Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases. There are extensive studies and reviews of the roles of ncRNAs in the regulation of DNA/histone modifications, chromatin structure, and gene expression [In eukaryotes, genomic DNA and its associated proteins, primarily histones, are organized into chromatin structural domains that affect the binding of transcription factors (TFs), recruitment of RNA polymerases (RNAPs) and the initiation and elongation of transcription . The advpression ,7,8,9,106A) and its RMPs are summarized in 5C) and its RMPs in Over 170 chemical modifications have been identified in RNA . The ass7G0pppN1 structure [7G) protects the pre-mRNA from degradation and is recognized by 5′ cap-binding complex (CBC). CBC binds the 5′ cap and interacts with transcription machinery and the RNA splicing complex as well as translation initiation factors and the 40S ribosomal subunit to co-ordinate the functions of transcription, splicing, RNA export and translation [In eukaryotes, a newly synthesized mRNA (pre-mRNA) must undergo three steps to become a mature mRNA, which include (1) capping at the 5′ end, (2) adding a poly(A) tail to the 3′ end and (3) splicing to remove introns . The 5′ tructure . Loaded nslation ,16.2′-O-methylation (Nm) is one of the most abundant RNA modifications and can occur on any base in all RNA species . In abou6A) is one of the most prevalent modifications in the 5′ cap of mRNAs [6A stabilizes mRNA transcripts through resistance to the mRNA-decapping enzyme DCP2 [6A of the first transcribed nucleoside adenosine to form the cap structure m7G(5′)ppp(5′)m6Am [6-methyltransferase that does not methylate adenosine residues in the RNA body and its function is the subject of ongoing research [N6-methyladenosine m6Am  m6Am . CAPAM/Presearch .6A is one of the most abundant modifications on mRNA and was first discovered in 1965 [6A exhibits tissue-specific distribution and is enriched near stop codons and in 3′ untranslated region (3’-UTRs) [6A is involved in nearly every aspect of the mRNA life cycle and cellular processes, as well as cell/organ development and disease pathogenesis [6A associated RMPs, i.e., writers, readers and erasers, are listed in S-adenosylmethionine (SAM) to the N6-amine of adenosine in the sequence motif RRACH  of RNA [m in 1965 . m6A exh3’-UTRs) . Found iogenesis ,13. The ) of RNA ,32,33. T) of RNA ,34  of RNA ,36, to r) of RNA ,37.6A modifications primarily include members of the YTH family proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs)  or regulating m6A-modified RNA splicing and degradation  [6A binding by targeting m6A-induced structural changes in RNA structure [6A readers have been identified, such as IGF2BP1-2 and Prrc2a, which stabilize and promote storage of m6A-modified mRNA, further affecting gene expression [6A erasers [Readers of RNA m(hnRNPs) A [38,39, YTHDC1) ,43,44. Structure . Additiopression . Finally erasers ,47,48. F erasers .5C) has been well-characterized in tRNA and rRNA [5C levels in mRNA remain low, reportedly ~0.43% by one early study [5C in mRNA is an obstacle in determining m5C function in transcription regulation [5C methyltransferases and readers has revealed some insight into this concept. RNA m5C is catalyzed by RNA cytosine methyltransferases (RCMTs)  B. This g in mRNA ,57. NSUN in mRNA .5C and is critical for exporting mRNAs that are methylated by NSUN2 [5C readers, include YTHDF2, and Y-box binding protein 1 (YBX1) [5C readers are known to selectively bind m5C to regulate transportation, ribosomal biogenesis, and mRNA stability (ALYREF has been shown to selectively bind mby NSUN2 . Other p1 (YBX1) ,61. Togetability B.5Cs to 5-hydroxymethylcytosines (5hmCs). Recently TET1/2 enzymes have been shown to share partial responsibility for RNA m5C demethylation to 5-hydroxymethylcytosine (hm5C) in tRNA to promote translation [Ten–eleven translocation (TET) enzymes are traditionally known to “reverse” DNA cytosine methylation by oxidizing mnslation  and to rnslation .RNA editing is another common RNA modification. There are two common forms of RNA editing in higher eukaryotes, known as adenosine-to-inosine (A-to-I) and cytosine to uracil (C-to-U) editing . Both A-A-to-I editing is carried out by adenosine deaminase acting on RNA (ADAR) enzymes 1–3 A, and thOccurring within regions of double-stranded RNA (dsRNA), A-to-I editing causes disruption and destabilization of dsRNA base pairing and affects the RNA interference pathway (RNAi), RNA splicing, and RNA secondary structures in a tissue-specific manner ,67,68.6A [ADAR1-mediated A-to-I editing of endogenous dsRNA is needed for normal development  and esse6A .C-to-U editing involves the hydrolytic deamination of a cytosine to a uracil base by a member of the AID/APOBEC enzyme family B. All meTranscription is the first step of an essential multistep bioprocess and consists of three major steps: initiation, elongation and termination . In euka1S2P3T4S5P6S7 (YSPTSPS) largely unphosphorylated during initiation [Chromatin remodeling begins with the interaction between sequence-specific transcription factors (TFs) and chromatin modifiers to “open” condensed chromatin (heterochromatin)  Figure , step 1,itiation   of gene promoters and enhancers in the human genome . In tranTCF21 transcriptional activation by generating an R-loop at the CpG-rich promoter of TCF21, enabling GADD45A binding to the R-loop and recruitment of the DNA demethylase TET1 [Antisense RNA transcripts promote the formation of R-loops, creating a local chromatin environment which facilitates the recruitment of transcription factors and chromatin modifiers to promote gene transcription ,101. Forase TET1 . On the ase TET1 . Co-tranase TET1 . R-loopsase TET1 . Mechaniase TET1 .In addition to gene promoters and enhancers, R-loops are highly enriched in retrotransposons , ribosomal RNA (rRNA), tRNA loci and mitochondria, and are crucial for regulating genome stability, stress response and mitochondrial function ,106,107.It is increasingly clear that the formation, distribution and removal of R-loops is tightly regulated during DNA transcription and replication.R-loops are involved in regulation of all stages of gene expression, from transcription initiation to termination, and can act as transcriptional activators or repressors at specific gene loci in different cell types and differentiation states. Regulatory R-loops are important for transcription elongation and termination, as well as telomere stability and DNA repair in normal physiological conditions. In contrast, dysregulated R-loops are associated with transcription elongation defects, DNA damage and genome instability in pathophysiological conditions, such as cancer ,105.7G 5′ capping of pre-mRNAs by binding to the E-box in the promoter of active genes, which recruits the capping enzyme complex composed of RNA guanylyltransferase and 5’-phosphatase (RNGTT) and CMTR1 [MYC is well-known to activate Myc-dependent transcription, but its underlying mechanisms remain incompletely understood ,114. As nd CMTR1 .Finally, MYC appears to be implicated in regulation of R-loop formation as well. Recent studies have shown that MYC can suppress R-loop formation in promoter-proximal regions through recruitment of BRCA1, which stabilizes the DCP2 de-capping enzyme complex and enables RNAPII escape from promoter pausing, leading to increased transcription elongation and gene expression  Figure .The 7SK small nuclear ribonucleoprotein (7SK snRNP) complex is composed of the core 7SK snRNPs and other proteins, including AFF1/4, HEXIM1/HEXIM2, LARP7, MePCE and hnRNPs . This coBRD4 is a member of the Bromodomains and Extraterminal (BET) family and contains two bromodomains that recognize acetylated lysine residues of histone tails ,122. BRDcis-elements and trans-factors through extensive interactions between splicing factors and the core transcription machinery [Over 90% of human genes undergo alternative splicing, which is carried out by splicing machinery called the spliceosome. The spliceosome contains more than 150 associated proteins and five uridine-rich small nuclear RNAs (snRNAs)—U1, U2, U4, U5 and U6  ,130. Nasachinery ,135,136.achinery . The U2 achinery . The U5/achinery ,140.trans-acting factors/complexes and cis-acting spicing elements/sequences, as well as transcription machinery and chromatin modifying complexes [cis exonic and intronic splicing elements, promoting spliceosome formation [Determination of the internal exons and introns of pre-mRNAs, i.e., alternative splicing, is carried out by the spliceosome through extensive interactions between omplexes ,146,147.ormation , while hormation   regulate transcription elongation and alternative splicing in tissue-specific patterns and sizes [Other nd sizes ,152. MYCnd sizes ,156,157,Intronic RNAs constitute most pre-mRNAs in vertebrate cells and were traditionally considered transcriptional “junk” or “waste” due to their complete degradation first by a unique debranching endonuclease, followed by exoribonucleases after splicing . HoweverTerminal exons are defined by the cleavage and polyadenylation complex (CPA), which is composed of the cleavage and polyadenylation specificity factor (CPSF) complex, cleavage factors (CFs) and the cleavage stimulation factor (CstF) complex. The CPSF complex recognizes the polyadenylation signal sequence AAUAAA and other nearby sequences  Figure B. The poMCL1, a gene encoding a longer form of antiapoptotic protein (MCL1L) and a shorter form of proapoptotic protein (Mcl-1S), resulting in exon 2 skipping and production of the short form of proapoptotic MCL1S [Earlier studies showed that nucleosome positioning contributes to definition of exons and introns, and the ratio between nucleosome occupancy within and upstream from the exons correlates with exon-inclusion levels ,164,165.ic MCL1S ,169. Altic MCL1S ,171. ZMYic MCL1S .Other studies have shown that DNA methylation and DNA modifiers are also involved in RNA splicing . Higher The advent of genome-wide sequencing technologies has revealed that 75% of the human genome is transcribed into RNA, but only a small percentage of transcribed RNA encodes proteins ,178. NonAs stated previously, ncRNAs were traditionally considered transcriptional “junk” or “waste”, but are now widely accepted to play an important role in regulation of transcription elongation and chromatin structure . It is wcis-acting models of ncRNAs include formation of R-loops and DNA-RNA triple helixes through Hoogsteen base-pairing interactions between the major groove of double-stranded DNA and single stranded RNA [cis-acting symmetrical triplex-forming motifs in lncRNAs favor the formation of RNA-DNA triplexes, which can affect RNA-RNA, RNA-protein, and RNA-DNA interactions and modulate chromatin structure [The nded RNA ,192,193.tructure . By formtructure ,195.in trans RNA-protein interactions. The role of RBPs in regulating chromatin structure and gene expression has been studied extensively [The most common mode of ncRNA-mediated chromatin organization is through ensively ,197. RBPensively ,199, RNAensively , double-ensively  and hnRNensively ,203. As ensively ,206,207.ensively ,209. MeCRecent genome-wide proteomic studies have identified many new RBPs ,210. Tho6A writers, readers and erasers, respectively, to regulate the level and distribution of m6A in a tissue-/differentiation-specific manner [6A in human tissues, which is significantly enriched in long intergenic non-coding RNA (lincRNA) and CpG-rich gene promoters, suggesting that m6A is positively correlated with gene expression homeostasis and has broad involvement in human development and disease [The METTL3/14 methyltransferases, YTH-domain-containing proteins (YTHDs)/HNRNPs, and