expressed and purified in the same manner as our previously published workPolyuridylic acid potassium salt (polyU: 800–1000 kDa) was ordered from Sigma Aldrich . All labeled RNA oligos used as markers in this study  were ordered from and synthesized by Sigma Aldrich.The polyethylene glycol used throughout the paper was PEG 8000, which is referred to in the maintext simply as PEG.54. α-synuclein was labeled with AlexaFluor488 C5 maleimide (Molecular Probes) and Hsp27 was labeled using AlexaFluor594 C5 maleimide (AF594) (Molecular Probes). The labeling reactions were carried out at 4 °C overnight in the dark, and the excess dye was removed by centrifugal filtration with a 3 K cutoff filter (Millipore). The purity of the labeled proteins was tested via SDS-PAGE and mass spectrometry. All samples showed a labeling efficiency >90% by UV-Vis measurements.α-synuclein and Hsp27 were all mono-labeled using cys-maleimide chemistry as described in previous work20 were labeled in a similar manner with the lyophilized powder resuspended in buffer containing excess AlexaFluor594 C5 maleimide (AF594) (Molecular Probes). The reaction took place overnight at 4 °C in the dark. The excess dye was removed by four rounds of acetone precipitation. Four times the sample volume of cold (−20 °C) acetone was added to the reaction mixture. The reaction was vortexed and incubated at −20 °C for 60 minutes. It was then centrifuged for 10 minutes, and the supernatant disposed of. After four rounds, the acetone was allowed to evaporate and the resulting purified labeled peptide pellet was dissolved in DEPC-treated water (Santa Cruz Biotechnology).RP3C and (GR)350 readings were taken. All experimental results were plotted and presented using OriginLab software.All samples were prepared in a buffer background of 10 mM TRIS-HCl, pH 7.5. All the turbidity measurements were taken on a NanoDrop 2000c Spectrophotometer (ThermoFisher) at room temperature. For all sequential titration experiments the initial samples (100 μL) were prepared to the conditions given in the figure legends. Salt was then added to the solution to the following concentration, the solution was mixed and allowed to equilibrate for 30 seconds, and 3 μL were taken out to measure the absorbance at 350 nm. For individual points, 5–10 μL samples were prepared under diverse salt conditions, mixed, allowed to equilibrate for 30 seconds, and then A2 salt solution was injected into the sample during imaging to the given bulk concentration. The same was done for the alternating EDTA/Mg2+ experiment. All images and videos were analyzed using Fiji software. Partitioning analysis was done using Zen software. Using the labeled proteins/peptides/RNA as partitioning markers, we made samples under the given conditions with 0.5 μM of labeled samples. Fluorescence intensity was measured within the dense phase and background fluorescence was measured in the dilute phase. The coefficient was calculated as a ratio of droplet intensity over background intensity. At least 15 droplets were analyzed in each sample. In the instances of high partitioning, samples had to be analyzed under increasing laser power to be able to measure background fluorescence. The fluorescence intensity was controlled for saturation, and the partitioning was corrected for laser power. Laser power control experiments were run to ensure that intensity increase was linear with the changes in laser power within the tested range for AF594 and FAM. All dyes  were controlled to ensure that both dye concentrations in the dilute and dense phase were within a linear fluorescence range. Since experimental conditions could result in variations of dye fluorescence quantum yields, which would result in changes to the calculated numbers, we define these numbers as apparent partition coefficients.All DIC and fluorescence microscopy images used in this study were taken on a Zeiss LSM 780 laser scanning confocal microscope. Samples were prepared on Lab-Tek chambered #1.0 borosilicate coverglass. The coverglass was treated with 10% TWEEN 20 for 30 minutes, rinsed with water, and allowed to dry prior to use to avoid sample sticking to the coverslip. Samples were imaged at room temperature using a 63x oil immersion objective . All samples, with conditions shown in the figure legends, were prepared with 0.5–1 μM of fluorescently labeled markers. For the droplet switching imaging experiment, 2 μL of concentrated MgCl2) and one was used as a reference marker. Experimental droplets were bleached using 10 iterative pulses of 100% laser power. Both the fluorescence recovery of the bleached droplet, and the changing fluorescence of the reference droplet due to photobleaching were collected and analyzed. The reference droplet was used as a baseline to calculate a normalized intensity. The normalized curves were plotted and fitted on OriginLab Software using an exponential fit (For fluorescence recovery after photobleaching (FRAP) experiments, the samples were prepared in the same manner as described above with conditions given in the figure legends. The {[6FAM]UGAAGGAC} RNA oligo was used as a marker for all FRAP experiments. For all FRAP images, one droplet was bleached (with a ROI between 3–25 µmSupplementary InformationSupplementary Movie 1Supplementary Movie 2


