C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the  Abnormal expansions of the GGGGCC  of FUS. In contrast, knockdown of endogenous caz, a Drosophila homologue of FUS, enhanced DPR aggregation and RNA foci formation, resulting in the enhancement of repeat-induced degeneration. Moreover, FUS was found to directly bind to G4C2 repeat RNA and modify its G-quadruplex structure as an RNA chaperone, resulting in the suppression of RAN translation in vitro. In addition, other G-quadruplex-targeting RBPs also suppressed RAN translation and G4C2 repeat-induced toxicity in our C9-ALS/FTD flies. These results strongly indicate that FUS regulates RAN translation and suppresses DPR toxicity through modulating the G-quadruplex structure of G4C2 repeat RNA. Our findings shed light on the regulatory mechanisms of RAN translation by G-quadruplex-targeting RBPs and propose novel therapeutic strategies for repeat expansion diseases by regulating RAN translation.Using Drosophila models of C9-ALS/FTD that express pathogenic length 42 or 89 G4C2 repeats  and confirmed that expanded G4C2 repeat sequences induce eye degeneration and motor dysfunction accompanied with the formation of RNA foci and the production of three types of DPRs 9 flies] and found that they did not show eye degeneration, motor dysfunction, RNA foci formation, or DPR aggregation 42 or 89, which show decreased eye size and loss of pigmentation 42 flies and resulted in lethality in (4C2G)89 flies, likely due to the toxicity of TDP-43 expression itself 89 and three RBPs that strongly suppressed eye degeneration. We found that coexpression of IGF2BP1 or hnRNPA2B1 significantly decreased G4C2 repeat RNA levels, whereas they were not altered upon coexpression of FUS 42 or 89 flies coexpressing FUS , which have been reported to eliminate its RNA-binding activity 89, suggesting that the RNA-binding activity of FUS is essential for its suppressive effects on G4C2 repeat-induced toxicity (4C2G)42 flies and confirmed that this phenotype was rescued by coexpression of FUS, but not by coexpression of FUS-RRMmut 42 42 , indicatf TDP-43 . Moreovef TDP-43 . These f4C2 repeat RNA. We performed RNA fluorescence in situ hybridization (FISH) of the salivary glands of fly larvae expressing (4C2G)89 and found that coexpression of FUS significantly decreased the number of nuclei containing RNA foci in (4C2G)89 flies, whereas it was not altered by coexpression of FUS-RRMmut (4C2 repeat RNA in (4C2G)89 flies were not altered by coexpression of FUS or FUS-RRMmut 89 revealed that coexpression of FUS significantly decreased the number of DPR aggregates in (4C2G)89 flies, whereas coexpression of FUS-RRMmut did not (4C2G)89 flies upon coexpression of FUS, but not FUS-RRMmut 44GR-GFP 42 or 89 89 flies 89. FISH analysis of the salivary glands revealed that knockdown of caz significantly increased the number of nuclei containing RNA foci in (4C2G)89 flies (4C2 repeat RNA in (4C2G)89 flies were not altered by the knockdown of caz (caz significantly increased the number of DPR aggregates in (4C2G)89 flies 89 89 , and tha4C2 repeat RNA by the filter binding assay. We found that His-tagged FUS binds to the (G4C2)4 RNA in a dose-dependent manner, but not to the control (AAAAAA)4 RNA (4C2)4 RNA, consistent with a previous study 4 RNA with the G-quadruplex structure in KCl buffer (D) = 1.5 × 10–8 M) and weakly to (G4C2)4 RNA with the hairpin structure in NaCl buffer (D = 1.3 × 10–7 μM). We also confirmed that FUS has poor binding affinity to (G4C2)4 RNA in LiCl buffer (D = 1.4 × 10–5 μM), which destabilizes the G-quadruplex structure (4C2)4 RNA (not shown). These results suggest the preferential binding of FUS to G4C2 repeat RNA with the G-quadruplex structure, which is consistent with a previous report showing preferential binding of FUS to G-quadruplex structured Sc1 and DNMT RNAs (4C2 repeat RNA. The circular dichroism (CD) spectrum of (G4C2)4 RNA in KCl buffer was found to exhibit a positive peak at approximately 260 nm and a negative peak at 240 nm (4C2)4 RNA (4C2)4 RNA. We also observed CD spectrum changes to some extent in the (G4C2)4 RNA upon the addition of FUS in NaCl buffer, but not in LiCl buffer, confirming an interaction between FUS and hairpin-structured (G4C2)4 RNA as well (4C2 repeat RNA by imino proton nuclear magnetic resonance (NMR). In KCl buffer, the NMR signals of the imino proton for the G-quadruplex structure of (G4C2)4 RNA were detected in the region around 10–12 ppm (4C2)4 RNA were decreased in an FUS concentration-dependent manner (4C2)4 RNA. Collectively, these results indicate that FUS directly binds to G4C2 repeat RNA, preferentially to its G-quadruplex form and modulates its higher-order structures. These structural alterations of G4C2 repeat RNAs by FUS did not require ATP or interactions with other proteins, suggesting its role as an RNA chaperone for G4C2 repeat RNA (We next confirmed the direct interaction of FUS with GAA)4 RNA , and Hisus study . We alsoRNA foci , consistRNA foci . Since Gructures , we nexte of Na+ . Surfacetructure , and wasNMT RNAs . ConsideNMT RNAs , we nextt 240 nm . Interes as well . We furt0–12 ppm , consist0–12 ppm . Upon tht manner , furtherpeat RNA .4C2 repeat RNA and its effects on RAN translation, we employed a cell-free in vitro translation assay using rabbit reticulocyte lysate. We designed a reporter construct containing the 80 G4C2 repeat sequence with the 5′ upstream intronic sequence of the C9orf72 gene and the Myc tag sequence in the GA reading frame at the 3′ downstream 89 flies without altering G4C2 RNA expression  domain involved in protein interactions, and exerts multifaceted functions, such as RNA transcription, RNA splicing, RNA transport, and formation of membraneless organelles, such as stress granules and nuclear paraspeckles via liquid–liquid phase separation . Recent 4C2 repeat RNA as an RNA chaperone and regulates RAN translation, resulting in the suppression of neurodegeneration in C9-ALS/FTD fly models. Recent advances in genome sequencing technology unveiled that such expansions of repeat sequences cause more than 50 monogenic human diseases . Female third-instar larvae were used for quantitative real-time polymerase chain reaction (PCR), RNA FISH, and immunohistochemistry experiments. The transgenic fly line bearing the GMR-Gal4 transgene has been described previously , elav-GeneSwitch (#43642), UAS-EGFP (#6874), UAS-DsRed (#6282), UAS-GFP-IR (inverted repeat) (#9330), UAS-(GR)36 (#58692), UAS-(GA)36 (#58693), UAS-(GR)100 (#58696), UAS-(GA)100 (#58697), and UAS-EWSR1 (#79592) were obtained from Bloomington Drosophila Stock Center. The transgenic fly line bearing UAS-caz-IR (#100291) was obtained from Vienna Drosophila Resource Center. The fly line with the caz null allele (2caz), UAS-LDS-(4C2G)44GR-GFP, and UAS-caz (UAS-FLAG-caz) and UAS-FUS-4 (UAS-FLAG-FUS) were kind gifts from Dr. Erik Storkebaum 50 sequences flanked at the 5′ end with an EagI recognition site and at the 3′ end with a PspOMI recognition site were subcloned into T-vector pMD20 (Takara Bio). To generate a longer repeat size, the pMD20-(4C2G)50 vector was digested with EagI and PspOMI, followed by ligation into the pMD20-(4C2G)50 vector linearized by digestion with EagI. This vector was digested with EcoRI and HindIII, and subcloned into the pcDNA3.1/myc-His(−)A vector (Thermo Fisher Scientific). We accidentally obtained the pcDNA3.1/myc-His(−)A-(4C2G)9 vector at this step. The pcDNA3.1/myc-His(−)A-(4C2G)n vector was digested with EcoRI and XbaI, and subcloned into the Drosophila pUAST vector. These constructs have no start codon sequence (ATG) upstream of the G4C2 repeat sequence (4C2G)n vectors were amplified with a recombinase-mutated SURE2 Escherichia coli strain (Agilent Technologies) at 28°C for 72 hr to prevent repeat length contraction. The number of G4C2 repeats in the pUAST-(4C2G)9 or 50 vectors was determined by sequencing. To determine the number of G4C2 repeats in the pUAST-(4C2G)89 vector, transposable element insertional mutagenesis using EZ-Tn5<KAN-2>Insertion Kit (Epicentre) and sequencing were performed. The entire sequence of the insert in the pUAST vector is shown in Artificially synthesized  was used. The human FUS or human TARDBP cDNA was subcloned into the pENTR/D-TOPO vector (Thermo Fisher Scientific). To generate the Gateway destination vector pUAST-DEST, we inserted the Gateway cassette A sequence (Thermo Fisher Scientific) into the pUAST vector. The pUAST-FUS or pUAST-TDP-43 vectors were generated using Gateway