FTO/ALKB family proteins function as mc manner ,46,216 , a chromatin-associated oncogenic TF important for embryonic stem cell (ESC) differentiation , is asso complex ,222 , including promoter-associated RNAs, enhancer RNAs and repeat RNAs [YTHDC1 can de-repress (activate) gene expression by physically interacting with and recruiting KDM3B demethylase to hyper-m H3K9me2 . YTHDC1 eat RNAs .6A was reported to promote translation of the SETD1A/B components, which increases the transcriptional activation histone mark H3K4me3 and recruitment of the erythroid-specific zinc finger transcription factor KLF1 to its target gene promoters [6A methyltransferase complex blocks erythropoiesis in human bone marrow hematopoietic stem/progenitor cells (HSPCs). The METTL3-METTL14-WTAP complex also functionally interacts with SMAD2/3 proteins, the key signal transducers and transcriptional modulators of the TGFβ pathway. SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions [6A methyltransferase complex, mediates the degradation of the pre-BAF155 mRNA to affect the chromatin remodeling function of the BAF (SWI/SNF-like) complex [mromoters . Inhibitecisions . In addi complex .5C is catalyzed by RCMTs, including NOL1/NOP2/SUN domain (NSUN) family enzymes and DNMT2 (5C in mRNAs and non-coding RNAs [5C in drug (5-azacitidine) resistant myeloid leukemia cells [5C eRNAs in mouse hepatoma cells and primary hepatocytes for adaptive metabolic responses [5C-marked enhancer RNAs of PGC-1α target genes to promote recruitment of transcription machinery and reinforce transcription of those genes , such as retinal noncoding RNA3 (RNCR3) [5C is present in a wide variety of RNA species, including cytoplasmic and mitochondrial ribosomal RNAs (rRNAs) and tRNAs, as well as mRNAs, eRNAs and other types of non-coding RNAs [5C in response to stress and stimuli, which suggests an important role of m5C and its RMPs in epigenetic gene regulation [5C and its RMP-mediated chromatin remodeling and gene regulation are yet to be elucidated.Heterozygous mutations in the X-linked gene encoding MeCP2 cause the neurological disorder Rett syndrome. MeCP2 interacts with more than 40 binding partners, including transcriptional regulators, chromatin modifiers and RNA- splicing factors . MeCP2 c (RNCR3) , muscle- (RNCR3) , and maj (RNCR3) . This Meing RNAs . There agulation . Howeverin cis by a nascent RNA folding back to its DNA template at the same transcription locus and in trans by ncRNAs at distant enhancers (R-loops can be formed both nhancers . R-loops6A-rich transcription termination sites (TTSs) [6A methyltransferase complex components dramatically reduces R-loop accumulation in m6A marked genes around TTSs, leading to disruption of proper transcription termination [6A reader protein YTHDC1 does not affect R-loop levels [A recent study showed that R-loops accumulate in ms (TTSs) . Depletimination . In contp levels .6A deposition leads to YTHDC1 recruitment and R-loop accumulation, promoting homologous recombination-mediated DNA repair [6A-containing R-loops are reported to accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle [6A reader YTHDF2 leads to increased R-loop levels, cell growth retardation, and accumulation of the DNA double-strand break marker γH2AX in mammalian cells, which suggests that YTHDF2 negatively regulates m6A and R-loop accumulation [6A and its RMPs regulate accumulation of R-loops and genomic stability in a position and context-dependent manner [ATM serine/threonine kinase (ATM) is recruited to DNA double-strand break (DSB) sites to phosphorylate several key proteins, leading to cell cycle arrest, DNA repair or apoptosis . SpecifiA repair . In humall cycle . Knockoumulation . Taken tt manner ,240,241.5C in tRNAs, consequently affecting protein translation [5C onto the DNA damage-associated mRNAs and contributes to R-loop formation. The m5C and DNMT2-mediated R-loops facilitate the homologous recombination (HR) and DNA repair of DSBs [R-loops have been found to be required for transcription-coupled homologous recombination, as they recruit Cockayne Syndrome Protein B (CSB) and RAD52 proteins, but not the canonical HR proteins BRCA1 and BRCA2, to sites of reactive oxygen species (ROS)-induced DNA damage . The fornslation ,245,246. of DSBs .3C in mRNA, while METTL2 and METTL6 contribute m3C in tRNAs [3C and R-loops in the nucleolus [Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo, suggesting that transcription-coupled RNA modifications may serve as DNA damage markers to regulate DNA repair . METTL8 in tRNAs . Recentlucleolus . The nucucleolus . Retentiucleolus ,250.6A in mammalian cells were shown to be negatively correlated, and depletion of m6A modification increases the association of m6A-depleted transcripts with ADAR enzymes, resulting in upregulated A-to-I editing on the same m6A-depleted transcripts [A-to-I editing is one of the most abundant RNA modifications in mammalian cells and is catalyzed by ADAR family proteins ADAR1 and ADAR2 A. A-to-Inscripts ,260.C-to-U editing is carried out by the APOBECs family proteins B. APOBECThe advent of new genome-wide sequencing technologies has uncovered abnormal RNA modifications and their associated RMPs in a variety of human diseases. Here we will briefly summarize the recent progress surrounding the roles of some major RNA modifications and their associated RMPs in four major disease categories: neuronal disorders, metabolic disorders, immune disorders/infections and cancer/leukemia .6A is highly enriched in neural tissue and plays an important role in neurogenesis and neuronal disorders [METTL3 or METTL14 prolongs the cell cycle of embryonic neural stem cells (NSCs)/radial glia cells (RGCs) and inhibits proliferation of NSCs/RGCs, resulting in delay of cortical neurogenesis into postnatal stages in embryonic mouse brains [6A and its writers on NSC self-renewal and neurogenesis have been linked to histone modifications [6A eraser protein FTO also plays a critical role in regulation of the proliferation and differentiation of adult neural stem cells (aESCs) [misorders ,266,267 e brains ,269. Theications ,271. METications . The m6A (aESCs) ,274.6A and its RMPs, specifically METTL3, YTHDF1 and FTO, play an important role in regulation of stress responses, formation of hippocampal memories, and development of neuropsychiatric diseases [6A levels have been shown to activate the TSC1-mTOR-Tau signaling pathway and increased apoptosis of dopaminergic neurons, which contributes to the pathogenesis of Alzheimer’s disease (AD) and Parkinson’s disease (PD), respectively [Recent studies show that mdiseases ,276,277.ectively ,279,280.RNA editing can alter codons and/or splicing sites in pre-mRNAs, resulting in non-functional transcripts or proteins. Earlier studies demonstrated that knockout (KO) of ADAR2 expression could substantially reduce the functional mRNA transcripts from the mutated AMPA receptor gene and rescue mice from mutations-induced lethality ,282. MorNm is one of the most abundant RNA modifications . Recentl5C and its RMPs regulate the function of three RNA species: rRNA, tRNA and mRNA [5C and its writer proteins, NSUNs, are involved in early mouse embryogenesis, especially neurogenesis [5C pattern in mRNA [5C writer protein, is highly expressed in early neuroepithelial progenitors of the developing human brain, and loss of NSUN2-mediated tRNA:m5C impairs differentiation and motility of NES cells in mouse models [NSUN2 gene cause aberrant tRNA:m5C and autosomal-recessive intellectual disability, called Dubowitz syndrome [Eukaryotic mand mRNA . m5C andogenesis , and the in mRNA . NSUN2, e models . Mutatiosyndrome ,295,296 6A and its RMPs play a critical role in Type 2 Diabetes (T2D) pathogenesis, and ablation of m6A levels by targeting METTL3 or METTL14 in mouse models recapitulates human T2D by inhibiting the Insulin/IGF1-AKT-PDX1 pathway in pancreatic β-cells, resulting in cell-cycle arrest and impaired glucose-stimulated insulin secretion (GSIS) [Recent studies show that mn (GSIS) . METTL14n (GSIS) ,299. WTAn (GSIS) . The zinn (GSIS)  , are known to ablate activation of dendritic cells (DCs) to inhibit innate immune system through TLRs [6A was recently shown to promote dendritic cell activation [6A in the mRNAs of SOCS family genes encoding the STAT signaling inhibitory proteins SOCS1, SOCS3 and CISH, leading to slower mRNA decay and increased protein expression, consequently inhibiting IL-7-mediated STAT5 activation in the Mettl3-deficient T cells [6A leads to suppression of IL-2-STAT5 signaling pathway, which controls Treg cell functions and regulates the tumor-killing functions of CD8 T cells [Recent studies demonstrate a critical role of mfections ,309. Thefections ,311. RNAugh TLRs . Specifitivation . METTL3 tivation . Depleti T cells . Another T cells . Yet fur T cells .6A levels in HIV-1 RRE due to silencing of METTL3/METTL14 or ALKBH5, respectively, lead to reduced or increased viral RNA export and replication suppression [Depletion of METTL14 showed a similar effect, stimulating accumulation of dsDNA- or virus-induced IFNB1 mRNA and inhibition of virus reproduction. Alternatively, ALKBH5 had the opposite effect . Since tpression  (Table 2Most A-to-I editing is predominantly located within mobile elements in non-coding parts of the human genome  and play7G (cap 0) and Nm of the first residue (cap 1) is vital for pre-mRNA processing and translation in multicellular eukaryotes and some viruses , and METTL3 loss leads to increased levels of phosphorylated AKT, promoting differentiation of hematopoietic stem/progenitor cells (HSPCs) [6A in the CEBPZ-targeting gene transcript, enhancing translation and thereby promoting leukemogenesis [Recent studies have demonstrated that RNA modifications and RMPs play a critical role in controlling cancer cell proliferation, metastasis, immune evasion and drug resistance ,330,331. (HSPCs)  , which results in decreased expression of the negative AKT regulator PHLPP2, increased expression of the positive AKT regulator mTORC2 and tumor cell proliferation [6A by METTL14 suppresses renal cancer cell migration and invasion through down-regulation of P2RX6 protein translation and ATP-P2RX6-Ca2+-p-ERK1/2-MMP9 signaling [XIST and miRNAs, respectively [In contrast to increased mchanisms . m6A wasferation . Increasignaling . METTL14ectively ,345. METectively . Howeverectively . Mechaniectively .6A writer proteins METTL3 and METTL14, the m6A reader proteins YTHDF1 and YTHDF2 can act as either an oncogene or a tumor suppressor. YTHDF1 promotes cancer cell growth through upregulation of MYC [6A-masked transcripts of lysosomal proteases. This binding increases lysosomal cathepsins translation, leading to inhibition of antigen presentation of wild-type dendritic cells and CD8+ T cell infiltration, and immune evasion by tumor cells in vivo [6A/METTL3-mediated tumor cell growth in glioblastoma (GBM) via regulation of nonsense-mediated mRNA decay (NMD) of serine- and arginine-rich splicing factors (SRSF) [6A-marked HINT2 mRNA, a tumor suppressor, which inhibits ocular melanoma progression [Like the mn of MYC   . Tumor cs (SRSF) , and HIFs (SRSF) . Howevergression , and YTHgression .6A writers and readers in cancer, less is known about the m6A demethylase, FTO. In breast cancer, FTO has been shown to enhance the PI3K/AKT signaling pathway and degrade BNIP3  mRNA transcripts, promoting cancer cell growth [6A levels in its mRNA transcripts, which in turn increase PGC-1α expression [In contrast to the plethora of studies on ml growth ,361. Parpression .Nm is one of the most common RNA modifications and plays a role in the pathogenic processes of various human diseases . Rare ca6A and its RMPs, the roles of A-to-I editing