Positive-sense single-stranded plant RNA viruses are obligate intracellular parasites that infect many agriculturally important crops. Most known plant RNA viruses are characterized by small genomes encoding a limited number of multifunctional viral proteins. Viral pathogens are considered to be absolutely dependent on their hosts, and viruses must recruit numerous host proteins and other factors for genomic RNA replication. Overall, the replication process depends on virus–plant protein-protein, RNA–protein and protein–lipid interactions. Recent publications provide strong evidence for the important role of chloroplasts in viral RNA synthesis. The chloroplast is considered to be a multifunctional organelle responsible for photosynthesis and for the generation of plant defense signaling molecules. High-throughput technologies (genomics and proteomics), and electron microscopy, including three-dimensional tomography, have revealed that several groups of plant RNA viruses utilize chloroplast membranes to assemble viral replication complexes (VRCs). Moreover, some chloroplast-related proteins reportedly interact with both viral proteins and their genomic RNAs and participate in trafficking these molecules to the chloroplast, where replication occurs. Here, we present the current knowledge on the important role of chloroplasts in the viral replication process. Positive-sense, single-stranded [(+)ssRNA] viruses are the main agents of many serious diseases of agriculturally important crops. Despite their relatively small genomes, these viruses that encode only a few proteins can efficiently replicate in their host cells . Virusesvia a neck-like opening and single- and/or double-membrane systems ; chloroplasts are also crucial for interorganellar signaling . Zhao etIn this review, we describe chloroplast–virus interactions on the basis of recent evidence highlighting the direct function of this organelle in virus RNA synthesis. We also list the most useful molecular approaches for studying viral RNA replication in this context.Alfamovirus genus, are also associated with the chloroplast envelope. However, further studies performed by Arabidopsis thaliana protoplasts showed that AMV replicates on tonoplast membranes.Although ss(+)RNA viruses display various genetic organization and expression strategies, they all replicate in a similar manner. All known plant ss(+)RNA viruses encode RNA-dependent RNA polymerase (RdRp) and ancillary replication factors. These viral proteins, which are produced in the early stage of infection, are involved in the assembly of VRCs and help to stabilize and facilitate optimal conditions for RNA synthesis. Viral and host proteins are selectively bound to the ss(+)RNA template and target subcellular membranes, which are later reorganized around the favorable microenvironment to form VRCs . Within The replication complex of the aforementioned virus families is associated with the outer chloroplast membrane, particularly with peripheral vesicles and cytoplasmic invaginations (CIs) on the surface. Among recently identified host factors participating in the viral replication process are several chloroplast and chloroplast-related proteins Table . Virus-iRecently published data indicate the role of the 140K/98K protein domains of TYMV, which possess two amphipathic helices (αA and αB) with a 41-residue internal sequence described as the chloroplast envelope-targeting domain (CTD) . Based ovia vesicles from ER-exit sites (ERESs) to the outer chloroplast membrane  attachment protein receptor) that are essential host factors for successful virus infection. For example, the cellular SNARE protein Syp71 directly mediates the fusion of vesicles with chloroplasts  showed that viral RNA and TGB3, which possesses a chloroplast-targeting signal, primarily accumulate near the surface of chloroplast membranes , with VRCs adjacent to the chloroplast membrane , and exonuclease XRN4, which all play a crucial role in the early stage of viral replication (Arabidopsis (via enzymatic activity by glutathione transferases (GSTs), and the 3′UTR-associated protein NbGSTU4 that in the presence of glutathione is able to neutralize oxidative stress inside chloroplasts, enabling efficient minus-strand RNA synthesis , followed by mass spectrometry (Tombusviridae family), potyviruses TuMV and TEV, and Bromoviridae, as exemplified by brome mosaic virus (in situ mapping are powerful tools that provide essential architectural information regarding VRC biogenesis and the function of these structures (Identification of host factors involved in virus replication may be achieved trometry . For ideic virus ,b. All tructures .In summary, the abovementioned research studies broaden our knowledge of chloroplasts and their important direct and indirect roles in the replication of several plant virus species. In recent years, increasing numbers of chloroplast and chloroplast-related proteins have been shown to be involved in the formation of membranous replication factories and RNA synthesis. However, it is worth noting that in many cases the mechanism of interactions between viral and the identified plant factors as well as their influence on the regulation of replication remains unknown. Importantly, it should be noted that despite being replication sites, chloroplasts are primarily implicated in plant defense against viruses. Therefore, molecular insights into pathways exploited by viral pathogens and the implementation of innovative powerful imaging techniques to study plant–virus interactions is of great importance. The results obtained might contribute to the development of new and effective antiviral strategies against existing and emerging plant virus diseases.MB and AO-S conceived the review. MB wrote a large part of the manuscript. AO-S critically reviewed and complemented the review. Both authors have made a substantial, direct and intellectual contributions to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ALS) and spinocerebellar ataxia (SCA) are caused by non‐coding nucleotide repeat expansions. Different pathogenic mechanisms may underlie these non‐coding repeat expansion disorders. While gain‐of‐function mechanisms, such as toxicity associated with expression of repeat RNA or toxicity associated with repeat‐associated non‐ATG (RAN) products, are most frequently connected with these disorders, loss‐of‐function mechanisms have also been implicated. We review the different pathways that have been linked to non‐coding repeat expansion disorders such as C9ORF72‐linked ALS/frontotemporal dementia (FTD), myotonic dystrophy, fragile X tremor/ataxia syndrome (FXTAS), SCA, and Huntington's disease‐like 2. We discuss modes of RNA toxicity focusing on the identity and the interacting partners of the toxic RNA species. Using the C9ORF72 ALS/FTD paradigm, we further explore the efforts and different methods used to disentangle RNA vs. RAN toxicity. Overall, we conclude that there is ample evidence for a role of RNA toxicity in non‐coding repeat expansion diseases.Several neurodegenerative disorders like amyotrophic lateral sclerosis ( RNA species or RAN (repeat‐associated non‐ATG) products.This review comprehensively delineates pathogenic mechanisms underlying various neurodegenerative disorders with special focus on disentangling toxicity associated with  ALSamyotrophic lateral sclerosisASOantisense oligonucleotideDPRdipeptide repeat proteinELISAenzyme‐linked immunosorbent assayFTDfrontotemporal dementiaFTLDfrontotemporal lobe degenerationFXTASfragile X tremor ataxia syndromeHDL‐2Huntington's disease‐like 2hnRNPheterogeneous nuclear ribonucleoproteinHREhexanucleotide repeat expansioniMNsinduced motor neuronsmRNPmessenger ribonucleoproteinPETpositron emission tomographyRANrepeat‐associated non‐ATGRBPRNA‐binding proteinrRBPrepeat RNA‐binding proteinSCAspinocerebellar ataxiaUPSubiquitin‐proteasome systemUTRuntranslated regionet al,et al,FMR1 loss of function  is the clinical dementia syndrome caused by frontotemporal lobe degeneration (FTLD) and is the second most common dementia after Alzheimer's disease (AD) in patients younger than 65 years  is an adult‐onset neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons  treatment resulting in decreased or dysfunctional C9ORF72 transcripts rescued the phenotype  and antisense DPRs  are formed with sense DPRs being more abundant than antisense and GA being the most frequently observed one  and lowest in affected tissue  in a tissue‐dependent manner  CGG repeats, do not develop any FXTAS features n), complicating the quest for the underlying mechanism. In the ATXN10, and a gain‐of‐function mechanism has been proposed due to two main observations. First, an ATXN10 loss‐of‐function mechanism is unlikely as ATXN10 transcript levels are unaltered in SCA10 patients  models overexpressing ATTCT repeat constructs exhibit phenotypes resembling SCA10 ) have not been assessed leaving the role of RAN toxicity in SCA10 unclear.SCA10 is caused by ATTCT repeats in the intron of PPP2R2B that encodes a subunit of the phosphatase PP2A. The repeat is located in the promoter region of one (of many) protein isoforms, leading to increased promoter activity upon repeat expansion  expression of TGGAA repeat constructs leads to toxicity that is suppressed by overexpression of the RNA‐binding proteins TDP‐43, hnRNPA2, and FUS, supporting an RNA toxicity gain‐of‐function mechanism . To specifically assess the contribution of RNA toxicity in the pathogenesis of C9 ALS/FTD, several approaches can be employed, each targeting a different step in the pathway leading to RNA toxicity Fig . We willC9ORF72 gain of function  models 30 in the eye or in adult neurons showed toxicity without detection of GR and GP , strongly arguing for a gain‐of‐function mechanism  altered subcellular distribution. This makes validation of rRBP mislocalization in C9ORF72 iMNs  with the highest level in cerebellar Purkinje cells .et al,et al,et al,First, analysis in other non‐coding repeat expansion disorders questions the pathogenic role of nuclear RNA foci. Instead of being stable aggregates of repeat RNA sequestering RNA‐binding proteins, work in myotonic dystrophy type 1 supports the notion that RNA foci are highly dynamic structures, with several RNA‐binding proteins themselves being involved in this dynamic process  is strikingly similar to the GGGGCC repeat expansion in C9 ALS/FTD. Moreover, SCA36 and C9 ALS/FTD are the only non‐coding repeat expansion disorders with significant motor neuron involvement  needs to be uncovered. Assessing whether RNA toxicity, alone or in addition to RAN toxicity, plays a substantial pathogenic role is crucial to predict the effectiveness of RAN directed therapies . At this moment, a multimodal therapeutic strategy aiming at all three possible mechanisms (or at least the two gain‐of‐function mechanisms) has the best potential of clinical success.As already pointed out, assessment of RNA toxicity in disease models is difficult since “non‐ATG repeat RNA constructs” have the intrinsic propensity to undergo RAN translation (cf. Box 1). Therefore, both repeat RNA and RAN proteins are present in most models, precluding a clean assessment of RNA toxicity. Unfortunately, even “RNA only constructs” are not optimal either (cf. Box 2). Different approaches to assess pure RNA toxicity, in a RAN‐devoid context, are highly needed. Possible paradigms include the use of specific RAN translation inhibitors or the induction of RAN peptide degradation. In this regard, further research into the exact mechanism underlying RAN translation will be crucial to develop these strategies. For the time being, an adequate assessment of RAN proteins in all models exploiting “non‐ATG repeat RNA constructs” is pivotal.et al,Another important issue is the need to assess all possible toxic repeat RNA species. Whereas research so far has been highly biased toward RNA foci, soluble repeat RNA has been given very little attention. Unfortunately, visualizing this soluble repeat RNA is very difficult. Approaches like MS2 tagging  of rRBP dysfunction should also be investigated .So far, most research has been performed in disease models. As C9ORF72 repeat expansion.Research in living patients is of highest disease relevance. Imaging approaches aiming to visualize RAN proteins  tracer) might provide valuable information and may be able to establish a temporal relationship with disease milestones. Also, fluid biomarkers  might be another approach to gauge the relation between these disease mechanisms and clinical aspects. These approaches should ideally be combined