recombination reactions (Thermo Fisher Scientific). The FUS RRM mutant construct (pUAST-FUS-RRMmut), in which leucine residues at positions 305, 341, 359, and 368 in the FUS protein were substituted to phenylalanine, was generated by PCR and the In-Fusion Cloning system (Takara Bio). To generate the other pUASTattB-RBP vectors, each cDNA encoding the RBP shown in UAS-(4C2G)n, UAS-FUS, UAS-FUS line 2, UAS-FUS-RRMmut, and UAS-TDP-43, the pUAST-(4C2G)n, pUAST-FUS, pUAST-FUS line 2, pUAST-FUS-RRMmut, and pUAST-TDP-43 vectors, respectively, were injected into fly embryos of the 1118w strain. To establish transgenic flies harboring the other UAS-RBP constructs including UAS-FUS line 3, pUASTattB-RBP vectors were injected into fly embryos of the attP40 strain. By employing site-specific transgenesis using the pUASTattB vector, each transgene was inserted into the same locus of the genome and was expected to be expressed at the equivalent levels. These transgenic flies were established using standard methods at BestGene Inc.To generate pUAST-UAS-(4C2G)9 or 42 transgenic flies was determined by genomic PCR using the forward (5′-AACCAGCAACCAAGTAAATCAAC-3′) and reverse (5′-TGTTGAGAGTCAGCAGTAGCC-3′) primers, which amplifies a part of the UAS-(4C2G)n sequence, including G4C2 repeat sequence, followed by sequencing using the forward (5′-GCCAAGAAGTAATTATTGA-3′) and/or reverse (5′-TCCAATTATGTCACACC-3′) primers.The number of repeats in 4C2 repeat RNA quantification of flies expressing (4C2G)n in ATGAATGGGAGCAGTGGTGG-3′) and reverse (5′-TGTTGAGAGTCAGCAGTAGCC-3′) primers were used. For G4C2 repeat RNA quantification of flies expressing (4C2G)89(H) and FUS, FUS-RRMmut, other RNA-binding proteins, or caz-IR in CCCAATCCATATGACTAGTAGATCC-3′) and reverse (5′- TGTAGGTAGTTTGTCCAATTATGTCA-3′) primers were used. Both of the abovementioned primer pairs recognize sequences downstream of the G4C2 repeats. For gal4 mRNA quantification, the forward (5′-TTGAAATCGCGTCGAAGGA-3′) and reverse (5′-GGCTCCAATGGCTAATATGCA-3′) primers were used 89(H) (4C2G)89(H) and EGFP (4C2G)89(H) and GFP-IR  according to the manufacturer’s instructions. First-strand cDNA was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN). Real-time PCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the Mx3000P Real-time quantitative PCR system (Agilent Technologies) or the CFX96 Real-Time PCR Detection System (Bio-Rad). For Gere used . Data weC2)89(H) , both  with a CCD camera DP21 (Olympus). Images shown are representative eye phenotypes of the fly crosses. Crosses were performed three times to validate the specific phenotypes. Eye size and pigmentation were quantified as previously reported . 5 or 10EGFP , those eFP , 1 (from 2 to 3.9 cm), 2 (from 4 to 5.9 cm), 3 (from 6 to 7.9 cm), 4 (from 8 to 9.9 cm), and 5 (higher than 10 cm). Five trials were performed in each experiment at intervals of 20 s. The assay was performed between 8:00 and 10:00. Climbing scores were calculated as an average of five trials.Drosophila medium (Wako) with RU486 (100 µg/mL) for the indicated periods. Every 2 or 3 d, flies were transferred to new medium with RU486. Climbing assays were performed at 0, 7, and 14 d after the start of RU486 treatment (Flies were crossed in the absence of RU486 (mifepristone) on standard fly food. 1-day-old adult male flies were transferred to Formula 4-24 Instant reatment .2C4)4 or Alexa 488-labeled (C2G4)4 locked nucleic acid (LNA) probe (5 nM) in hybridization buffer. These LNA probes were synthesized by GeneDesign Inc. After the hybridization, samples were washed once for 5 min in 4× SSC at 80°C, three times for 20 min each in 2× SSC and 50% formamide at 80°C, three times for 40 min each in 0.1× SSC at 80°C, and once for 5 min in PBS containing 0.5% Triton X-100 (PBT) at RT. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) or 2'-(4-ethoxyphenyl)–5-(4-methyl-1-piperazinyl)–2,5'-bi-1-H-benzimidazole, trihydrochloride (Hoechst 33342). Stained samples were mounted in SlowFade Gold antifade reagent (Thermo Fisher Scientific) and observed under a Zeiss LSM710 or LSM880 confocal laser-scanning microscope.Female third-instar larvae were dissected in ice-cold phosphate-buffered saline (PBS). Salivary glands were fixed with 4% paraformaldehyde (PFA) (pH 7.0) in PBS for 30 min and incubated in 100% methanol. Fixed samples were rehydrated in 75% (v/v), 50%, and 25% ethanol in PBS, and rinsed in PBS and distilled water (DW). Samples were then treated with 0.2 N HCl/DW for 20 min at room temperature (RT) and rinsed in DW. Next, the samples were permeabilized with 0.2% Triton X-100 in PBS for 10 min, rinsed in PBS for 5 min, fixed again in 4% PFA in PBS for 20 min, then washed twice for 5 min each in PBS, and incubated twice for 15 min each in 2 mg/mL glycine/PBS. After the acetylation treatment, samples were incubated for 1 hr at 37°C in hybridization buffer consisting of 50% formamide, 2× saline sodium citrate (SSC), 0.2 mg/mL yeast tRNA, and 0.5 mg/mL heparin. For hybridization, samples were incubated overnight at 80°C with a 5′ end Alexa 594-labeled . RNA foci-positive nuclei in more than 30 cells per salivary gland were counted, and the percentage of nuclei containing RNA foci in the salivary gland was calculated. Ten salivary glands were analyzed for each genotype.Female third-instar larvae were dissected in ice-cold PBS. Eye imaginal discs and salivary glands were fixed with 4% PFA in PBS for 30 min and washed three times with PBT. After blocking with 5% goat serum/PBT, the samples were incubated overnight at 4°C with a rat monoclonal anti-poly(GR) antibody , a mouse monoclonal anti-poly(GA) antibody , a rabbit polyclonal anti-poly(GA) antibody , a rabbit polyclonal anti-poly(GP) antibody , or a rabbit polyclonal anti-FUS antibody  at 1:1000 dilution as the primary antibody. After washing three times with PBT, the samples were incubated with an Alexa 633-conjugated anti-rat antibody , or an Alexa 488-conjugated or Alexa 555-conjugated anti-rabbit antibody  at 1:500 dilution as the secondary antibody. After washing three times with PBT, nuclei were stained with DAPI or Hoechst 33342. Stained samples were mounted in SlowFade Gold antifade reagent and observed using confocal laser-scanning microscopes .The number of DPR aggregates in the eye discs was quantitatively measured using ZEN imaging software (Zeiss) and ImageJ , as foll4C2G)89(H) and either EGFP, FUS, or FUS-RRMmut using the GMR-Gal4 driver were collected and stored at –80°C. Samples were prepared using a previously reported method (4C2G)89(H) and EGFP 44GR-GFP and either FUS or FUS-RRMmut using the GMR-Gal4 driver were homogenized in 100 µL of sodium dodecyl sulfate (SDS) sample buffer using a pestle, boiled for 5 min, and centrifuged at 10,000 × g for 3 min at 25°C. 5 µL of each supernatant were run on a 5–20% gradient polyacrylamide gel (Wako) and then transferred onto an Immun-Blot polyvinylidene fluoride membrane (Bio-Rad). Membranes were blocked with 5% skim milk in PBS containing 0.1% Tween-20 (PBST) or PVDF Blocking Reagent for Can Get Signal (TOYOBO) for 2 hr at RT, and then incubated overnight at 4°C with a rabbit polyclonal anti-FUS antibody , a rat monoclonal anti-poly(GR) antibody , a Living Colors EGFP mouse monoclonal antibody , or a mouse monoclonal anti-actin antibody  at 1:1000 dilution as the primary antibody. After washing three times with PBST, membranes were incubated for 2 hr at RT with either HRP-conjugated anti-rat, anti-rabbit, or anti-mouse antibody  at 1:5000 dilution as the secondary antibody, washed three times with PBST, treated with SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific), and imaged using the LuminoGraphII imaging system (ATTO). Data were normalized by setting the average values of samples from flies expressing FUS (LDS-(4C2G)44GR-GFP and DsRed as 100  of the plasmid vector pET-15b (Novagen) (E. coli BL21 (DE3), the expression of His-FUS proteins was induced by culturing the transformed cells in the presence of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 20°C for 20 hr. Cells were lysed by ultrasonication in PBS (pH 7.4) containing 2% (v/v) Triton X-100, 1 M NaCl, DNase I, MgSO4, and ethylenediaminetetraacetic acid-free cOmplete Protease Inhibitor Cocktail (Roche Applied Sciences). After centrifugation at 20,000 × g for 30 min at 4°C, the pellets were redissolved in a buffer (pH 7.0) containing 6 M guanidine hydrochloride (GdnHCl), 50 mM Tris, and 1 M NaCl. His-FUS proteins in the pellets were purified by Ni2+ affinity chromatography. In