and ADARs are also multifaceted. ADAR1 is overexpressed in many cancer types, including lung, liver and esophageal cancers and chronic myeloid leukemia (CML), and it acts predominantly as an oncogene. In contrast, ADAR2 expression is reduced in glioblastoma, in which ADAR2 acts as a tumor suppressor [A-to-I editing and ADARs are known to contribute to tumorigenesis . Like m6ppressor ,372,373.ppressor . In antippressor . ADAR1 pppressor . Recentlppressor ,378,379 ADAR2 is essential for editing several miRNAs, such as miR-222/221 and the miR-21 precursor in vivo and in vitro, most of them oncogenic miRNAs . However5C is mediated by NSUN RNA methyltransferase family proteins  is a well-known tumor proliferation associated nucleolar protein ,384 and 5C in mammalian cells and is upregulated in various cancer types [5C deposition in the 3’ UTR of hepatoma-derived growth factor (HDGF). HDGF stabilizes the mRNA transcripts marked with m5C, which is selectively bound by YBX1 protein [5C in CDK1 mRNA promotes CDK1 translation, leading to tumor cell proliferation [NSUN2 is responsible for the majority of mRNA:mer types ,389. NSUer types . In blad protein ,392. NSUferation ,394.RNA modifications and RMPs are distributed widely in various subcellular organelles, such as nuclear particles, ribosomes and mitochondria, and identifying the role of RNA epigenetics in coordinating the functions of these subcellular organelles is likely to play an important part in more accurately characterizing multiple cellular functions and disease processes. It is increasingly clear that the role of RNA epigenetics is multifaceted and context-dependent in chromatin remodeling and gene expression. Although great progress has been made in elucidating the many roles of RNA epigenetics physiologically, the detailed mechanisms by which RNA modifications and RMPs control cancer progression and drug resistance remain largely unknown. There is still little known about how selective hyper- or hypo-regulation of RNA modifications in various RNA species contributes to tumor progression and therapeutic response/resistance. Great potential exists for the use of RNA modifications and RMPs as diagnostic/prognostic tools and therapeutic targets in future cancer therapy. However, given the large number of RNA modifications, an enormous amount of work lies ahead to fully understand the pathophysiological function of RNA modifications and their RMPs in cancer.


N = 92; aged 8–9 years; 48 boys); comparing those with no attentional difficulties and severe inattention. We hypothesized that 1) choice would increase reading comprehension and enjoyment 2) choice would increase the reading comprehension and enjoyment of children both with severe inattention and no attentional difficulties 3) choice effects would be significantly greater for children with severe inattention than those with no attentional difficulties. Children participated in a reading intervention that included a Choice  and a No Choice (control) condition. Child inattention was measured via a Virtual Reality Continuous Performance Task  and Teacher Ratings. Choice significantly increased reading comprehension, but not enjoyment compared with no choice. Choice improved the reading comprehension of children with both severe inattention and no attentional difficulties. Choice did not benefit the reading of severely inattentive children more than that of children with no attentional difficulties. These findings underline the educational benefits of choice for young readers both with severe inattention and no attentional difficulties, which are further discussed drawing on existing theory and research.It has been proposed that enhancing motivation supports the learning of children with Attention Deficit Hyperactivity Disorder (ADHD). Less is known if inattentive children with no ADHD diagnosis may similarly benefit, when being motivated to engage in an academic task. Using a repeated-measures design, this study investigated the effects of text choice as an intrinsic motivator on the reading comprehension and enjoyment of Year 4 children attending mainstream primary schools (The online version contains supplementary material available at 10.1007/s10802-021-00835-8. Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common childhood neurodevelopmental conditions characterized by severe degrees of inattention and/or hyperactivity/impulsivity that are inconsistent to the child’s developmental level  and ADHD co-occur in both community and clinical samples  and behavioral interventions (e.g. parent training and positive reinforcement) are designed to reduce diagnosed inattention and/or hyperactivity/impulsivity, rather than target specifically learning  in behavioral tasks  for both neurotypical children and children with ADHD, with these effects being more salient for the latter group , Smith et al.  including choice. In a correlational study with 302 adolescents with and without diagnosed ADHD  than their neurotypical peers . Year 4 children were sought as reading motivation and enjoyment are more likely to decline towards the end of primary phase , data on the virtual reality task (N = 6) and reading data (N = 5) could not be collected due to child absence or technical difficulties. Ninety-two children were finally included in the study. Additional information about the participant characteristics was provided by teachers. The majority of children were of Black ethnic background , others had a White ethnic background , others an Asian ethnic background , others a Mixed ethnic background  and others had any other ethnic background . Of the 92 participating children, 12 children (13%) were eligible to free school meals, 16 children were on pupil premium (17.4%) and 49 children (53.3%) had English as an Additional Language (EAL).Using the G* Power 3.1 software, an a priori analysis showed that a minimum sample size of 42 children was necessary to report medium effects . Cronbach’s alpha was previously reported to be above 0.9 for both test sections ensuring high reliability  were selected as we were interested in the attentional difficulties specifically, rather than hyperactivity/impulsivity. Omission errors occur when the child fails to respond on trials, which require a response. These are indicative of poor sustained attention. RTV has been associated with ADHD, poor word reading, reading fluency and reading comprehension in children with ADHD , r ranging between 0.6 and 0.8 in a mixed sample of 57 children with ADHD  in the presence of realistic classroom distractors and not for clinical purposes  showing high reliability  were rotated and administered across both conditions, counterbalancing the condition order.This study received ethical approval by the Research and Ethics Committee at UCL, Institute of Education. Information letters and opt-in consent forms were sent via teachers to parents/carers before the study. The study began in December 2017 and lasted until January 2019. In the first school visit, children completed the NGRT first and then the MRQ. In the second visit, children of each classroom were randomly assigned to the Choice or No Choice condition. In the first classroom, children participated in the experimental condition (Choice) with the control condition (No Choice) following on a different date (third visit). In the second classroom, children took part in the control condition first with the experimental one taking place after (third visit). Children completed all the tasks individually. The reading tasks were administered in children’s regular classroom during morning sessions that lasted approximately 1 h each. The three visits were between two to seven days apart. Further visits took place in-between the three main visits, during which children completed the AULA task individually.In the Choice  condition, children received two A4 envelopes. Stapled to the front of the envelope they found a different cover page for each story, a different prologue for each story and two different child reviews. The choice was a perceived choice; the actual stories inside the envelopes were the same; only the titles, prologues and reviews were different. Perceived choice enables to control for background knowledge and individual/topic interest. Children had 5 min to read through the stapled material to ensure more meaningful choice. The number of stories was restricted to two as limitless choice could be burdensome and potentially demotivating , the assumption of normality is not necessary . Thus, parametric tests were employed. Descriptive statistics and bivariate correlations among all study variables were initially explored. In order to address Hypothesis 1, we used a repeated measures ANOVA with Choice as the within-subjects variable. For Hypothesis 2 and 3, we followed two analytical methods. First, we applied a whole-sample analysis in order to preserve the continuous nature of the Attention variables, by checking the correlations between the difference scores in Reading comprehension and Enjoyment and the Attention measures to test for any relationships between Attention and Choice. Greater difference scores would indicate that children achieved greater Reading comprehension and Enjoyment in Choice than No Choice condition and vice versa. Use of difference scores could help infer an interaction from the correlation; significant positive correlations between the difference scores in Reading comprehension and Attention variables and between the difference scores in Enjoyment and Attention variables would suggest that there is an interaction effect such that severely inattentive children would benefit more from Choice than No Choice than children with no attentional difficulties. This method aligns with dimensional approaches to ADHD  still met power calculation requirements as indicated by the power analysis (N ≥ 42). Table SD above the mean for Omission errors and more than one SD above the mean for RTV). Such congruence between the statistics reported in our study and previous research provides further support for the reliability of our trichotomization method.After this, three mixed 2 × 3 repeated measures ANOVA tests with a within-subjects variable (Choice) and a trichotomized between- subjects variable  for each of the three attention variables  were employed to test the three hypotheses. Contrary to the median split method, trichotomization is a more refined method that enables to discretise a continuous variable (inattention as measured by Conners 3 and AULA) into three groups based on percentiles and then compare the upper and lower third group  = –0.40, p < 0.01 and a weak negative correlation between the NGRT and Teacher-rated Inattention, r(92) = –0.21, p < 0.05. Therefore, more inattentive children as assessed by AULA were more likely to achieve poor reading scores on the NGRT. Such correlations were not found for RTV. Bivariate correlation analysis showed a weak negative correlation between RTV and MRQ, r(92) = –0.27, p < 0.05. Thus, less motivated children as shown by the MRQ were more likely to experience lapses in attention as measured by AULA. Similar correlations were not found between the Omission errors and MRQ and Teacher-rated Inattention and MRQ. Surprisingly, no correlation was found between the NGRT and MRQ. Children also achieved higher, but not significantly different Reading comprehension scores for Story A across both conditions, M = 9.04, SD = 3.94 compared with Story B, M = 8.42, SD = 3.31, p = 0.122 (two-tailed). Similarly, children achieved higher, but not significantly different Enjoyment scores for Story A across both conditions, M = 39.01, SD = 7.93 compared with Story B, M = 38.22, SD = 6.44, p = 0.319.Table Hypothesis 1: Choice will increase the reading comprehension and enjoyment of children compared with no choice.F = 6.18, p = 0.015, M = 9.22, SD = 3.68 than No Choice condition, M = 8.25, SD = 3.55. Similarly, a repeated measures ANOVA tested whether children reported greater Enjoyment in the Choice than No Choice. There was no significant difference in Enjoyment scores across the conditions, F = 3.48, p = 0.065, A repeated measures ANOVA showed that there was a two-tailed statistically significant difference, ores see .Hypothesis 2: Choice will increase the reading comprehension and enjoyment of children with both severe inattention and no attentional difficulties compared with no choice.Hypothesis 3: There will be an interaction between choice and attention, such that choice will increase particularly the reading comprehension and enjoyment of severely inattentive children than with those with no attentional difficulties.Positive correlations were not reported between difference scores for both Reading comprehension and Enjoyment and Attention , showing that more inattentive children did not achieve greater Reading comprehension and Enjoyment in Choice than No Choice Table .Hypotheses 2 and 3. There was a main effect of Choice, F = 6.17, p = 0.015, F = 5.68, p = 0.005, p = 0.004); however, both groups did better in Choice  compared with No Choice . There was no significant interaction between Choice and Teacher-rated Inattention, F = 0.12, p = 0.891, F = 6.04, p = 0.016, F = 7.74, p = 0.001, p = 0.001). Taken these results together, children with severe attentional difficulties achieved lower Reading comprehension scores than children with no attentional difficulties, however, both groups had better reading comprehension in Choice  compared with No Choice . There was no interaction between Choice and Omission errors, F = 0.89, p = 0.915, F = 6.14, p = 0.015, M = 9.30, SD = 3.84 for children with no attentional difficulties; M = 8.16, SD = 3.74 for children with severe inattention) than No Choice . There was no main effect of RTV, F = 1.83, p = 0.167, F = 1.19, p = 0.310, A mixed 2 × 3 ANOVA was employed with Choice as the within-subjects variable and trichotomized Teacher-rated Inattention as the between-subjects variable to address Choice, F,89 = 6.1Hypotheses 2 and 3  = 3.37, p = 0.070, F = 0.35, p = 0.703, F = 0.06, p = 0.941, F = 3.58, p = 0.062, F = 1.10, p = 0.338, F = 2.90, p = 0.060, F = 3.39, p = 0.069, F = 2.82, p = 0.065, F = 0.41, p = 0.664, A mixed 2 × 3 ANOVA was employed with Choice as the within-subjects variable and trichotomized Teacher-rated Inattention as the between-subjects variable to address We tested the hypotheses that 1) eight- to- nine year old children in mainstream schools would show increased reading comprehension and enjoyment when given a (perceived) choice of a story rather than no choice; 2) choice effects would be present for all children, irrespective of the severity of inattention ; and 3) choice effects would be significantly stronger for children with severe inattention than those with no attentional difficulties. Children participated in a reading intervention with two conditions, a Choice where they selected one out of two stories and a No Choice where they were assigned a story. They completed a virtual reality task designed to assess inattention in a classroom setting and measures of baseline reading ability and motivation. Teachers rated children’s inattention.Hypothesis 1 showed that all children achieved greater reading comprehension, when they had the option to choose their story compared with no choice. These findings replicated Fridkin’s study Testing of p < 1) with children achieving greater enjoyment scores in Choice than No Choice. In theoretical terms, choice has been viewed as a trigger of situational interest, which represents the first phase of interest development . Results remained consistent across all the analyses for each of the different measures of inattention. The partial confirmation of Hypothesis 2 suggests that choice can improve the reading comprehension (not the reading enjoyment) of all children compared with no choice, including those children with attentional difficulties, a risk factor for reading difficulties  and research in ADHD  and baseline reading motivation (MRQ). Similar findings were also reported in Fridkin’s study . These fIn conclusion, this is the first study to explore empirically and report that choice is an important motivational practice that increases the reading comprehension of children with severe inattention and those with no attentional difficulties alike. Choice did not significantly increase enjoyment, although there was a trend with children achieving greater enjoyment scores in Choice than No Choice. Choice did not increase the reading comprehension and enjoyment of the severely inattentive children independently. These findings do not fully support previous studies that found more salient effects of motivational practices (e.g. extrinsic motivators) for the behavior of children with ADHD. These novel findings extend our knowledge about the importance of autonomy-supportive practices, such as choice provision, for the reading of all children in mainstream classrooms. They also provide a starting point to further examine choice effects in young readers with varying attentive profiles  to establish generalizability, with the aim of informing the reading practices of educators. Considering teachers’ high workload, stress and limited freedom over classroom learning due to the exam-oriented character of school education Ofsted, , inclusiSupplementary file1 (DOCX 48 KB)Below is the link to the electronic supplementary material.


Emerging evidence suggests that RNA editing is associated with stress, neurological diseases, and psychiatric disorders. However, the role of G-to-A RNA editing in chronic social defeat stress (CSDS) remains unclear. We herein identified G-to-A RNA editing and its changes in the ventral tegmental area (VTA), a key region of the brain reward system, in CSDS mouse models under emotional stress (ES) and physiological stress (PS) conditions. Our results revealed 3812 high-confidence G-to-A editing events. Among them, 56 events were significantly downregulated while 23 significantly upregulated in CSDS compared to controls. Moreover, divergent editing patterns were observed between CSDS mice under ES and PS conditions, with 42 and 21 events significantly upregulated in PS and ES, respectively. Interestingly, differential RNA editing was enriched in genes with multiple editing events. Genes differentially edited in CSDS included those genetically associated with mental or neurodevelopmental disorders, especially mood disorders, such as FAT atypical cadherin 1 and solute carrier family 6 member 1. Notably, changes of G-to-A RNA editing were also implicated in ionotropic glutamate receptors, a group of well-known targets of adenosine-to-inosine RNA editing. Such results demonstrate dynamic G-to-A RNA editing changes in the brain of CSDS mouse models, underlining its role as a potential molecular mechanism of CSDS and stress-related diseases. RNA editing is the change of RNA nucleotide sequence that occurs at the transcriptional level . CanonicHNRNPK) gene in colorectal cancer and the glutamate ionotropic receptor kainate type subunit 3, GRIK3) gene in the fetal human brain , depression, and other stress-related diseases. Recent studies have shown that social defeat stress could induce changes in RNA editing . Dick etChronic social defeat stress could be induced in mice under emotional stress (ES) condition by witnessing traumatic events and insulating effects of physical stress (PS) . Interes1. The samples used for RNA editing event discovery consisted of adult C57BL/6J male mouse brain VTA from CSDS mouse models under ES or PS conditions, and controls (N = 3 per group) (GSE36005) (Raw RNA-Seq read data were downloaded from NCBI Gene Expression Omnibus (GEO) databaseSE36005) . As descSE36005) , 2019.The raw sequencing data obtained above were first analyzed by FASTQC for quality control. Reads that passed quality control were aligned and mapped to the mouse genome (UCSC mm10) using RNA STAR (version 2.7.0e) , and gen2 (3 (RNA-Seq data from CSDS and control mice (GSE36005) were used for discovery of RNA editing events. Single nucleotide variants (SNV) were called from the GATK re-calibrated BAM files by using VarScan (version 2.4.3) . The var2 . SNVs th2 (3 : (1) locFeatureCounts was used to obtain the pseudo-counts of gene expression from the BAM files , and traPrincipal component analysis (PCA) of G-to-A RNA editing events was performed using the function Prcomp in R (version 3.6.3), and visualized using the ggplot2 (version 2.2.1) package.4  and kyoko encyclopedia of genes and genomes (KEGG) pathways were conducted using Enrichr4 . Enrichmt-test between groups. P-values < 0.05 were considered to be significant. The correlation coefficient (r) between RNA editing levels was calculated using Spearman correlation analysis. Fisher’s exact test was used to analyze enrichment of differentially edited genes.Levels of RNA editing or gene expression were compared using Student’s G-to-A RNA editing events in 2553 genes in the mouse VTA RNA-Seq data , TMF1 regulated nuclear protein 1 , and prothymosin alpha  , and another Fat1 missense event at Fat1:chr8:45025105  were observed in controls only. Both of the two Fat1 G-to-A changes were predicted to be deleterious to the protein function , tripartite Motif Containing 2 , and apolipoprotein D  (Grin2b) and subunit associated protein 1 (Grina) could be the two most evident glutamate receptors subjected to G-to-A RNA editing in CSDS   . Notablyfunction . In cont 0.0082) . In addictively) . These RTo help understand the biological impact of G-to-A RNA editing in CSDS, enrichment analysis of genes with RNA editing enriched in either controls or CSDS was further compared. Intriguingly, the results in Although both ES and PS mice were reported to exhibit similar defects of depression- and anxiety-like behavior, ES mice experienced CSDS by only witnessing emotional events without direct physical interaction. Previously Warren’s study indicated possible differences in transcript expression between the two CSDS models. We thus went on to compare the G-to-A RNA editing patterns between the two CSDS paradigms.t-test identified 63 events with significantly different editing levels between ES and PS, among which 42 were upregulated in ES and 21 were upregulated in PS , c-Maf inducing protein , and multivesicular body subunit 12b . The top three events that significantly upregulated in PS were found in SSX family member 2 interacting protein , DAZ interacting zinc finger protein 3 , and dystrophin related protein 2 . PCA with these 63 events showed that ES cluster separately from PS, with higher intra-group variance in ES, with PC1 contributing 80.2% to the total variance,  and HECT, UBA And WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1) genes, were found to be the top two genes with the largest counts of editing events. Moreover, differential G-to-A RNA editing was also observed in the two genes. As illustrated in Fisher’s exact test implicated that differential G-to-A RNA editing between control and CSDS or between PS and ES were dramatically enriched in genes with more than one editing event .It was noted that a large proportion of genes in the mouse VTA RNA-Seq were observed to contain multiple G-to-A RNA editing events. The top ten genes with the largest counts of editing events are shown in The rodent model of CSDS has been widely used in the study of stress-related diseases such as major depression and PTSD, providing an important and useful