with prospective long‐term follow‐up studies of presymptomatic individuals carrying a post‐mortem material was positively excluded. Contribution of RAN toxicity in SCA10 and SCA36 is still largely elusive as the presence of RAN proteins in post‐mortem tissue has not reliably been established yet. Loss of function could eventually only play a significant pathogenic role in SCA12, where an “alteration‐of‐function” instead of a loss‐of‐function is suspected.Three possible mechanisms might underlie pathogenesis in non‐coding repeat expansion disorders; RNA toxicity, RAN toxicity, and loss of function. With the currently available information, we conclude that RNA toxicity might contribute to the disease mechanism in all diseases. However, it has not been assessed in SCA12 so far. RAN toxicity seems possible in all diseases except SCA12 as presence of RAN protein inclusions in Data so far have been inconclusive in determining the exact contribution of RNA toxicity, RAN toxicity, and loss of function in the pathogenesis of non‐coding repeat expansion disorders. As all non‐coding repeat expansion disorders probably share a similar underlying pathogenesis, a holistic research approach concerning these disorders needs to be implemented. Pathogenic differences and commonalities between these disorders are the key to unravel the exact contribution of RNA toxicity, RAN toxicity, and loss‐of‐function. Of interest, several rRBPs are shared between different repeat RNA interactomes, with MBNL1, CUGBP1, PURA, HNRNPA2, HNRNPK, SRSF1, and SRSF2 being shared by at least two disorders.in vitro and in vivo models, DPRs could be involved. While DPRs have been modeled extensively, research into RNA toxicity is still limited, mainly due to technical challenges in modeling RNA toxicity. Despite these methodological problems, we conclude that evidence favoring the existence of RNA toxicity is increasing and that this toxicity could induce alterations in splicing, nucleolar function, mRNA nuclear export, cytoplasmic RNA transport, and autophagy. Further research to establish the exact role of RNA toxicity in C9 ALS/FTD needs to be structured along the pathogenic cascade of repeat RNA toxicity. Such research will be crucial in guiding clinical research to develop new therapeutic approaches for C9 ALS/FTD.While C9 ALS/FTD mainly seems to be a gain‐of‐function disease, it is currently unclear whether this is mediated by DPR and/or RNA toxicity. Given the toxicity in several BS performed the literature search and wrote the manuscript. LVDB and WR discussed the literature and co‐wrote the manuscript.The authors declare that they have no conflict of interest.


Crassostrea virginica, in Chesapeake Bay and elsewhere have been limited by shell availability, necessitating the use of alternative structures as subtidal reefs, yet these have rarely been evaluated quantitatively. We quantified population structure, density, abundance and biomass of eastern oyster and hooked mussel, Ischadium recurvum, on a concrete modular reef (75 m2 surface area over 5 m2 of river bottom) deployed subtidally at 7 m depth in the Rappahannock River, Virginia during October, 2000. After nearly 5 y (May 2005), we took 120 stratified random samples over the reef. The reef was heavily colonized by 28-168 oysters and 14-2177 mussels m-2 surface area. These densities translate to 1085 oysters and 8617 mussels m-2 river bottom, which are the highest recorded for artificial oyster reefs. Size structure of oysters reflected four year classes, with over half of oysters more than 1 y old and of reproductive age. Oyster biomass (1663 g dry mass m-2 river bottom) and condition index were equally high, whereas parasite prevalence and intensity were low. Oyster density correlated positively in a sigmoid fashion with mussel density up to high densities, then declined. This modular reef is one of the most successful artificial reefs for eastern oyster and hooked mussel restoration, and details features that are conducive for successful settlement, growth and survival in subtidal habitats.Restoration efforts with native eastern oyster,  Crassostrea virginica) along the Atlantic and Gulf of Mexico coasts and Olympia oyster (Ostrea lurida) along the Pacific coast have declined by 88% in biomass and by 64% in reef area over the past 200 y [Native oyster reefs have been functionally extirpated worldwide , 2. For st 200 y , 4. ThesUnfortunately, the preferred substrate for oyster restoration, natural oyster shell, has become a limiting resource, which has spurred the use of alternative reef substrates such as concrete structures. European countries have been experimenting with various types of subtidal artificial reefs over the past few decades –9. OftenIschadium recurvum as a function of various features of the artificial reef, which can serve as a model for the use, performance and monitoring of artificial reefs in restoration efforts.To ensure that effort and funds are expended judiciously for restoration, credible evaluation of alternative reef structures is critical. For subtidal shell and intertidal restoration reefs, there is ample evidence of their productive performance in restoring the eastern oyster –14. In cIn October 2000, a substantial rebar-reinforced concrete modular reef system was deployed subtidally at 7 m depth near the mouth of the Rappahannock River, a western-shore tributary of Chesapeake Bay . The desDue to logistical constraints, we were only able to sample the top three modules of the modular reef. However, a commercial diver indicated that the lowest two modules appeared equivalent in oyster and mussel abundance to the upper three. The three modules were secured simultaneously with straps by a commercial diver and brought to the surface by a crane aboard a commercial barge for sampling . To accePermission to sample the reef was given by United States Navy Captain (Retired) Robert W. Jensen (deceased). The field sampling did not involve