brief, the His-FUS proteins were mixed with Profinity IMAC Ni2+-charged resin (Bio-Rad) for 30 min at 20°C. Then, FUS proteins bound to the resin were washed with wash buffer , and eluted with elution buffer . For preparation of soluble, refolded FUS, FUS proteins (200 μM) in the elution buffer were diluted 20-fold with a buffer (pH 7.0) containing 50 mM Tris, 100 mM NaCl, 10% (v/v) glycerol, and 5 mM Tris (2-carboxyethyl) phosphine (buffer A), which, however, produced significant amounts of precipitate. This insoluble material was removed by centrifugation at 20,000 × g for 10 min at 4°C, resulting in the recovery of soluble FUS proteins in the supernatant fraction. Protein concentrations were spectroscopically determined from the absorbance at 280 nm using the following extinction coefficients: 71,630 cm−1 M−1 for both FUS and FUS-RRMmut.For preparation of FUS proteins with an N-terminal His tag (His-FUS and His-FUS-RRMmut), cDNAs of the human FUS protein (wild type or RRM mutant) from pUAST-Novagen) . After t4C2)4, 10 nM biotin-(AAAAAA)4, or 10 nM biotin-(UUAGGG)4 (telomeric repeat-containing RNA: TERRA) were incubated with soluble FUS proteins  in buffer A with 0.4 U/µL RNase inhibitor . Biotinylated (AAAAAA)4 and TERRA are negative and positive controls, respectively. After an hour at RT, the mixture was filtered through a nitrocellulose membrane  overlaid on a nylon membrane  in a 96-well slot-blot apparatus (ATTO) (Biotinylated RNAs were synthesized by GeneDesign Inc. 10 nM biotin-(Gs (ATTO) . After eFUS (WT) and FUS-RRMmut genes flanked at the 5′ end with an NdeI recognition site and at the 3′ end with a XhoI recognition site was amplified by PCR from pUAST-FUS and pUAST- FUS-RRMmut, respectively. PCR fragments were digested with NdeI and XhoI. These fragments were ligated into the cloning sites of the plasmid vector pET-21b (Novagen) between NdeI and XhoI. After transfection of the plasmids into E. coli BL21 (DE3), expression of the FUS protein was induced by culturing the transformed cells in the presence of 0.5 mM IPTG at 37°C for 6 hr. Cells were harvested by centrifugation and suspended with buffer B –1-piperazineethanesulfonic acid [HEPES]-NaOH [pH 7.0], 300 mM NaCl, 1 mM dithiothreitol [DTT], 1 mM ethylenediaminetetraacetic acid [EDTA], 0.1% Tween-20, and 0.1% benzamidine hydrochloride) containing 1.5 mg/mL lysozyme, and stored for 30 min on ice. Cell lysates were sonicated, and insoluble protein was collected by centrifugation. The pellet was solubilized in buffer C . After centrifugation, supernatants were loaded onto a DE52  open column. The flow-through fraction was loaded onto a CM52  open column. The flow-through fraction of DE52 was applied to a CAPTO S column , and the flow-through fraction was collected using the ÄKTAexplorer 10S/100 system . The flow-through fraction was applied to a Mono S column . Proteins were fractionated with a 0–500 mM linear gradient of NaCl in buffer D  using ÄKTA explorer 10S/100 system. The FUS fraction was eluted at 150–200 mM NaCl. For refolding, the eluted peak fraction was diluted fivefold using refolding buffer , and stored overnight at RT. The solution was concentrated using a centrifugal filter  to 1–2 mg/mL, and then dialyzed against buffer E , and stored frozen at −80°C.For preparation of the FUS proteins, the human 4C2)4 RNA was analyzed using a Biacore T200 instrument . (G4C2)4 RNAs biotinylated at the 5′ end in 10 mM HEPES pH 6.8 and 500 mM MCl  was injected over the streptavidin-coated surface of a sensor chip SA . The amount of immobilized RNA was as follows: 240 resonance unit (RU) in KCl, 363 RU in NaCl, or 319 RU in LiCl buffer condition. Binding experiments were performed using the single-cycle kinetics method. The running buffer used was 20 mM HEPES (pH 6.8), 1 mM MgCl2, 0.05% Tween-20, and 150 mM KCl, NaCl, or LiCl. FUS was diluted in the running buffer and injected sequentially over the RNA-immobilized sensor surface in increasing concentrations . Sensorgrams were obtained at 25°C, 30 µL/min flow rate, 60 s of contact time, and 120 s of dissociation time.The binding of FUS to (G4C2)4 RNA was synthesized by GeneDesign Inc and dissolved in 20 mM HEPES (pH 6.8), 18.75 mM NaCl, 10 mM MgCl2, 0.625% glycerol, 0.625 mM β-cyclodextrin, and 0.0625 mM EDTA with 150 mM KCl, NaCl, or LiCl. RNA samples containing 150 mM KCl were first heated at 95°C for 5 min and then cooled to RT to form the G-quadruplex structure. The other samples were not heated. FUS (1 μM) was added to the RNA sample (4 μM) and mixed before recording the spectrum. CD spectra were recorded at a speed of 50 nm min–1 and a resolution of 1 nm, and 10 scans were averaged.CD spectra were measured at 25°C using a spectropolarimeter model J-820 (JASCO).  at 25°C. (G4C2)4 RNA dissolved in 20 mM HEPES (pH 6.8), 150 mM KCl, 18.75 mM NaCl, 10 mM MgCl2, 0.625% glycerol, 0.625 mM β-cyclodextrin, and 0.0625 mM EDTA was first heated at 95°C for 5 min and then cooled to room temperature to form the G-quadruplex structure. The RNA (10 μM) was mixed with FUS at molar ratios (RNA:FUS) of 1:0, 1:0.2, 1:0.4, and 0:1. The samples were then prepared at a final concentration of 10% D2O before recording their spectra. 1H NMR data were acquired using simple single 90° hard-pulse excitations following solvent signal suppression with a jump-and-return pulse scheme. Free induction decay data  were collected by repeating the scans (2600 times) with an interscan delay of 2.5 s. All NMR data were processed using Topspin 3.6 software (Bruker BioSpin).All one-dimensional C9orf72 intron1-(4C2G)80 vector was digested with HindIII and NotI to obtain the fragment C9orf72 intron1-(4C2G)80 and subcloned into the pcDNA5/FRT vector (Thermo Fisher Scientific). To add the T7 promoter upstream of the C9orf72 intron 1 sequence in this pcDNA5/FRT-C9orf72 intron1-(4C2G)80 vector, a forward primer including T7 promoter sequences with the 5′-terminal region of C9orf72 intron 1 flanked at the 5′ end with an HindIII recognition site, and a reverse primer recognizing the 3′-terminal region of C9orf72 intron 1 sequences including a BssHII recognition site were designed, and used to amplify a fragment containing C9orf72 intron 1 with a T7 promoter by PCR. Then, this fragment was subcloned into the pcDNA5/FRT-C9orf72 intron1-(4C2G)80 vector digested by HindIII and BssHII. In addition to the T7 promoter, the Myc tag in the GA frame downstream of (4C2G)80 was introduced into this vector.For preparation of the C9-RAN reporter plasmid, the pEF6-XbaI. Linearized DNA was in vitro transcribed using mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) according to the manufacturer’s instructions. T7 reactions were carried out at 37°C for 2 hr, treated with TURBO DNaseI for 15 min at 37°C to remove the DNA template, and then polyadenylated with E. coli Poly-A Polymerase (NEB) for 1 hr at 37°C. Synthesized mRNAs were purified by LiCl precipitation. The size and quality of the synthesized mRNAs were verified on a denaturing RNA gel.The reporter plasmids were linearized with C9orf72 intron1-(4C2G)80 with a Myc tag in the GA frame were in vitro translated with Flexi Rabbit Reticulocyte Lysate System (Promega) according to the manufacturer’s instructions. Translation reactions were performed with 10 ng/μL mRNA and contained 30% rabbit reticulocyte lysate, 10 μM amino-acid mix minus methionine, 10 μM amino-acid mix minus leucine, 0.5 mM MgOAc, 100 mM KCl, and 0.8 U/μL Murine RNAse Inhibitor (NEB). FUS or FUS-RRMmut at each concentration  was preincubated with mRNA for 10 min to facilitate the interaction between FUS protein and G4C2 repeat RNA, and added for translation in the lysate. Samples were incubated at 30°C for 90 min before termination by incubation on ice. 10 μL of samples were analyzed by 13% SDS-polyacrylamide gel electrophoresis and western blotting using a mouse monoclonal anti-c-Myc antibody  as the primary antibody.mRNAs of t-test was used for Statistical parameters including the definitions and exact values of n , distributions, and deviations are stated in the figures and corresponding figure legends. Multiple-comparison tests using one-way ANOVA with Tukey’s post hoc analysis were performed for As the sample sizes used in this study were similar to previous publications , statist important study demonstrates that the human FUS protein, which is implicated in ALS and related conditions, interacts with RNAs containing GGGGCC repeats and can regulate their translation by altering three-dimensional structures caused by