tool for the understanding of these diseases. The current study for the first time systematically investigated G-to-A RNA editing in the brain VTA in CSDS mice at the transcriptome-level, and demonstrated dynamic changes of G-to-A RNA editing associated with CSDS as well as divergent editing patterns found under ES or PS conditions.HNRNPK in colorectal cancer  of the RNA editing variants found, while missense, stop-gain and stop-lost variants accounted for another 68.2% of the coding variants. This may imply that G-to-A RNA editing could have a potential impact on the sequences of protein expressed in VTA. Such findings thus underscore the potential role of G-to-A editing in the brain.Ventral tegmental area is a key region of the dopaminergic brain reward system, and its role in mood and anxiety disorders has been implicated by emerging evidence . Therefol cancer  and WT1  samples . For neuan brain . Moreovean brain  with decan brain . In the Htr2c) in CSDS mice. In line with such existing evidence, our results revealed changes of G-to-A editing events in CSDS mice, suggesting that CSDS could have dramatic effects on G-to-A editing in the brain. Moreover, divergent G-to-A editing patterns contributed to the difference between CSDS paradigms induced under ES and PS conditions. As a result, gene list enrichment further suggested that such changes may lead to altered gene functions or pathways in the brain.Recently, RNA editing has been reported to be involved in CSDS. For example, by using target sequencing, Fat1. It encodes a member of the cadherin superfamily. Two missense events in Fat1 were exclusive found in controls but not CSDS . The Slc6a1 transporter removes GABA, the primary inhibitory neurotransmitter in the CNS, from the synaptic cleft, and maintains low extracellular levels of GABA. Mutations of SLC6A1 were reported in a variety of neurological and psychiatric disorders, myoclonic atonic epilepsy, intellectual disability  together with increased G-to-A RNA editing in the in the 3′-UTR. Such findings, taken together, pointed to a potential effect of G-to-A RNA editing on Slc6a1 expression and GABAergic system in CSDS.It was noteworthy that a number of the genes with differential G-to-A editing in CSDS had been reported to be associated with neurological or psychiatric diseases, or functions as key components in neurons . In the not CSDS , which mpression . Likewissability , 1, autisability , as wellsability . Associaehaviors . Moreove anxiety . In lineGrin2b) and glutamate ionotropic receptor NMDA type subunit associated protein 1 (Grina) could be the two most significant glutamate ionotropic receptors subjected to G-to-A RNA editing in CSDS. In addition, G-to-A RNA editing was also found in kainate receptors and AMPA receptors  receptors, N-methyl-D-aspartate (NMDA) receptors, and kainate receptors. By far, Non-NMDA glutamate receptors have been known to be important targets for A-to-I RNA editing. Interestingly, our results implicate that NMDA including glutamate ionotropic receptor NMDA type subunit 2b   . Moreovedisorder . TherefoMap1b gene encodes a protein member of the microtubule-associated protein family involved in essential microtubule assembly in neurogenesis. Loss-of-function mutations of MAP1B were reported in patients with intellectual disability (Huwe1, which encodes an E3 ubiquitin ligase, and plays a key role in cerebral cortex neurogenesis (G-to-A RNA editing was not evenly distributed among expressed genes in the transcriptome. A number of genes were found to contain more than one editing event, suggesting that these genes might be editing hotspots. Moreover, genes with multiple editing events showed a significantly higher possibility to be differentially edited in CSDS compared to controls. These genes are mostly involved in development and function of the nervous system, or associated with neurological and mental disorders , 3. For sability . Anotherogenesis . Mutatioogenesis .In conclusion, the current study identified high-confidence G-to-A RNA editing in the VTA transcriptome of adult male mouse brains, and revealed dynamic changes in different CSDS models. With a number of stress/mood disorder-related genes revealed to be involved in altered G-to-A RNA editing, especially those with multiple editing events, our findings thus provide new insight into the understanding of the role played by brain G-to-A RNA editing in CSDS and its related diseases.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Ethics Committee of Jiangnan University.JT and C-YR performed the bioinformatic analysis. Z-YW improved the data analysis pipeline. JX and J-HC conceived the project and planned the experiments. FZ participated in the discussion. All authors contributed to the final manuscript.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.


In this study, the impact of the structural context of the TABS in RNA1 on the TA–TABS interaction and sg mRNA transcription was investigated using in vitro and in vivo approaches. The results (i) generated RNA secondary structure models for the TA and TABS, (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA–TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner. These findings advance our understanding of transcriptional regulation in RCNMV and provide novel insights into the origin of the TA–TABS interaction.Many positive-sense RNA viruses transcribe subgenomic (sg) mRNAs during infections that template the translation of a subset of viral proteins. Red clover necrotic mosaic virus (RCNMV) expresses its capsid protein through the transcription of a sg mRNA from RNA1 genome segment. This transcription event is activated by an RNA structure formed by base pairing between a  Tombusviridae . R. RDianth(1.5 kb) B 13]. R. RDianth(1.5 kb) A 16]..Dianthovtrans-acting base pairing interaction between short sequences located near the end of the RdRp-coding region in RNA1 (termed the trans-activator binding site or TABS) and at a central position within the movement protein ORF in RNA2 (termed the trans-activator or TA) (Transcription of the RCNMV sg mRNA involves a r or TA) A 13]. T. Ttrans-r or TA) A,B. The r or TA) B. The TAr or TA) A, which r or TA) A 21]. T. Ttrans-The full-length cDNA clones of wild-type RCNMV RNA1 and RNA2 genome segments (kindly provided by Tim Sit and Steven Lommel)  were useUncapped viral RNAs were in vitro-transcribed from SmaI-linearized RCNMV RNA1 and RNA2 constructs using the T7-FlashScribeTM transcription kit (Cellscript) as described previously . RNA conSHAPE  was performed on the TABS and TA regions of full-length RNA1 and RNA2 genomes, respectively, as described previously . Briefly5 protoplasts were transfected with infectious viral transcripts (3 μg of RNA1 and/or 1 μg of RNA2) using PEG-CaCl2. Transfected protoplasts were incubated under constant light (60 watt) at 17 °C for 22 h. Total nucleic acids were extracted using phenol-chloroform isoamyl alcohol purification. Following ethanol precipitation, one-sixth of the total volume was separated in 2% agarose gels and products were transferred to nylon membrane. Plus-strand viral RNA accumulation was detected via northern blotting using 32P-ATP radiolabeled DNA oligonucleotide probes that are complementary to the 3′ end of RNA1  and RNA2 , as described previously [32P-UTP labeled riboprobes (nt 3621–3890) complementary to the 5′ end of negative-sensed RNA1. Viral RNA accumulation was measured by radioanalytical scanning using a Typhoon FLA 9500 variable mode imager  and quantified by Quantity One Software (Bio-Rad). Infections and northern blotting analyses were repeated for a minimum of three trials to generate averages and corresponding standard error of the means (SEMs).Cucumber protoplasts were isolated from 6-day-old cotyledon leaves and isolated as described previously . Approxieviously . Minus-s2, 100 mM KCl, 3.8% glycerol) [2) [Full-length RNA1 and RNA2 genomes subjected to EMSA were in vitro-transcribed from the viral constructs described above. EMSAs were performed using modified methods from a previously described protocol . Brieflylycerol) ,28,29. Rrol) [2)  and imagPreviously, nuclear magnetic resonance spectroscopy and computer modelling was used to generate a structural model for the 34 nt long TA bound to an 8 nt TABS oligoribonucleotide . The samThe results predicted the presence of the TA as part of a multihelical RNA structure A. WithinInterestingly, examination of the TA for CRSV revealed that, in order for it to include an AIL and a terminal loop complementary to its TABS, it would have to adopt two mutually-exclusive conformations C. NotablExamination of the predicted structural context of the TABS in RCNMV RNA1 indicated that its 5′-half is base paired within the lower portion of a 5′-adjacent SL, termed TABS-proximal SL (TP-SL) B. The reThe observation that the 5′-half of the TABS is predicted to be paired within the base of the TP-SL in all three dianthoviruses suggested that the TABS, within its genomic context, may not be readily available for pairing with the TA D. This iTo determine if the RNA1 mutations that altered TP-SL/TABS pairing also affected sg mRNA levels during infections, these mutants were transfected into protoplasts in the absence or presence of RNA2. In each coinfection, the level of sg mRNA accumulation was calculated relative to the accumulation level of its cognate RNA1. Consistent with the EMSA results, co-transfections of RNA2 with RNA1 mutant 177 moderately reduced relative sg mRNA levels (79%), with mutant 178 notably enhanced levels (168%), and with mutant 180 essentially abolished accumulation (3%) C. Co-tracis-acting, attenuation RNA structure. Indeed, we proposed previously that RNA1 likely expressed its sg mRNA via a simpler cis-acting attenuation RNA structure before evolving to its current more sophisticated trans-mechanism [cis-acting attenuation structure via the introduction of a few substitutions into the TABS’s structural context and (ii) the trans-TA–TABS interaction does not contain any unique structural features that are essential for activating sg mRNA transcription.The presence of the 5′-half of the TABS within TP-SL prompted us to consider whether further pairing of the 3′-half of the TABS with the sequence immediately 5′-proximal to TP-SL could potentially generate a SL-based, echanism . Examinacis-acting, attenuation RNA structure and later, while in association with RNA2, evolved to a trans-acting, TA-dependent mechanism; perhaps involving an intermediate stage where both modes of transcriptional activation were partially active (trans-system provided clear benefits not afforded by the cis-system: (i) the coordination of CP production with RNA1 and RNA2 abundance [cis-acting attenuation RNA structure.Lastly, these results demonstrate that, as alluded to previously , it is py active E. The ulbundance  and (ii)bundance ,31. Thus