endangered or protected species. The two living individuals  in one of the manuscript photos have given written informed consent (as outlined in PLOS consent form) to publish the photo. The third individual (R. W. Jensen) is deceased but gave verbal permission.2. When the quadrat did not fit on a particular sample plot, as for plots in the holes of the module, we measured the actual area of the sample. A total of 120 samples was collected; 10 samples were taken from each of the 12 module-face combinations.The modular reef was sampled using a stratified random sampling design . Two strUpon removal of the three modules, it became apparent that the lifting straps had removed epifauna at each strap-reef interface. Sample plots that were impacted by the straps were discarded and the next random plot selected. Epifauna were removed from each plot with hand scrapers, placed in large trays, stored in large freezer bags on ice, and transported to the laboratory for processing.Samples were processed in increments of 24 samples (3 modules x 4 faces x 2 replicates). Each sample was thawed and rinsed over a 1-mm mesh sieve. Bivalve (oyster and mussel) and sponge volume were measured using volumetric displacement. Shell height (SH), width, and depth were measured for all bivalves, living and dead. For oysters, SH was considered as the distance from the umbo to the farthest end of the shell.All internal tissues were collected for each oyster in pre-weighed aluminum weigh boats for dry mass (DM) and ash-free dry mass (AFDM) measurements. All 108 oysters and 138 of the 924 mussels from the first 24 samples were processed for DM and AFDM. The oysters and selected mussels represented the full range of SH values. The DM data for oysters and mussels were used in a length-weight regression to estimate biomass over the entire five-module reef, assuming the size structure produced from all 120 samples was consistent with the size structure produced from the first 24 samples.CI1 [CI2 [CI3 [Condition Indices  were calwith CI1 , CI2 [21CI2 [CI3 .CI1 and CI2, shell cavity volume is equal to the difference between the mass of the whole oyster (g) and the mass of the empty valves (g) [CI1 considered the mass of the empty shells immediately after shucking whereas CI2 used the mass of the shells after a period of drying [For lves (g) , 21. CI1f drying , 21. Forf drying . Of the -1 in spatfall. Based on the initial estimates of spatfall, oyster size and growth, we developed five potential cases for the number of cohorts for the 2001 through 2004 age classes  one cohort y-1 for 2001 through 2003, and two cohorts in 2004; (iii) one cohort y-1 in 2001 and 2003, and two cohorts y-1 in 2002 and 2004; (iv) one cohort in 2003, and two cohorts y-1 in 2001, 2002, and 2004; and, (v) one cohort each in 2001, 2002 and 2004, and none in 2003. We eliminated the 2003 cohort in case (v) because spatfall and oyster abundance were extremely low in 2003 [We analyzed size and age structure for all 120 oyster samples (523 oysters), whereas for mussels we only described size structure, using 24 of the 120 samples (924 mussels). To determine size- and age-class peaks of eastern oyster, we first defined the number of annual cohorts for 2001 through 2004 from spatfall and benthic samples , and gro classes : (i) one in 2003 . We did The five cases were used along with the respective nominal mean size of each cohort  in the RPerkinsus marinus and MSX Haplosporidium nelsoni were determined. Some other parasites and pathogens commonly found in oyster tissue, but not generally associated with serious disease and mortality, were noted. These included Nematopsis, Rickettsia-like organisms, Sphenophyra-like ciliates, Stegotricha ciliates, and viral gametocytic hypertrophy.Thirty large oysters (75.6-125.2 mm SH) were haphazardly sampled from the different module faces for pathology tests performed within 2 weeks of sampling. Samples were brought back live on ice to the Pathology Laboratory at VIMS. Presence and concentration of Dermo -2 reef surface area ranging from 28-168 and from 14-2177, respectively.The modular reef was heavily colonized by eastern oysters of multiple year classes, hooked mussels, and redbeard sponges . OystersX2 test, p < 0.0001), the factor Face was significant , and it was selected as the best-fitting model. Oyster density was significantly higher on the top Face, at 159.1 individuals m-2 surface area, than on all other faces (p < 0.0001), which did not differ significantly from each other  and whose average densities were below 60 individuals m-2 surface area (p > 0.156).Oyster and mussel density were analyzed using four linear models with Module and Face as fixed factors . For oysace area . The meaX2 test, p < 0.0001), the interaction effect Module x Face was significant , and it was selected as the best-fitting model. Irrespective of module, mussel density was significantly higher on the Top face, at 1660.1 individuals m-2 surface area, than on all other faces (p < 0.001). Across all modules, mussel density was higher on Hole faces  than on Side and Bottom faces , and significantly so in three of six cases . Mussel density on Side and Bottom faces did not differ significantly for any module . The mean fraction of dead mussels  was low with a mean of 0.06, and was usually significantly higher on the Top face than on the other three faces .For mussel density, the only model fitting the data was the interaction model; all others had weighted probabilities << 0.001 . The int2 of river bottom for both a single module and for the modular reef composed of five stacked modules . Approximately 58% of all oysters were 1+ y of age  and reproductive  many older (ages 2 and 3), larger oysters survived and likely produced some oysters living to 5 or 6 y, and (ii) a high proportion of mortality occurred for age 2 and 3 oysters, but not as much for age 0 oysters once they had reached > 20 mm SH . Similar10transformed dry mass (DM) as a