these repeats. The study is carefully executed and the data provide convincing evidence for its major claims. This work will likely be of interest to researchers studying RNA binding proteins, and to those working on ALS and related diseases.This  Drosophila fly models, and the mechanism appears to include modulating the repeat structure and mitigating RAN translation. They suggest FUS, as well as a number of other G-quadruplex binding RNA proteins, are RNA chaperones, meaning they can alter the structure of the expanded repeat sequence to modulate its biological activities.Overall this is a nicely done study with nice quantitation.This is a carefully performed and well documented study to indicate that the FUS protein interacts with the GGGGCC repeat sequence in  Drosophila. Both FUS and G4C2 repeats of >25 are associated with ALS/FTD spectrum disorders. Thus, these data are important for increasing our understanding of potential interactions between multiple disease genes.Fuijino et al provide interesting data describing the RNA-binding protein, FUS, for its ability to bind the RNA produced from the hexanucleotide repeat expansion of GGGGCC (G4C2). This binding correlates with reductions in RNA foci formation, the production of toxic dipeptides and concomitant reductions in toxic phenotypes seen in (G4C2)30+ expressing  In this manuscript Fujino and colleagues used C9-ALS/FTD fly models to demonstrate that FUS modulates the structure of (G4C2) repeat RNA as an RNA chaperone, and regulates RAN translation, resulting in the suppression of neurodegeneration in C9-ALS/FTD. They also confirmed that FUS preferentially binds to and modulates the G-quadruplex structure of (G4C2) repeat RNA, followed by the suppression of RAN translation. The potential significance of these findings is high, since C9ORF72 repeat expansion is the most common genetic cause of ALS/FTD, especially in Caucasian populations and the DPR proteins have been considered the major cause of the neurodegenerations.1. While the effect of RBP as an RNA chaperone on (G4C2) repeat expansion is supposed to be dose-dependent according to (G4C2)n RNA expression, the first experiment of the screening for RBPs in C9-ALS/FTD flies lacks this concept. It is uncertain if the RBPs of the groups "suppression (weak)" and "no effect" were less or no ability of RNA chaperone or if the expression of the RBP was not sufficient, and if the RBPs of the group "enhancement" exacerbated the toxicity derived from (G4C2)89 RNA or the expression of the RBP was excessive. The optimal dose of any RBPs that bind to (G4C2) repeats may be able to neutralize the toxicity without the reduction of (G4C2)n RNA.2. In relation to issue 1, the rescue effect of FUS on the fly expressing (G4C2)89 (FUS-4) in Figure 4-figure supplement 1 seems weaker than the other flies expressing both FUS and (G4C2)89 in Figure 1 and Figure 1-figure supplement 2. The expression level of both FUS protein and (G4C2)89 RNA in each line is important from the viewpoint of therapeutic strategy for C9-ALS/FTD.3. While hallmarks of C9ORF72 are the presence of DPRs and the repeat-containing RNA foci, the loss of function of C9ORF72 is also considered to somehow contribute to neurodegeneration. It is unclear if FUS reduces not only the DPRs but also the protein expression of C9ORF72 itself.4. In Figure 5E-F, it cannot be distinguished whether FUS binds to GGGGCC repeats or 5' flanking region. Same experiment should be done by using FUS-RRMmut to elucidate whether FUS binding is the major mechanism for this translational control. Authors should show that FUS binding to long GGGGCC repeats is important for RAN translation.5. It is not possible to conclude, as the authors have, that G-quadruplex-targeting RBPs are generally important for RAN translation , without showing whether RBPs which do not affect to (G4C2)89 RNA levels lead to decreased DPR protein level or RNA foci. The following is the authors' response to the original reviews.Reply to Public Reviews:Reply to Reviewer #1:Drosophila fly models, and the mechanism appears to include modulating the repeat structure and mitigating RAN translation. They suggest FUS, as well as a number of other G-quadruplex binding RNA proteins, are RNA chaperones, meaning they can alter the structure of the expanded repeat sequence to modulate its biological activities.This is a carefully performed and well-documented study to indicate that the FUS protein interacts with the GGGGCC repeat sequence in Response: We would like to thank the reviewer for her/his time for evaluating our manuscript. We are very happy to see the reviewer for highly appreciating our manuscript.1. Overall this is a nicely done study with nice quantitation. It remains somewhat unclear from the data and discussions in exactly what way the authors mean that FUS is an RNA chaperone: is FUS changing the structure of the repeat or does FUS binding prevent it from folding into alternative in vivo structure?Response: We appreciate the reviewer’s constructive comments. Indeed, we showed that FUS changes the higher-order structures of GGGGCC [G4C2] repeat RNA in vitro, and that FUS suppresses G4C2 RNA foci formation in vivo. According to the established definition of RNA chaperone, RNA chaperones are proteins changing the structures of misfolded RNAs without ATP use, resulting in the maintenance of proper RNAs folding . Thus, we consider that FUS is classified into RNA chaperone. To clarify these interpretations, we revised the manuscript as follows.(1) On page 10, line 215-219, the sentence “These results were in good agreement with our previous study on SCA31 showing the suppressive effects of FUS and other RBPs on RNA foci formation of UGGAA repeat RNA as RNA chaperones …” was changed to “These results were in good agreement with … RNA foci formation of UGGAA repeat RNA through altering RNA structures and preventing aggregation of misfolded repeat RNA as RNA chaperones …”.(2) On page 17, line 363-366, the sentence “FUS directly binds to G4C2 repeat RNA and modulates its G-quadruplex structure, as evident by CD and NMR analyses , suggesting its functional role as an RNA chaperone.” was changed to “FUS directly binds to G4C2 repeat RNA and modulates its G-quadruplex structure as evident by CD and NMR analyses , and suppresses RNA foci formation in vivo , suggesting its functional role as an RNA chaperone.”Reply to Reviewer #2:Drosophila. Both FUS and G4C2 repeats of >25 are associated with ALS/FTD spectrum disorders. Thus, these data are important for increasing our understanding of potential interactions between multiple disease genes. However, further validation of some aspects of the provided data is needed, especially the expression data.Fuijino et al. provide interesting data describing the RNA-binding protein, FUS, for its ability to bind the RNA produced from the hexanucleotide repeat expansion of GGGGCC (G4C2). This binding correlates with reductions in the production of toxic dipeptides and reductions in toxic phenotypes seen in (G4C2)30+ expressing Response: We would like to thank the reviewer for her/his time for evaluating our manuscript and also for her/his important comments that helped to strengthen our manuscript.Some points to consider when reading the work:1. The broadly expressed GMR-GAL4 driver leads to variable tissue loss in different genotypes, potentially confounding downstream analyses dependent on viable tissue/mRNA levels.Response: We thank the reviewer for this constructive comment. In the RT-qPCR experiments , the amounts of G4C2 repeat transcripts were normalized to those of gal4 transcripts expressed in the same tissue, to avoid potential confounding derived from the difference in tissue viability between genotypes, as the reviewer pointed out. To clarify this process, we have made the following change to the revised manuscript.gal4 transcripts in the same sample” was changed to “The amounts of G4C2 repeat transcripts were normalized to those of gal4 transcripts expressed in the same tissue to avoid potential confounding derived from the difference in tissue viability between genotypes”.(1) On page 30, line 548-550, the sentence “The amounts of G4C2 repeat transcripts were normalized to those of 2. The relationship between FUS and foci formation is unclear and should be interpreted carefully.Response: We appreciate the reviewer’s important comment. We apologize for the lack of clarity. We showed the relationship between FUS and RNA foci formation in our C9-ALS/FTD fly, that is, FUS suppresses RNA foci formation , and knockdown of endogenous caz, a Drosophila homologue of FUS, enhanced it conversely . We consider that FUS suppresses RNA foci formation through altering RNA structures and preventing aggregation of misfolded G4C2 repeat RNA as an RNA chaperone. To clarify these interpretations, we revised the manuscript as follows.