The health and condition of the world’s reefs are in steep decline. This has triggered the development of fledgling micro-scale coral reef restoration projects along many reef coastlines. However, it is increasingly recognised that the scale and productivity of micro-scale coral gardening projects will be insufficient to meet the growing global threats to reefs. More recently, efforts to develop and implement restoration techniques for application at regional scales have been pursued by research organisations. Coral reefs are mostly located in the unindustrialised world. Yet, most of the funding, and scientific and engineering method development for larger-scale methods will likely be sourced and created in the industrialised world. Therefore, the development of the emerging at-scale global reef restoration sector will inevitably involve the transfer of methods, approaches, finances, labour and skills from the industrialised world to the unindustrialised world. This opens the door to the industrialised world negatively impacting the unindustrialised world and, in some cases, First Nations peoples. In Western scientific parlance, ecological imperialism occurs when people from industrialised nations seek to recreate environments and ecosystems in unindustrialised nations that are familiar and comfortable to them. How a coral reef ’should’ look depends on one’s background and perspective. While predominately Western scientific approaches provide guidance on the ecological principles for reef restoration, these methods might not be applicable in every scenario in unindustrialised nations. Imposing such views on Indigenous coastal communities without the local technical and leadership resources to scale-up restoration of their reefs can lead to unwanted consequences. The objective of this paper is to introduce this real and emerging risk into the broader reef restoration discussion. Most of the world’s coral reefs are located in unindustrialised nations, many of which are located in the Southern Hemisphere, and the global decline of coral reef condition is now well-documented . Few reeTo date, the management of coral reefs in the industrialised world has focused on managing anthropogenic activities undertaken directly on, or adjacent to, reefs . CommonlFirstly, local reef managers, as well as the global reef research and advocacy community, are not well placed to directly control, diffuse and mitigate worldwide global emissions. In many cases, local reef managers struggle to control activities occurring in their own reef catchments . Secondl2 [Given this situation in both the industrialised and unindustrialised world, it is understandable that a number of communities, researchers and reef stakeholders have begun trials for restoring reefs (a summary of these current practices can be found in ). The ma2 , and rec2 . Operati2 .Achieving such a scale will require substantial investment in method research and development  , the conThis can bring forth a one-way flow of knowledge and organic materials, including potentially corals, that is often forced upon Indigenous communities and First Nations peoples, be it deliberate or undeliberate, leading to a phenomenon known in Western academia as ecological imperialism. This phenomenon has been occurring for centuries. In the nineteenth century, there was a dramatic increase in ecological imperialism, with a similar transfer of knowledge and materials to that of today. This left many nations with drastically altered landscapes and biodiversity and has led to the ongoing social and cultural impacts on First Nations peoples , 22. Theth century as Europeans began to invade the American continent in earnest, with the first colony established in North America in Virginia in 1607.Colonialism was practiced by the ancient empires that expanded and subsequently contracted over many centuries. The expansion of the Roman Empire and establishment of Viking strongholds across northern Europe are well-known examples. These colonisations are often celebrated as leading to the spread of architecture, language and culture, often to the detriment of remembrance of the violence inflicted upon colonised peoples. Modern Colonialism commenced in the late 15Between1820 and 1930, over 50 million Europeans colonised a relatively small number of countries including the United States, Canada, Australia, New Zealand, and South American countries such as Argentina and Uruguay . This reFor some, ecological imperialism is a modern form of colonialism in which the populations in postcolonial eras continue to have an immense impact on unindustrialised countries . For FirPasser domesticus) were introduced to North America by the nostalgic Englishman Nicolas Pike in 1850, and camels were introduced to outback Australia during the same period [Colonisers justified the persistent push to import the familiar plants and animals from the homeland given the unfamiliarity of newly colonised landscapes and climates, as well as their lack of knowledge of how to harness the local flora and fauna for food, fibre, clothes and transportation. Colonisers proactively sought to make their new home like their old home based on their Western concept of what a ‘natural’ landscape should look like. English house sparrows  or ecological services . Methods to deliver these outcomes might include asexual and sexual methods of propagation and managing corals at a range of life-stages from larvae to reproducing adults . Method There are two fundamental approaches to upscaling the global reef restoration activity. The first approach involves replicating micro-scale operations such as coral gardening over and over. This can be thought of as the ‘domestic’ development model of restoration. This is how the global agricultural sector developed over almost the entire history of human civilisation–simply applying more and more labour to more and more land using the same small-scale methods . By contReef restoration activities are undertaken by people. Therefore, scaling is dependent not only on access to technology but also access to labour to apply the technology. In the micro-scale replication or domestic model of upscaling, labour skill requirements, and hence productivity, is generally low. For example, the labour productivity of current methods of reef restoration is estimated to be at least two orders of magnitude less than current industrial practices in agriculture . TherefoReef restoration requires labour, technology and deployment infrastructure. The mobilisation of these resources requires financing. At present, many reef restoration programs are funded through international aid programs or through existing tourism operations that are financially supported through the industrialised world . With fuThe four ecological imperialism risk pathways identified above are enabled by the Western concept of social licence to operate (SLO), which in itself is a concept that can be abhorrent to First Nations peoples. SLO is commonly viewed as a way to reduce business risk by gaining social acceptance from local and regional communities . The oriIt has been argued that the very concept of social licence is a Western concept that is not well understood or practiced in many unindustrialised nations; despite the fact that the concept was developed to allow foreign-owned companies to operate in unindustrialised nations . TherefoMany of the negative consequences of colonisation and ecological imperialism influence what is commonly known as the Global South. The Global South is defined by the Brandt line as an economic zone that splits the world into developed, developing and least developed countries . MoreoveTherefore, taking an approach that includes social licence but excludes cultural relativism runs a high risk of losing trust amongst stakeholders and, in many cases, is in principle unethical. This reinforces the risk pathways whereby there is lack of alignment in terms of the desired restored biological assemblage or community structure, associated trade-offs of ecosystems goods and services delivered by the restored reef system , and traditional cultural norms and values.All four of the attributes identified above  are essential for at-scale restoration to be achieved. However, of relevance to this discussion, the framework these pathways are operated within  will largely determine the scale of ecological imperialism effects and ongoing colonisation. The logic of this is as follows:For the domestic model of upscaling:Current practices of reef restoration follow the domestic development model. These are largely funded by international NGOs and larger individual tourism operators who are often owned by non-local organisations.The NGOs  generally seek funding from industrialised-world philanthropists, and larger tourism operations also seek visitors from wealthy nations.NGOs also commonly utilise volunteer labour from industrialised nations for reef restoration operations.For the industrialised model of upscaling (yet to be developed):These industrialised approaches are in the very early phase of development . HoNo matter which development pathway is pursued, if Western-developed reef restoration practices are to be globalised than they will create both the demand for restored reefs (through tourism), and the supply of restoration methods and services to unindustrialised nations. In other words, a transfer of demand, resources and knowledge from industrialised to unindustrialised nations is likely to occur. This is, therefore, the vector through which ecological imperialism, and ongoing colonialism, can be activated. This is also precisely the mechanism of ecological imperialism through colonisation during the 1800s during which demand, technology, labour and financing were transferred from the industrialising nations to the unindustrialised world. Human culture and landscapes of the new world replicated the old world to the ongoing detriment of First Nations peoples. There is a current risk ecological imperialism will occur again with the scaling up of reef restoration, in which industrialised nations, knowing or unknowingly, will pressure First Nations peoples to develop designer reefs and reef communities that replicate the attributes and expectation of ecological and human communities in those industrialised nations. By creating a new activity, and possibly an industrialised reef restoration support sector in unindustrialised nations, there is a real risk of Westernisation of not only the underwater ecological communities but also the human reef communities, including First Nations peoples, which practitioners of reef restoration should be aware of and strive to avoid. Below, we outline a number of suggestions on how this may be achieved.One of the major bottlenecks in gaining consensus for global greenhouse gas emissions targets has been the challenge of working in the face of injustices whereby unindustrialised nations understand that the old-world industrial collective of nations has both created the anthropogenic climate change problem and has also contributed more than its fair share of the global atmospheric carbon budget , workingFrom a First Nations perspective, even clinically identifying and labelling perceived past misdemeanors as ecological imperialism can be abrasive, as if by labelling we are consigning it to past misbehaviours. However, we have clearly not learned as we are embarking upon a new crusade to develop and impose Western-science-developed knowledge in approaches to reef restoration onto the unindustrialised world, where most coral reefs are located. For example, the first question to consider in any reef restoration program is often, ‘What should the attributes of the restored reef be?’