function of log10transformed shell height of oysters. The regression was highly significant , the factor Face was significant , and it was selected as the best-fitting model. Similar to the results for oyster density, DM was significantly higher on the Top face, with 219.0 g DM m-2 surface area, than on all other faces (p < 0.005). In contrast to oyster density, the Bottom and Hole faces had similarly high DM values of 97.5 and 91.8 g DM m-2 surface area, respectively, and which did not differ significantly . DM on the Side face was 54.9 g DM m-2 surface area and significantly lower than that on all other faces .Oyster DM was also analyzed using four linear models with Module and Face as fixed factors . Similar2 of river bottom for both a single module and for the modular reef . The mean CI values for CI1 to CI3 were 12.2, 8.8 and 6.0, respectively, which are all near the upper end of reported CI values [Sixty-two oysters throughout the full size range were processed to produce the three CIs Eqs  to 3. ThI values .Nematopsis (1), Rickettsia-like organisms (1), Sphenophyra-like ciliates (11), Stegotricha spp. ciliates (1), and viral gametocytic hypertrophy (1).Of the 30 large oysters processed for parasite assessment, none was infected with MSX and 30% were infected with Dermo. Of the nine oysters infected with Dermo, none had a serious infection (four infections were light and five were rare). The following pathogens were found in one or more oysters: The key findings of our study, which we believe is one of the most thorough evaluations of subtidal artificial oyster reefs , 17, rel-2 river bottom, which are the highest recorded for subtidal, artificial oyster reefs [-2 river bottom. These extremely high densities were the product of densities per unit surface area and the total reef surface area per m2 of river bottom, which reflected the complex architectural design of the modular reef. By comparison, seven systems of subtidal, artificial oyster reefs constructed of concrete and other similar materials, and deployed throughout the Gulf of Mexico from Alabama through Texas averaged about 15 age 0 and 85 adult oysters m-2 [-2 and on nearby shell reefs up to 611 m-2 [-2 [After nearly 5 y of deployment at 7 m in the Rappahannock River, the reef was heavily colonized by an average of 1041 oysters and 8450 mussels mer reefs . Of the ters m-2 . Total a 611 m-2 . In cont m-2 [-2 , though -2 [-2 river bottom [Oyster densities of spat and adults on the modular reef were comparable to those on the most successful restored shell reefs for the eastern oyster , which averaged 683 adults and 344 spat m-2 , and hig-2 , 12. Oysr bottom , and on In addition, oyster density and mussel density were significantly and positively correlated with a sigmoid relationship such that the modular reef provided suitable habitat for another suspension-feeding bivalve, thereby augmenting ecosystem services of the reef. Hence, this artificial reef is an extremely effective structure for restoring eastern oyster and hooked mussel populations.-2 river bottom. Collectively, oyster density, biomass, and age structure far surpassed the targets for successful oyster reef performance developed for Chesapeake Bay restoration reefs, specifically 50 oysters, 50 g dry mass, and multiple year classes m-2 river bottom [Population size structure of eastern oyster on the modular reef also reflected a persisting, successful reef due to its being composed of four year classes, over half of which (59%) were of reproductive age. Biomass of eastern oyster was equally high, and averaged 1663 g dry mass mr bottom .The fractions of dead oysters and mussels were relatively low at 0.31 and 0.06, respectively. Comparisons of live and dead oyster age structure indicated that many older (1 to 3+ y of age), larger oysters survived, that a high proportion of mortality occurred for age 2 and 3 oysters, but that mortality was low for age 0 oysters once they reached > 20 mm SH. Similarly, many mussels survived to 3+ y of age, such that most mortality occurred when mussels were somewhere between 2 and 3 y of age. The notable survival was reflected in the high condition index and low disease incidence, which when combined with exceptional biomass and abundance, indicate that the modular reef is an excellent model of effective artificial reefs for eastern oyster restoration.-2 surface area, respectively. For oysters, all other faces did not differ significantly from each other, with average densities below 60 individuals m-2 surface area. Our results are consistent with those of a study in the Gulf of Mexico, which indicated that vertical surfaces were favored over horizontal surfaces by oyster larvae during periods of higher sedimentation, but that the reverse was true when sedimentation was low [Oyster and mussel densities were significantly higher on the horizontal Top face, at 159 and 1660 individuals m was low . Our mod was low .Sampling the separate reef modules on deck of a barge allowed us to detect a substantial portion of oysters and mussels that would not have been easily detected underwater by SCUBA divers. In particular, oysters and mussels inhabiting the Hole and Bottom faces would be very difficult to see underwater, potentially biasing density estimates low by up to 50%. Hence, effective evaluation of structurally complex artificial reefs requires attention to the architectural features of specific reefs.The modular reef performed exceptionally well in an unstructured habitat dominated by muddy sand. Thus, the gains in secondary production at that site were extremely high and the potential for the reintroduction of critical three-dimensional habitats to areas of low native oyster and hooked mussel production are promising. This increased capacity for metapopulation expansion and the associated ecosystem services, such as improved water clarity, due to filter feeders  aligns directly with the needs of native oyster and ecosystem restoration.