(1) On page 10, line 215-219, the sentence “These results were in good agreement with our previous study on SCA31 showing the suppressive effects of FUS and other RBPs on RNA foci formation of UGGAA repeat RNA as RNA chaperones …” was changed to “These results were in good agreement with … RNA foci formation of UGGAA repeat RNA through altering RNA structures and preventing aggregation of misfolded repeat RNA as RNA chaperones …”.(2) On page 17, line 363-366, the sentence “FUS directly binds to G4C2 repeat RNA and modulates its G-quadruplex structure, as evident by CD and NMR analyses , suggesting its functional role as an RNA chaperone.” was changed to “FUS directly binds to G4C2 repeat RNA and modulates its G-quadruplex structure as evident by CD and NMR analyses , and suppresses RNA foci formation in vivo , suggesting its functional role as an RNA chaperone.”Reply to Reviewer #3:In this manuscript Fujino and colleagues used C9-ALS/FTD fly models to demonstrate that FUS modulates the structure of (G4C2) repeat RNA as an RNA chaperone, and regulates RAN translation, resulting in the suppression of neurodegeneration in C9-ALS/FTD. They also confirmed that FUS preferentially binds to and modulates the G-quadruplex structure of (G4C2) repeat RNA, followed by the suppression of RAN translation. The potential significance of these findings is high since C9ORF72 repeat expansion is the most common genetic cause of ALS/FTD, especially in Caucasian populations and the DPR proteins have been considered the major cause of the neurodegenerations.Response: We would like to thank the reviewer for her/his time for evaluating our manuscript. We are grateful to the reviewer for the insightful comments, which were very helpful for us to improve the manuscript.1. While the effect of RBP as an RNA chaperone on (G4C2) repeat expansion is supposed to be dose-dependent according to (G4C2)n RNA expression, the first experiment of the screening for RBPs in C9-ALS/FTD flies lacks this concept. It is uncertain if the RBPs of the groups "suppression (weak)" and "no effect" were less or no ability of RNA chaperone or if the expression of the RBP was not sufficient, and if the RBPs of the group "enhancement" exacerbated the toxicity derived from (G4C2)89 RNA or the expression of the RBP was excessive. The optimal dose of any RBPs that bind to (G4C2) repeats may be able to neutralize the toxicity without the reduction of (G4C2)n RNA.Response: We appreciate the reviewer’s constructive comments. We employed the site-directed transgenesis for the establishment of RBP fly lines, to ensure the equivalent expression levels of the inserted transgenes. We also evaluated the toxic effects of overexpressed RBPs themselves by crossbreeding with control EGFP flies, showing in Figure 1A. To clarify them, we have made the following changes to the revised manuscript.(1) On page 8, line 166-168, the sentence “The variation in the effects of these G4C2 repeat-binding RBPs on G4C2 repeat-induced toxicity may be due to their different binding affinities to G4C2 repeat RNA, and their different roles in RNA metabolism.” was changed to “The variation in the effects of these G4C2 repeat-binding RBPs on G4C2 repeat-induced toxicity may be due to their different binding affinities to G4C2 repeat RNA, and the different toxicity of overexpressed RBPs themselves.”.(2) On page 29, line 519-522, the sentence “By employing site-specific transgenesis using the pUASTattB vector, each transgene was inserted into the same locus of the genome, and was expected to be expressed at the equivalent levels.” was added.2. In relation to issue 1, the rescue effect of FUS on the fly expressing (G4C2)89 (FUS-4) in Figure 4-figure supplement 1 seems weaker than the other flies expressing both FUS and (G4C2)89 in Figure 1 and Figure 1-figure supplement 2. The expression level of both FUS protein and (G4C2)89 RNA in each line is important from the viewpoint of therapeutic strategy for C9-ALS/FTD.Response: We appreciate the reviewer’s important comment. The FUS-4 transgene is expected to be expressed at the equivalent level to the FUS-3 transgene, since they are inserted into the same locus of the genome by the site-directed transgenesis. Thus, we suppose that the weaker suppressive effect of FUS-4 coexpression on G4C2 repeat-induced eye degeneration can be attributed to the C-terminal FLAG tag that is fused to FUS protein expressed in FUS-4 fly line. Since the caz fly expresses caz protein also fused to FLAG tag at the C-terminus, we used this FUS-4 fly line to directly compare the effect of caz on G4C2 repeat-induced toxicity to that of FUS.3. While hallmarks of C9ORF72 are the presence of DPRs and the repeat-containing RNA foci, the loss of function of C9ORF72 is also considered to somehow contribute to neurodegeneration. It is unclear if FUS reduces not only the DPRs but also the protein expression of C9ORF72 itself.Response: We thank the reviewer for this comment. We agree that not only DPRs, but also toxic repeat RNA and the loss-of-function of C9ORF72 jointly contribute to the pathomechanisms of C9-ALS/FTD. Since Drosophila has no homolog corresponding to the human C9orf72 gene, the effect of FUS on C9orf72 expression cannot be assessed. Our fly models are useful for evaluating gain-of-toxic pathomechanisms such as RNA foci formation and RAN translation, and the association between FUS and loss-of function of C9ORF72 is beyond the scope of this study.4. In Figure 5E-F, it cannot be distinguished whether FUS binds to GGGGCC repeats or the 5' flanking region. The same experiment should be done by using FUS-RRMmut to elucidate whether FUS binding is the major mechanism for this translational control. Authors should show that FUS binding to long GGGGCC repeats is important for RAN translation.Response: We would like to thank the reviewer for these insightful comments. Following the reviewer’s suggestion, we perform in vitro translation assay again using FUS-RRMmut, which loses the binding ability to G4C2 repeat RNA as evident by the filter binding assay , instead of BSA. The results are shown in the figures of Western blot analysis below. The addition of FUS to the translation system suppressed the expression levels of GA-Myc efficiently, whereas that of FUS-RRMmut did not. FUS decreased the expression level of GA-Myc at as low as 10nM, and nearly eliminated RAN translation activity at 100nM. At 400nM, FUS-RRMmut weakly suppressed the GA-Myc expression levels probably because of the residual RNA-binding activity. These results suggest that FUS suppresses RAN translation in vitro through direct interactions with G4C2 repeat RNA.Unfortunately, RAN translation from short G4C2 repeat RNA was not investigated in our translation system, although the previous study reported the low efficacy of RAN translation from short G4C2 repeat RNA .Author response image 1.(A) Western blot analysis of the GA-Myc protein in the samples from in vitro translation.(B) Quantification of the GA-Myc protein levels.We have made the following changes to the revised manuscript.(1) Figure 5F was replaced to new Figures 5F and 5G.(2) On page 14-15, line 326-330, the sentence “Notably, the addition of FUS to this system decreased the expression level of GA-Myc in a dose-dependent manner, whereas the addition of the control bovine serum albumin (BSA) did not .” was changed to “Notably, upon the addition to this translation system, FUS suppressed RAN translation efficiently, whereas FUS-RRMmut did not. FUS decreased the expression levels of GA-Myc at as low as 10nM, and nearly eliminated RAN translation activity at 100nM. At 400nM, FUS-RRMmut weakly suppressed the GA-Myc expression levels probably because of the residual RNA-binding activity .”.(3) On page 15, line 330-332, the sentence “Taken together, these results indicate that FUS suppresses RAN translation from G4C2 repeat RNA in vitro as an RNA chaperone.” was changed to “Taken together, these results indicate that FUS suppresses RAN translation in vitro through direct interactions with G4C2 repeat RNA as an RNA chaperone.”