. However, in order to answer this, we must have an understanding of, at minimum, how local stakeholders value reefs and how they should participate in the design, management and implementation of at-scale activities. This is particularly the case when restoration may become a major community activity and source of employment. Whilst seemingly straightforward, given the complexities of likely research and development and implementation supply chains for capital, labour and technology, the management of these issues can be problematic. This is especially the case for small communities such as in the Small Island Nations. With regards to this first question above, the scientific (mostly Western) community has developed ecological design principles for reef restoration. However, as argued here, these may not necessarily reflect the view of individual communities, and Western science is only beginning to learn how to find a pathway through the parallel, but largely independent, if not mutually exclusive, approaches to Western and Indigenous resource management.Fortunately, this is not a new problem. Over recent decades, foreign governments and increasingly the international NGO sector have been ramping up ‘in country’ activities to work with local stakeholders to achieve development and sustainability outcomes. It is now recognised that many of these earlier aid-type programs were essentially following the colonisation pathway–seeking to nudge unindustrialised world communities and settlements into Westernised versions . For exaMore recent in-country activities undertaken by foreign governments and the global NGO sector are increasingly culturally sensitive and relevant. However, this approach is challenging and resource intensive. Anne Ross  describeEpistemological barriers often relate to the way that the knowledge of Indigenous communities is expressed. Barriers continue to arise when Western systems disregard the relevance of social, cultural and spiritual forms of Indigenous knowledge, and when Western property rights, including intellectual property rights, impose over Indigenous peoples’ rights . The WesIn summary, global at-scale reef restoration is in its infancy, although micro-scale restoration efforts are underway. However, the preconditions for scaling up reef restoration activities, many of which will be led by industrialised nations, also create an environment whereby unintentional and unwanted ecological imperialism and ongoing colonisation may result. Therefore, a key message from this thought-piece is that organisations embarking on the at-scale restoration journey need to be aware of the possible consequences of ongoing colonialization through ecological imperialism and the potential for new colonialism. In its simplest form, managing this risk consists of recognizing when plans and discussions undertaken in research organisations in the industrialised work drift into ecological imperialism, especially through the risk pathways identified here. The next level of consideration includes actively involving not only local research organisations where they exist, especially local communities in unindustrialised nations in restoration projects, but empowering them to take the lead on specifics of the restoration outcomes sought, including which species to restore. In fact, non-local participants should consider  to take the reverse approach by seeking methods to incorporate Western-style technocratic approaches into established Indigenous management systems. Increasingly Western research programs develop projects in partnership with local institutions. However, it can be argued that, in many cases, this consists of allowing local labour to participate in delivering Western research programs rather than viewing them as equal partners if not research and operations directors . There i


Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity. The genetic code can be modified at different levels, namely nucleotides, codons, and amino acids. At each of these levels, unnatural components can be used. Thus, unnatural amino acids can be encoded by unnatural nucleotides within the three-letter codons ,2,3,4,5,The development of unnatural base pairs (UBPs) is one of the most remarkable advances in synthetic biology. UBPs, which function as a third base pair in replication, transcription, and translation, expand the genetic alphabet and can be used in various applications such as site-specific fluorescence labeling, nucleic acid immobilization, and DNA and RNA analysis. Originally synthesized to expand the genetic alphabet, UBPs have become powerful tools for monitoring the structural changes in nucleic acids and their interactions with other molecules ,20,21,22All UBPs obtained to date can be divided into two groups: (1) UBPs, which form hydrogen bonds between bases (hydrogen complementarity) and (2) UBPs with hydrophobic interactions within the pair  ,34,35,36Bacteriophage T7 RNA polymerase (T7 RNAP) is one of the widely used polymerases for incorporating UBPs into RNA. It is a one subunit protein with 883 amino acid residues (98 kDa). T7 RNAP is capable of performing a full transcription cycle without involving any additional protein factors . For a b2) cell-free transcription–translation system, in which we determined the activity of mutant T7 RNA polymerases by the luminescence of synthesized nanoluciferase (Nluc). As a result, we evaluated the transcription efficiency of unnatural templates with different mutants of T7 RNA polymerases. In our system mutant T7 RNAPs used natural ribonucleotides to transcribe DNA templates containing UBPs. Furthermore, we revealed high sensitivity and simplicity of the developed method and demonstrated the ability of the studied mutants of T7 RNA polymerase to recognize UBPs. Additionally, we demonstrated that the approach we developed for determining the activity of RNA polymerases is applicable to a wide range of UBPs.Nevertheless, presently, no simple methods are available for determining the activity of mutant forms of RNA polymerase, synthesizing RNA on a template containing UBPs. In the present study, we proposed a new method based on a double-coupled  luciferases induce luminescence in the presence of substrate immediately after their translation because they do not need a maturation time and they leave the ribosome in a catalytically active form ; (2) amoTo investigate the transcriptional efficiency of T7 RNAP, we constructed a plasmid encoding the human beta-globin 5′UTR, T7 RNAP coding sequence containing a 6xHis tag, and a subsequent TEV protease site at the N-terminus b. The glAlong with wild-type T7 RNA polymerase (T7wt RNAP), we obtained by site-directed mutagenesis and tested the functional activity of 10 previously published mutant T7 RNAPs with different specificities . As a ne2 system, we added 1–10 ng/µL of linear DNA coding T7 RNAP to TNT WGE along with ribonucleotide triphosphates (NTPs), SP6 RNA polymerase, and 1 ng/µL DNA encoding Nluc. We observed the maximum luminescence signal using 10 ng/µL linear DNA coding T7 RNAP. The optimized concentrations of the coupled2 system are summarized in For the initial testing of the approach, we first used a Nluc coding template containing only natural dNTPs. To determine the optimal conditions for the coupled2 system. The T7 RNAP translation was controlled by western blotting with anti-His-tag antibodies  from the plasmid pAR1219 using primers T7_HindIII_F and T7_XhoI_R . The resThe linear templates for T7 RNAP and Nluc were obtained by PCR with the primers shown in ® T7 Coupled Wheat Germ Extract System  was used to perform the transcription and translation of wt and mutant T7 RNAP and then to detect their activity in the Nluc DNA transcribing. Ten T7 RNAP mutants . Western blots of lysates were carried out in 3 replicates and the average amounts of synthesized T7RNAPs were determined.2, it was increased in the reaction mixture from 2 mM to 2.4 mM. Amplification was performed in a 50 μL mixture using Q5 DNA polymerase  and 25 pmol of each primer (glob-Nluc forward primer and Nam-Seq1 reverse primer). The amplification program consisted of 1 cycle of initial denaturation at 98 °C for 30 s of the plasmid with the Nluc gene; 25 amplification cycles—primer annealing at 60 °C for 10 s, elongation at 72 °C for 80 s and denaturation at 98 °C for 10 s. At the final stage, one cycle of DNA elongation was performed at 72 °C for 5 min. Thereafter, the 200 bp PCR product was purified in 1% agarose gel using a Qiagen kit . This PCR product was added to the second PCR to produce a full-length PCR product with a reverse primer Nluc-A50 complementary to the 3′ end of the noncoding region comprising the T50 sequence. The volume of the mixture was 50 μL and it contained 25 μL (½ V) of previously purified PCR product. Amplification program: 1 cycle—98 °C, 30 sec, initial denaturation; 28 cycles—98 °C, denaturation for 10 sec. Annealing of the primer 69 °C, 10 s. Elongation at 72 °C for 160 s. Final elongation—5 min at 72 °C. Since the NaM-5SICS pair is specific [To obtain templates comprising the unnatural nucleotide dNaM in the second position of the V29 codon, a reverse primer containing the required substitution was synthesized  . In the specific ,40, it wPair of unnatural nucleotides 5-Me-isodC–isodG does not reveal enough specificity which leads to the accumulation of errors with every round of amplification of approximately 2% ,29,59. TAmplification of fragments was performed in 50 μL of the mixture using Q5 DNA polymerase  and 25 pmol of each primer. First pair: glob-Nluc forward primer and isoC reverse primer; second pair: isoG forward primer, Nluc-A50 reverse primer. In such a case, the working chain for the T7 RNAP contains isoC. For a template with isoG decoding, the same steps were carried out, but the first pair contained the isoG reverse primer, and the second pair contained the isoC forward primer. The amplification program consisted of 1 cycle of initial denaturation of the plasmid with the Nluc gene at 98 °C for 30 s; 25 amplification cycles—primer annealing at 60 °C for 10 s, elongation at 72 °C for 80 sec and denaturation at 98 °C for 10 s. At the final stage, 1 cycle of DNA elongation was performed at 72 °C for 5 min. Then, PCR products of 200 bp and 600 bp were purified in 1% agarose gel using a Qiagen kit .2 cell-free system.In the second round of amplification, we mixed these two PCR products without adding flanking primers and performed 10 amplification cycles without using unnatural nucleoside triphosphates . The vol


The genetics of human disease serves as a robust and unbiased source of insight into human biology, both revealing fundamental cellular processes and exposing the vulnerabilities associated with their dysfunction. Over the last decade, the genetics of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have epitomized this concept, as studies of ALS-FTD-causing mutations have yielded fundamental discoveries regarding the role of biomolecular condensation in organizing cellular contents while implicating disturbances in condensate dynamics as central drivers of neurodegeneration. Here we review this genetic evidence, highlight its intersection with patient pathology, and discuss how studies in model systems have revealed a role for aberrant condensation in neuronal dysfunction and death. We detail how multiple, distinct types of disease-causing mutations promote pathological phase transitions that disturb the dynamics and function of ribonucleoprotein (RNP) granules. Dysfunction of RNP granules causes pleiotropic defects in RNA metabolism and can drive the evolution of these structures to end-stage pathological inclusions characteristic of ALS-FTD. We propose that aberrant phase transitions of these complex condensates in cells provide a parsimonious explanation for the widespread cellular abnormalities observed in ALS as well as certain histopathological features that characterize late-stage disease. In eukaryotic cells, a large fraction of intracellular RNA is associated with RNA-binding proteins in ribonucleoprotein (RNP) granules, which are complex assemblies that arise via biomolecular condensation and can greatly influence the distribution and utilization of RNA . These cEvidence accumulated over many years from the disciplines of genetics, cell biology, and pathology has revealed that disturbances in RNA metabolism are important contributors to both the initiation and progression of important neurodegenerative diseases, most prominently amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . These dAlthough ALS and FTD are clinically distinct, insights from human genetics, patient pathology, and investigations into molecular mechanisms have established that the most common forms of these diseases represent two extremes of a broader disease continuum, termed ALS-FTD, that in some cases also extends to muscle tissue to cause inclusion body myopathy (IBM) .ALS, also known as Lou Gehrig's disease, is a fatal neurodegenerative disease that affects motor neurons in the brain and spinal cord. ALS can strike as early as adolescence, but is typically diagnosed in the sixth decade of life (mean age of 55 yr), when patients experience progressive deterioration of voluntary muscle function that generally culminates in death three to five years after symptom onset . In the FTD represents a clinically and pathologically heterogeneous group of dementias characterized by progressive atrophy of the frontal and/or temporal lobes, manifesting in changes in behavior and personality or deficits in language functions . As defiA rare, related syndrome is multisystem proteinopathy (MSP), a dominantly inherited, pleiotropic, degenerative disorder that can affect muscle, bone, and/or the central nervous system. MSP can manifest clinically as ALS, FTD, IBM, Paget's disease of bone (PDB), or as multisystem disease . There iSOD1 or FUS, which result in inclusions that are TDP-43-negative but positive for SOD1 or FUS, respectively  show early deposition  and/or defects in translation attributable to a toxic gain of function (replicated by exogenous addition of disease gene product). With regard to the former, several studies of TDP-43 have demonstrated evidence of loss of function in the nucleus related to its splicing activity, particularly in splicing of cryptic exons , with doBiomolecular condensates constitute a diverse group of intracellular structures that govern a variety of cellular functions, spanning a broad range of complexities and sizes . RNP graFound throughout the cell, including the nucleus, cytoplasm, and distant processes of neurons, RNP granules are implicated in governing many aspects of RNA metabolism from biogenesis to splicing, localization, translation, and eventual degradation. The mechanisms proposed to underlie governance of these activities largely fall into one of two categories: either (i) sequestering constituents to limit their activity in the nucleoplasm or cytoplasm , which mA role for RNP granules in RNA metabolism has been defined more extensively for some RNP granules than for others. For instance, the nucleolus is well known as the site of ribosome biogenesis—a function whose biophysical connection to phase separation has now been defined —and the Defining the precise functions supported by other RNP granules has been more challenging. Nuclear speckles, for example, have been implicated in mRNA splicing, transcription termination, and nuclear export of mRNA, but these RNP granules have proven difficult to manipulate experimentally, as not a single factor has been identified without which speckles do not form . NonetheHowever, RNP granule “function” need not be limited to circumstances in which a granule actively promotes the catalysis of a biochemical reaction. Rather, in many cases RNP granules appear to serve as a temporary storage site for specific mRNAs and RNA-binding proteins, with significant functional consequences. For example, germ granules repress mRNA expression by sequestration of certain maternal transcripts in association with repressors  and alsoStress granules and RNA transport granules share similarities with germ granules in that their RNA content is enriched in nontranslating mRNAs, and this may have functional significance. Indeed, RNA transport granules not only control the spatiotemporal distribution of mRNAs, but also retain these mRNAs in a condensed state where they are inactive but poised for bursts of local protein synthesis in response to stimuli, such as activity-dependent local translation in synaptic compartments . Stress Indeed, the concept of functional sequestration of specific molecules into RNP granules is intimately linked to the concept of buffering, in which the bioavailability of a given constituent is limited by sequestering excess molecules beyond a specific concentration threshold into the dense phase . In thisAs reviewed elsewhere in this issue, RNP granules arise by liquid–liquid phase separation (LLPS), a process wherein certain components of a mixed phase de-mix to produce a second, more concentrated liquid phase that remains in dynamic equilibrium with the less concentrated liquid phase. Early insights into this process were derived from the serendipitous discovery in 2012 that intrinsically disordered low-complexity domains (LCDs) in many RNA-binding proteins can self-assemble into fibrils characterized by cross-β sheet content and the suggestion that this higher-order assembly contributes to the formation of RNP granules . SubsequAlthough the condensation and liquid behavior of droplets of individual RNA-binding proteins are highly reminiscent of the behavior of RNP granules where these proteins reside, simple reconstituted systems have limited ability to reveal the mechanisms of assembly of complex RNP granules in living cells, in which hundreds of constituents give rise to a bewildering number of interactions that collectively drive assembly. Indeed, whereas condensates are enriched in proteins that undergo monocomponent LLPS, the phase separation of behavior of individual proteins in vitro gives little information about the role of a given constituent in condensate assembly, material properties, or function in living cells. In one particularly germane example, ALS-causing mutations in hnRNPA1 have no impact on its phase separation behavior in vitro, but lead to dynamical arrest of RNP granules in living cells . Similarpercolation threshold, which is the threshold achieved when the sum and duration of all interactions are sufficient to create a system-spanning network  the concentrations of the individual constituent molecules, (ii) the number of interactions per molecule, and (iii) the lifetime of those interactions . This peractions . By logiThis model is consistent with empirical observations that each type of RNP granule represents a unique biomolecular network with specific topology. Moreover, the forces underlying granule assembly are distributed unevenly across this network of interacting molecules—that is, some nodes within a given network have greater influence on granule assembly than others. For example, our recent study of stress granules defined a core protein–RNA network and correlation between the network centrality of a given node and its contribution to assembly . ImportaOur current understanding of the etiology and pathogenesis of ALS-FTD derives from decades of investigation of the underlying genetics and histopathology of this spectrum of diseases, supplemented by investigations in model systems. The view arising from these observations is that the dysfunction and ultimate demise of motor axons reflects their status as a single point of failure within a complex physiological motor system that comprises multiple cell types, including both neurons and glia. Whereas cell-autonomous defects in motor neurons are certainly central to disease, abnormalities in other cell types are important and perhaps essential contributors, including interneurons, astrocytes, oligodendrocytes, microglial cells, and perhaps muscle cells. Thus, “motor neuron disease” represents the culmination of multiple types of insults impacting multiple types of cells.Although a plethora of defects in biological processes have been recorded in disease models, the most consistently observed and most proximal to the disease-initiating mutations are disturbances of RNA metabolism, including post-transcriptional processing, RNA export from the nucleus, trafficking of RNAs, biogenesis of noncoding RNAs, the synthesis of ribosomal subunits, translation, and degradation of RNAs . Among tThe recognition that abnormal condensation contributes to ALS-FTD arises from the appreciation that many ALS-FTD-causing mutations fall into two general classes. In the first, mutations directly impact constituents of RNP granules, changing their interactions in a manner that alters the assembly and material properties of the condensates in which they reside. In the second, mutations occur in factors that exert higher-order regulation over these same constituents, with the same eventual consequences to granule dynamics. Below we review three examples of the first class—mutations in the LCDs of RNA-binding proteins, neomorphic peptides that exploit common modes of interaction underlying condensation, and pathological amplification of RNA–RNA interactions through expansion of GC-rich RNA sequences B–D—folloAs noted above, some of the earliest insights into the phase separation behavior of biomolecules highlighted a central role for the LCDs of RNA-binding proteins in mediating fibril assembly in vitro . ImmediaThe manner in which LCD mutations disturb RNP granule assembly and dynamics is strikingly similar across different disease genes. For hnRNPA1 and hnRNPA2B1, LCD mutations accelerate the formation of labile fibrils in vitro and alter the dynamics of stress granule assembly in cells , an obseAmong these, TIA1 provides a striking example of how a single point mutation in a single constituent of stress granules is sufficient to slow the dynamics of disassembly and cause the accumulation of nondynamic granules . NotablyCaenorhabditis elegans neurons  are knoALS-FTD-causing mutations that impair the dynamism of stress granules and RNA transport granules are further linked to impaired local translation, as demonstrated by studies of mutations in FUS  and TDP-Granule hardening may also have loss-of-function consequences. One prominent example is the sequestration of TDP-43 in poorly dynamic RNP granules in the cytoplasm of neurons, which culminates in gradual redistribution of this splicing factor from a predominantly nuclear location to cytoplasmic foci . As a reThe second hypothesis, not mutually exclusive with the first, derives from evidence that the late-stage proteinaceous pathology in ALS-FTD may arise, at least in part, from the evolution of poorly dynamic condensates . AccordiSOD1 and C9ORF72 transcripts are currently in development for SOD1-related and C9ORF72-related diseases, respectively, because substantial depletion of these gene products appears achievable without unacceptable adverse effects  are also found in the sporadic ALS-FTD population. Thus, to the extent that defects in condensation may be an important driver of ALS-FTD in both the monogenic and sporadic populations, it remains possible that reversing an ongoing defect in a condensate could mitigate and perhaps even reverse disease progression.The largest unmet need is found in sporadic ALS-FTD cases that are not linked to a specific genetic defect. Substantial evidence suggests a shared pathomechanism in monogenic and sporadic cases of disease, not the least of which is the common feature in late-stage disease of accumulation of RNA-binding protein inclusions composed predominantly of TDP-43. Additional evidence derives from appreciation that genetic modifiers of certain inherited forms of ALS-FTD  can provide an entry point for therapeutic manipulation . Indeed,C (Beyond ALS-FTD, a role for pathological phase transitions is increasingly implicated in other neurodegenerative diseases. The idea that defects in biomolecular condensation may contribute to neurodegeneration more broadly arises from the observation that other important disease-associated proteins, namely tau , α-synucC , are als