Here, we demonstrate that most of the thirteen main m6A RNA methylation regulators are differentially expressed among gliomas stratified by different clinicopathological features in 904 gliomas. We identified two subgroups of gliomas (RM1/2) by applying consensus clustering to m6A RNA methylation regulators. Compared with the RM1 subgroup, the RM2 subgroup correlates with a poorer prognosis, higher WHO grade, and lower frequency of IDH mutation. Moreover, the hallmarks of epithelial-mesenchymal transition and TNFα signaling via NF-κB are also significantly enriched in the RM2 subgroup. This finding indicates that m6A RNA methylation regulators are closely associated with glioma malignancy. Based on this finding, we derived a risk signature, using seven m6A RNA methylation regulators, that is not only an independent prognostic marker but can also predict the clinicopathological features of gliomas. Moreover, m6A regulators are associated with the mesenchymal subtype and TMZ sensitivity in GBM. In conclusion, m6A RNA methylation regulators are crucial participants in the malignant progression of gliomas and are potentially useful for prognostic stratification and treatment strategy development.N6-methyladenosine (m RNA modhylation ,11,13. Akaryotes ,15.6A methylation regulators consist of ‘writers’ such as methyltransferase like 3 (METTL3), METTL14, WT1-associated protein (WTAP), KIAA1429, RNA binding motif protein 15 (RBM15) and zinc finger CCCH domain-containing protein 13 (ZC3H13), ‘readers’ such as YTH domain-containing 1 (YTHDC1), YTH domain-containing 1 (YTHDC2), YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) and heterogeneous nuclear ribonucleoprotein C (HNRNPC), and ‘erasers’ such as fat mass- and obesity-associated protein (FTO) and α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) [6A RNA methylation regulators has dramatically increased our understanding of the function and mechanism of m6A modification in the posttranscriptional regulation of gene expression [RNA modification, similar to DNA and protein modification, is dynamically regulated by methyltransferases (‘writers’), binding proteins (‘readers’), and demethylases (‘erasers’) . The pro(ALKBH5) –21. The pression ,15,22,236A modification not only plays a vital role in oocyte and central nervous system development [6A RNA methylation regulators are closely associated with malignant progression in various kinds of cancer [IDH-mutant leukemia and plays an anti-tumor role through the FTO/m6A/MYC/CEBPA signaling pathway [IDH mutation is also a major driver of mutation in glioma, and gliomas with IDH mutation have a better prognosis than IDH wildtype gliomas. The m6A RNA methylation regulators were also reported to play pivotal roles in glioma stem-like cell maintenance and radio-resistance [6A RNA methylation regulators in gliomas with different clinicopathological characteristics, their function in glioma malignant progression, and their prognostic value.melopment ,16 but aelopment ,22,24,25f cancer ,22,26,27 pathway . IDH mut6A RNA regulators in 904 gliomas with RNA sequencing data from the Chinese Glioma Genome Atlas (CGGA) (n = 309) and The Cancer Genome Atlas (TCGA) (n = 595) datasets. We provided the expression data of each m6A modification regulator with regard to different clinicopathological features. We found that the expression of m6A RNA methylation regulators plays important roles in the malignant progression of gliomas, and a signature with seven selected m6A RNA methylation regulators was designed to stratify the prognosis of gliomas.In this study, we systematically analyzed the expression of thirteen widely reported m6A RNA methylation regulator in tumorigenesis and development, we systematically investigated the relationships between each individual m6A RNA methylation regulator and the pathological features of gliomas, including WHO grade, IDH status, and 1p/19q codeletion status. The expression level of each m6A RNA methylation regulator and WHO grades are presented as heatmaps  and IDH-wildtype GBM (139). In the LGG with mutant-IDH, we observed that the expressions of YTHDF2 and WTAP were highly correlated with the status of 1p/19q codeletion (1p/19q codel).We then studied the relationship between mas LGG,  and gliomas LGG, , respectoth CGGA  and TCGAoth CGGA  datasets dataset . However6A methyltransferase and knockdown of METTL16 could result in approximately 20% decrease of m6A [IDH status in LGG (P<0.001) and GBM (P<0.05), it did not have prognostic value in any pathological subgroup of gliomas (data not shown). In addition, the expression information was also not included in the TCGA dataset, so METTL16 was not included in the following section of this study.We noticed that the expression of both METTL3 and METTL14 were not correlated with the WHO grade of gliomas. Considering METTL16 is also a putative me of m6A , we also6A RNA methylation regulators were very low  in gliomas  frequencies of the thirteen m gliomas , indicat6A RNA methylation regulators, k = 3 seemed to be an adequate selection with clustering stability increasing from k = 2 to 10 in the CGGA datasets  and 1p/19q codel status (P < 0.0001). The RM2 subgroup mainly contains gliomas with an older age at diagnosis, glioblastoma (GBM) phenotype, classic or mesenchymal subtypes, IDH-wildtype status and 1p/19q noncodel status  to compare the transcriptional profile between RM1 and RM2 subgroups. The results showed a clear distinction between them . We iden6A RNA methylation regulators in gliomas. We performed a univariate Cox regression analysis on the expression levels in the CGGA dataset  Cox regression algorithm to the eleven prognosis-associated genes in the CGGA dataset, which we used as a training set . Seven g6A RNA methylation regulators in high- and low-risk patients in the CGGA dataset (IDH status (P < 0.001), 1p/19q codel status (P < 0.001), TCGA subtypes (P < 0.001), and RM1/2 subgroups (P < 0.001). We also examined the association between the risk scores and each clinicopathological feature. We observed that risk scores are significantly different between patients stratified by WHO grade, TCGA subtype, age, IDH status, 1p/19q codel status, and RM1/2 subgroups, but not by gender, in both the CGGA (IDH-mutant status (AUC = 86.3%) and glioma 1p/19q codel status (AUC = 82.3%). Furthermore, the predictive efficiency