.FUS (WT) gene flanked at the 5¢ end with an _Nde_I recognition site and at the 3¢ end with a _Xho_I recognition site was amplified by PCR from pUAST-FUS.” was changed to “For preparation of the FUS proteins, the human FUS (WT) and FUS-RRMmut genes flanked at the 5¢ end with an _Nde_I recognition site and at the 3¢ end with a _Xho_I recognition site was amplified by PCR from pUAST-FUS and pUAST- FUS-RRMmut, respectively.”.(4) On page 37, line 720-723, the sentence “For preparation of the FUS protein, the human (5) On page 41, line 816-819, the sentence “FUS or BSA at each concentration  was added for translation in the lysate.” was changed to “FUS or FUS-RRMmut at each concentration  was preincubated with mRNA for 10 min to facilitate the interaction between FUS protein and G4C2 repeat RNA, and added for translation in the lysate.”.5. It is not possible to conclude, as the authors have, that G-quadruplex-targeting RBPs are generally important for RAN translation , without showing whether RBPs that do not affect (G4C2)89 RNA levels lead to decreased DPR protein level or RNA foci.Response: We appreciate the reviewer’s critical comment. Following the suggestion by the reviewer, we evaluate the effect of these G-quadruplex-targeting RBPs on RAN translation. We additionally performed immunohistochemistry of the eye imaginal discs of fly larvae expressing (G4C2)89 and these G-quadruplex-targeting RBPs. As shown in the figures of immunohistochemistry below, we found that coexpression of EWSR1, DDX3X, DDX5, and DDX17 significantly decreased the number of poly(GA) aggregates. The results suggest that these G-quadruplex-targeting RBPs regulate RAN translation as well as FUS.Author response image 2.G4C2)89 and the indicated G-quadruplex-targeting RBPs.(A) Immunohistochemistry of poly(GA) in the eye imaginal discs of fly larvae expressing ((B) Quantification of the number of poly(GA) aggregates.We have made the following changes to the revised manuscript.(1) Figures 6E and 6F were added.(2) On page 6-7, line 135-137, the sentence “In addition, other G-quadruplex-targeting RBPs also suppressed G4C2 repeat-induced toxicity in our C9-ALS/FTD flies.” was changed to “In addition, other G-quadruplex-targeting RBPs also suppressed RAN translation and G4C2 repeat-induced toxicity in our C9-ALS/FTD flies.”.(3) On page 15, line 344-346, the sentence “As expected, these RBPs also decreased the number of poly(GA) aggregates in the eye imaginal discs .” was added.(4) On page 15, line 346-347, the sentence “Their effects on G4C2 repeat-induced toxicity and repeat RNA expression were consistent with those of FUS.” was changed to “Their effects on G4C2 repeat-induced toxicity, repeat RNA expression, and RAN translation were consistent with those of FUS.”(5) On page 16, line 355-357, the sentence “Thus, some G-quadruplex-targeting RBPs regulate G4C2 repeat-induced toxicity by binding to and possibly by modulating the G-quadruplex structure of G4C2 repeat RNA.” was changed to “Thus, some G-quadruplex-targeting RBPs regulate RAN translation and G4C2 repeat-induced toxicity by binding to and possibly by modulating the G-quadruplex structure of G4C2 repeat RNA.”Drosophila models.” was changed to “We further found that G-quadruplex-targeting RNA helicases, … ,also suppress RAN translation and G4C2 repeat-induced toxicity without altering the expression levels of G4C2 repeat RNA in our Drosophila models.”.(6) On page 19, line 417-421, the sentence “We further found that G-quadruplex-targeting RNA helicases, including DDX3X, DDX5, and DDX17, which are known to bind to G4C2 repeat RNA , also alleviate G4C2 repeat-induced toxicity without altering the expression levels of G4C2 repeat RNA in our Reply to Recommendations For The Authors:1. It is not clear from the start that the flies they generated with the repeat have an artificial vs human intronic sequence ahead of the repeat. It would be nice if they presented somewhere the entire sequence of the insert. The reason being that it seems they also tested flies with the human intronic sequence, and the effect may not be as strong (line 234). In any case, in the future, with a new understanding of RAN translation, it would be nice to compare different transgenes, and so as much transparency as possible would be helpful regarding sequences. Can they include these data?Response: We thank the editors and reviewers for this comment. We apologize for the lack of clarity. We used artificially synthesized G4C2 repeat sequences when generating constructs for (G4C2)n transgenic flies, so these constructs do not contain human intronic sequence ahead of the G4C2 repeat in the C9orf72 gene, as explained in the Materials and Methods section. To clarify the difference between our C9-ALS/FTD fly models and LDS-(G4C2)44GR-GFP fly model , we have made the following change to the revised manuscript.G4C2)44GR-GFP construct was presented in Figure 3—figure supplement 1.(1) Schema of the LDS-n flies were presented in Figure 1—figure supplement 1—source data 1.(2) The artificial sequences inserted in the pUAST vector for generation of the . Thus, we consider that FUS is classified into RNA chaperone. To clarify these interpretations, we revised the manuscript as follows.(1) On page 10, line 215-219, the sentence “These results were in good agreement with our previous study on SCA31 showing the suppressive effects of FUS and other RBPs on RNA foci formation of UGGAA repeat RNA as RNA chaperones …” was changed to “These results were in good agreement with … RNA foci formation of UGGAA repeat RNA through altering RNA structures and preventing aggregation of misfolded repeat RNA as RNA chaperones …”.(2) On page 17, line 363-366, the sentence “FUS directly binds to G4C2 repeat RNA and modulates its G-quadruplex structure, as evident by CD and NMR analyses , suggesting its functional role as an RNA chaperone.” was changed to “FUS directly binds to G4C2 repeat RNA and modulates its G-quadruplex structure as evident by CD and NMR analyses , and suppresses RNA foci formation in vivo , suggesting its functional role as an RNA chaperone.”Besides these RNA chaperones, we observed the expression of IGF2BP1, hnRNPA2B1, DHX9, and DHX36 decreased G4C2 repeat RNA expression levels. In addition, we recently reported that hnRNPA3 reduces G4C2 repeat RNA expression levels, leading to the suppression of neurodegeneration in C9-ALS/FTD fly models . We speculate these RBPs could be involved in RNA decay pathways as components of the P-body or interactors with the RNA deadenylation machinery , possibly contributing to the reduced expression levels of G4C2 repeat RNA. To clarify these interpretations, we revised the manuscript as follows.(3) On page 18, line 392-398, the sentences “Similarly, we recently reported that hnRNPA3 reduces G4C2 repeat RNA expression levels, leading to the suppression of neurodegeneration in C9-ALS/FTD fly models . Interestingly, these RBPs have been reported to be involved in RNA decay pathways as components of the P-body or interactors with the RNA deadenylation machinery , possibly contributing to the reduced expression levels of G4C2 repeat RNA.” was added.4. What is the level of the G4C2 repeat when they knock down caz? Is it possible that knockdown impacts the expression level of the repeat? Can they show this (or did they and I miss it)?Response: We thank the editors and reviewers for this comment. The expression levels of G4C2 repeat RNA in (G4C2)89 flies were not altered by the knockdown of caz, as shown in Figure 4G.5. A puzzling point is that FUS is supposed to be nuclear, so where is FUS in the brain in their lines? They suggest it modulates RAN translation, and presumably, that is in the cytoplasm. Is FUS when overexpressed now in part in the cytoplasm? Is the repeat dragging it into the cytoplasm? Can they address this in the discussion? If FUS is never found in vivo in the cytoplasm, then it raises the point that the impact they find of FUS on RAN translation might not reflect an in vivo situation with normal levels of FUS.Response: We appreciate these important comments. We agree with the editors and reviewers that FUS is mainly localized in the nucleus. However, FUS is known as a nucleocytoplasmic shuttling RBP that can transport RNA into the cytoplasm. Indeed, FUS is reported to facilitate transport of actin-stabilizing protein mRNAs to function in the cytoplasm . Thus, we consider that FUS binds to G4C2 repeat RNA in the cytoplasm and suppresses RAN translation in this study.6. When they are using 2 copies of the driver and repeat, are they also using 2 copies of FUS? These are quite high levels of transgenes.Response: We thank the editors and reviewers for this comment. We used only 1 copy of FUS when using 2 copies of GMR-Gal4 driver. Full genotypes of the fly lines used in all experiments are described in Supplementary file 1.7. In Figure5-S1, FUS colocalizing with (G4C2)RNA is not clear. High-magnification images are recommended.Response: We appreciate this constructive comment on the figure. Following the suggestion, high-magnification images are added in Figure 5—figure supplement 1.8. I also suggest that the last sentence of the Discussion be revised as follows: Thus, our findings contribute not only to the elucidation of C9-ALS/FTD, but also to the elucidation of the repeat-associated pathogenic mechanisms underlying a broader range of neurodegenerative and neuropsychiatric disorders than previously thought, and it will advance the development of potential therapies for these diseases.Response: We appreciate this recommendation. We have made the following change based on the suggested sentence.(1) On page 20-21, line 455-459, “Thus, our findings contribute not only towards the elucidation of repeat-associated pathogenic mechanisms underlying a wider range of neuropsychiatric diseases than previously thought, but also towards the development of potential therapies for these diseases.” was changed to “Thus, our findings contribute to the elucidation of the repeat-associated pathogenic mechanisms underlying not only C9-ALS/FTD, but also a broader range of neuromuscular and neuropsychiatric diseases than previously thought, and will advance the development of potential therapies for these diseases.”.Authors’ comment on previous eLife assessment:Drosophila expressing human FUS in vivo, and the recombinant human FUS protein in vitro. To validate that FUS functions as an endogenous regulator of RAN translation, we additionally evaluated the function of Drosophila caz protein as well. We are afraid that the first sentence of the eLife assessment, that is, “This important study demonstrates that the Drosophila FUS protein, the human homolog of which is implicated in amyotrophic lateral sclerosis (ALS) and related conditions, …” is somewhat misleading. We would be happy if you modify this sentence like “This important study demonstrates that the human FUS protein, which is implicated in amyotrophic lateral sclerosis (ALS) and related conditions, …”.We thank the editors and reviewers for appreciating our study. We mainly evaluated the function of human FUS protein on RAN translation and G4C2 repeat-induced toxicity using


Correction: Environ Health 21, 99 (2022)https://doi.org/10.1186/s12940-022-00912-5Following the publication of the original article , a mistaThe daily mean temperature data was mislabelled during the analysis—the temperature data in the West was mislabelled as North, the temperature data in the East was mislabelled as West and the temperature data in the North was mislabelled as East.All changes after and before the correction are presented Table Figures


Adenosine-to-inosine RNA editing is essential to prevent undesired immune activation. This diverse process alters the genetic content of the RNA and may recode proteins, change splice sites and miRNA targets, and mimic genomic mutations. Recent studies have associated or implicated aberrant editing with pathological conditions, including cancer, autoimmune diseases, and neurological and psychiatric conditions. RNA editing patterns in cardiovascular tissues have not been investigated systematically so far, and little is known about its potential role in cardiac diseases. Some hints suggest robust editing in this system, including the fact that ADARB1 (ADAR2), the main coding-sequence editor, is most highly expressed in these tissues. Here we characterized RNA editing in the heart and arteries and examined a contributory role to the development of atherosclerosis and two structural heart diseases -Ischemic and Dilated Cardiomyopathies. Analyzing hundreds of RNA-seq samples taken from the heart and arteries of cardiac patients and controls, we find that global editing, alongside inflammatory gene expression, is increased in patients with atherosclerosis, cardiomyopathies, and heart failure. We describe a single recoding editing site and suggest it as a target for focused research. This recoding editing site in the IGFBP7 gene is one of the only evolutionary conserved sites between mammals, and we found it exhibits consistently increased levels of editing in these patients. Our findings reveal that RNA editing is abundant in arteries and is elevated in several key cardiovascular conditions. They thus provide a roadmap for basic and translational research of RNA as a mediator of atherosclerosis and non-genetic cardiomyopathies. The human genetic code is highly preserved, yet RNA editing is a process that changes it in RNA molecules. This may alter the resultant proteins or the properties of the RNA strands themselves, leading to changes in their special structure and affecting their interaction with the immune system. Unedited RNA may be highly immunogenic, and studies of recent years demonstrated that dysregulated editing causes an inflammatory response. We show that human RNA editing is especially active in the arteries and investigate its role in cardiovascular diseases such as atherosclerosis and cardiomyopathies. These diseases have an underlying inflammatory component that eventually leads to the loss of normal structure and function. We show that RNA editing is increased in patients and draw a line connecting it with a concomitant increase in inflammatory pathways. We also detected specific coding editing sites, mainly in the IGFBP7 gene, where aberrant editing is present, resulting in an altered protein product. We delineate these changes and suggest they may contribute to or are a marker of the underlying pathology. Our findings indicate that RNA editing is a key player in several cardiovascular diseases. As a result of editing, the RNA sequence is modified from its genomic blueprint, and genomically-encoded adenosines on the RNA molecule are deaminated into inosines [RNA editing is an endogenous post-transcriptional process, catalyzed by the Adenosine deaminases acting on RNA (ADAR1 and ADAR 2) enzymes. ADAR1 has 2 isoforms, p110 and Editing occurs in both coding and non-coding sequences of the genome. Editing of a coding sequence may result in amino-acid substitution in the protein product ("recoding"), similar to genomic mutations. Some recoding events have an enormous functional impact. For example, recoding of the Q/R site within the Glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) gene is essential to prevent an inborn fatal phenotype in mice ,4, and eAlu elements reside close to one another in the same RNA transcript but with the opposite orientation. Double-stranded RNA molecules are potent immune stimulators that the intracellular sensor melanoma differentiation-associated protein 5 (MDA5) and mitochondrial antiviral signaling protein (MAVS) recognize [However, virtually all the editing occurs in non-coding repetitive elements, e.g., the human Alu sequences (Alu editing). The current view is that the primordial role of RNA editing is to prevent the identification of endogenous double-stranded RNA structures by the innate immune system. Such structures can be formed when two repetitive ecognize .Editing introduces mismatches between the two RNA strands and disrupts otherwise perfect complementation, the hallmark of viruses –9. This Exceptionally high levels of A-to-I editing were demonstrated in arteries –16, and atherosclerotic-related diseases such as ischemic heart disease (IHD) and cardiomyopathy (ICM), are the most common causes of morbidity and mortality in the western world . They arIt is increasingly appreciated that atherosclerosis is also a result of vascular inflammation driven by the IFN, IL-1-IL-6 pathway. A number of large-scale clinical trials ,19, demoAlus is associated with heightened interferon activity and disease prevalence. Common genetic variants that are associated with RNA editing were recognized to be enriched in GWAS signals of autoimmune and immune-related diseases, including CAD, and accounted for a high fraction of disease heritability.A very recent, pivotal work by Li et al  has demoWe hypothesized that since RNA editing is associated with inflammation, aberrant editing might contribute to the development of either atherosclerosis or non-genetic cardiomyopathies. We