is better than that of the WHO grade and age , age (P < 0.001) and WHO grade (P = 0.005) remained significantly associated with the OS in the multivariate regression  and hepatocellular carcinoma [6A methylation regulators are linked to mis-regulated RNAs in tumors, and the same m6A methylation regulators may have distinct functions in different tumors.Among the marcinoma ,26; the arcinoma ; the eraarcinoma ; and thearcinoma . These f6A RNA methylation regulators in gliomas with different clinicopathological features. As an m6A methylation writer, the expression of METLL3 was decreased in the mesenchymal subtype or IDH-wildtype gliomas. WTAP expression was significantly increased in high-grade, mesenchymal subtype, IDH-wildtype, 1p/19q noncodel and elderly glioma patients, indicating potential functions of WTAP in glioma malignancy. For the m6A methylation readers, the expression of HNRNPC, YTHDF1, and YTHDF2 was significantly increased in high-grade gliomas. Furthermore, the expression of YTHDF2 is highly correlated with 1p/19q codeletion (1p/19q codel) status. For m6A methylation erasers, although both ALKBH5 and FTO were reported as oncogenes in GBM [IDH-wildtype status, 1p/19q noncodel and older ages at diagnosis. Unlike ALKBH5, FTO could mediate the demethylation of both internal m6A and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA [6Am rather than of m6A [6A RNA methylation regulators is closely associated with malignant clinicopathological features in gliomas. Moreover, these findings are also useful for developing novel therapeutic methods through characterizing the expression of each individual m6A methylation regulator in gliomas, as chemicals targeting m6A methylation are considered a new method for cancer therapy [In this section, we comprehensively analyzed the expression of all ms in GBM , their e in mRNA . Recentln of m6A . This al therapy ,40.6A RNA methylation regulators were also correlated with the biological processes and signaling pathways of glioma malignant progression. The role of RNA m6A methylation in cancer has only been ascertained in recent years. Several biological processes and signaling pathways that it affects have been identified, including tumor stem cell growth; self-renewal, and tumorigenesis [6A/MYC/enhancer-binding protein alpha signaling pathway [6A RNA methylation regulators is significantly associated with biological processes, such as extracellular matrix, angiogenesis, cell division, response to hypoxia, inflammatory response, immune response and others, as well as with signaling pathways, such as ECM-receptor interaction, p53, cell cycle, PI3K-Akt, TNF, NFκB and others.This study uncovered that the migenesis ,26; RNA igenesis ; DNA damigenesis ,41; the igenesis ; and the pathway . Here, w6A RNA methylation regulators can be used as a prognostic maker is an important topic of research [6A RNA methylation regulators was found to be of value. In addition, we also determined the prognostic value of the signature for WHO grades II, III, and IV gliomas in both the CGGA and TCGA dataset. Moreover, the risk score also had prognostic value in gliomas stratified by WHO 2016 grades . A similar scenario was also observed in the multivariate COX analysis. This may be caused by the strong correlation between the risk signature and IDH status.Whether the expression level of mresearch . In this6 grades . As we o6A RNA methylation regulators in gliomas  and 595 glioma patients from TCGA (http://cancergenome.nih.gov/). For the RNA-seq data, reads per kilobase of transcript per million mapped reads values were obtained for the CGGA samples [The RNA-seq transcriptome data and corresponding clinicopathological information were obtained for 309 glioma patients from CGGA . We used PCA with the R package for R v3.4.1 to study the gene expression patterns in different glioma groups. GO and KEGG pathway enrichment analyses were performed with the Database for Annotation, Visualization, and Integrated Discovery (http://david.abcc.ncifcrf.gov/home.jsp) to functionally annotate genes that are differentially expressed in different groups. Interactions among m6A RNA methylation regulators were analyzed using the STRING database (http://www.string-db.org/). GSEA was performed to investigate the functions correlated with different subgroups of gliomas.To investigate the function of m6A RNA methylation regulators, we performed univariate Cox regression analyses of their expression in the CGGA dataset. From this, we identified eleven genes significantly associated with survival (P < 0.05), which we selected for further functional analysis and development of a potential risk signature with the LASSO Cox regression algorithm [To determine the prognostic value of mlgorithm –46. Finai is the coefficient, and where Coef6A RNA methylation regulators in gliomas with different WHO grades and TCGA subtypes, and t-tests were used to compare the expression levels in gliomas for age, gender, IDH status, and 1p/19q codel status.One-way ANOVA was used to compare the expression level of m6A RNA methylation regulators or were divided into high- and low-risk groups using the median risk score (derived from the risk signature) as the cutoff value. Chi-square tests were used to compare the distribution of gender, WHO grade, TCGA subtype, IDH status, and 1p/19q codeletion status between the two risk groups.Patients were clustered into two groups by consensus expression of mTo compare the risk scores of the signature for gliomas with different clinicopathologies, a one-way ANOVA or t-test was performed to compare the risk scores in patients grouped by clinical or molecular-pathological characteristics. Univariate and multivariate Cox regression analyses were performed to determine the prognostic value of the risk score and various clinical and molecular-pathological characteristics.IDH-mutant status and 1p/19q codeletion status were tested with operating characteristic (ROC) curves.The prediction efficiency of the risk signature, WHO grade and age for 3-year survival, RM1/2 groups, https://www.r-project.org/), SPSS 16.0  and Prism 7 .The Kaplan–Meier method with a two-sided log-rank test was used to compare the OS of the patients in the RM1/2 groups or in the high- and low-risk groups. All statistical analyses were conducted using R v3.4.1 (Supplementary FiguresSupplementary Tables