additionally speculated that a single editing disruption at a critical recoding site may adversely affect cellular function and lead to a disease, as reported with FLNA editing and cardiomyopathy .p150 expression, resulting in elevated editing in cardiovascular pathologies.Here we analyzed RNA sequencing data from the GTEx project, encompassing thousands of human samples, as well as additional datasets of cardiomyopathy patients. We observed an increase in both intronic and recoding editing in atherosclerosis patients, with a particular increase in Insulin-like growth factor binding protein 7 (IGFBP7) gene editing. Similarly, we observed increases in intronic editing in patients with various types of cardiomyopathies. We have confirmed that these pathologies are accompanied by increased expression of inflammatory genes . FinallyGTEx patients were classified according to their medical records and the medical examiners’ diagnoses. The atherosclerosis group contained all patients who died from or had a history of myocardial infarction or ischemic heart disease. The cerebrovascular group contained patients who either died from or had a history of cerebrovascular accident (CVA), intracranial hemorrhage (ICH), or cerebral aneurysm. The control group contained all other individuals not included in the atherosclerosis and cerebrovascular groups, with the exception of patients who died from an overwhelming infection who were removed from the analysis.Transcriptomic data quality was evaluated using the FastQC quality control tool version 0.10.1 . The reaWe used Salmon tool version 0.11.2 to quantify the transcript levels of the RNA-seq data in units of transcripts per million (TPM)  using deAlu repeats [Alu elements, we used the Alu Editing Index (AEI) method version 1.0 [Alu over adenosine and guanosine at the same genomic locus. A higher index indicates a greater percentage of editing activity in a sample. To clean the editing signal further, we analyzed an additional subset of 3,031 genomic Alus that are located within 3’ UTR of genes, have a minimum length of 250bp, and have an oppositely oriented Alu on the same 3’ UTR.Most editing in humans takes place within  repeats . To meassion 1.0 . BrieflyWe used the REDIToolKnown script to measure A-to-I RNA editing levels in coding editing sites . The scrTo evaluate IFN signaling activity modifications, we first calculated the gene expression levels of the interferon genes using the Salmon tool version 0.11.2  with defWe used “Trimmomatic” (version 0.33) with the parameters: -phred33 LEADING:3 TRAILING:3 MINLEN:20 to remove the Illumina trueseq adapters from each ribo-seq fastq format file and a “bowtie” aligner (version 1.2.3) with default parameters to index the human hg38 genome and align fastq files to SAM format. To avoid contamination, we used “FastqScreen” (version 0.14.0) to filter out reads that aligned to the rRNA transcriptome. In order to optimize alignment, we added the -v 2 parameter, which permits a maximum of 2 mismatches in each read while aligning. We sorted the SAM with “Picard” (version 2.5.0) SortSam with the parameter SORT_ORDER = coordinate. We indexed the Bam file using Picard BuildBamIndex and default parameters.All values in graphs are expressed as the mean ± SEM. Normality was tested with the Shapiro-Wilk normality test. If the data exhibited a normal distribution, we performed pairwise testing with two-sided, unpaired, Student’s t-test, and multiple group comparisons with a 2-way ANOVA followed by Tukey post-test. For non-normally distributed data or N < 30, we performed pairwise testing using the two-sided non-parametric Mann-Whitney U test with multiple group comparisons by the non-parametric Kruskal-Wallis test, followed by Dunn’s post-test. Corrections for multiple comparisons were made by the Benjamini–Hochberg false discovery rate (FDR) multiple testing procedure.All tests and analyses were performed using “R” version 3.5.3 (R Core Team 2014) and Rstudio, an integrated development environment for R, version 1.3.1093. Figures were drawn with R, Microsoft Excel, and Adobe Illustrator (version 24.0.1).A major resource of human RNA-seq data is the GTEx cohort , which iAlu editing was the highest in the arteries , but not in the heart . However, the scope of editing in coding sequences is unknown.A previous analysis of the extensive GTEx dataset , which iHere we analyze the same dataset to compare the level of editing within the coding region across tissues. We extended the previously published Recoding Editing Index  to a CodBoth the arteries and the brain have highly edited recoding sites. However, unlike the brain, the sites in the arteries are strongly expressed, leading to a much higher number of edited transcripts.To illustrate this point, we compared the total number of edited transcripts, averaged over donors. Summing over the 314 evaluated coding sites see , the numInterestingly, the editing profile across all 314 coding sites reveals tissue-specific patterns that largely reflect the anatomy and function of cardiovascular tissues. We ran a PCA based hierarchical clustering analysis and found that arteries cluster separately from the LV and LAA as two distinct groups. Within the arteries, the aorta and coronaries share a similar pattern that is distinct from that of the tibial artery . This suAn additional difference we discovered between the brain and the cardiovascular tissues is that cardiovascular editing is highly centralized- i.e., a small number of sites account for the majority of recoding editing. To illustrate this point, we inspected the distribution of editing in specific genes. We found that the five most edited targets in cardiovascular tissues are IGFBP7, FLNA, NEIL1, CCNI, and SRP9. Together, these five sites account for 92%, 91%, 90%, 64%, and 68% of all coding editing activity in the aorta, coronary arteries, tibial artery, LV, and LAA, respectively. The markedly different behavior of recoding activity between the tissues is due to both elevated expression and higher editing levels of the abovementioned few recoding sites. In contrast, the top five edited sites in the cerebellar hemisphere account for only 29% of the editing activity .Recoding in cardiovascular tissues is focused on a few editing sites, in stark contrast to the complex recoding profile in the brain. This difference may be partly attributed to differences in the cellular composition of each tissue. Brain tissues exhibit diverse cellular populations , while tNotably, mutations and altered expression of IGFBP7 and FLNA have been observed in various cardiovascular diseases ,33–39.A medical record of each GTEx donor is available upon request. We used this information to further analyze the GTEx cohort and divided its donors into three groups of interest: patients with atherosclerosis (n = 104), patients with cerebrovascular disease , and controls . Linear regression analysis revealed that the Alu editing is significantly correlated to both ADAR1 expression and the ISG score, with a positive interaction term (p for interaction = 0.004)  .Alu editing  in disease. Analyses were performed separately for each of the cohorts. We found that coding editing was increased in atherosclerotic cardiac diseases and decreased in cerebrovascular diseases, reminiscent of what we observed in  editing . 34 codiAlthough the cohort’s sizes of ICM and DCM were relatively small, we could still detect three differentially edited sites within the IGFBP7, Copper-exporting P-type ATPase (COPA), and Conserved oligomeric Golgi complex subunit 3 (COG3) transcripts that exhibited a substantial increase . These sIn order to examine whether the recoded RNA transcripts were in fact being translated into proteins, bearing a potential functional impact, we analyzed ribosome profiling data  of left Cardiac markers of injury greatly facilitate the management of ischemic and heart failure patients, and novel markers are constantly sought after. IGFBP7 can be sampled from the peripheral blood and serum levels of this protein increase in atherosclerosis and heart failure with preserved ejection fraction and correlate with cardiac function ,52–55.Our analysis demonstrates that the editing of IGFBP7 is markedly increased with cardiac or vascular injury . Integrating both IGFBP7 serum levels and edited isoform fraction may thus further increase its diagnostic performance without the need for sequencing, rendering it an accessible and potentially useful novel clinical marker. This can be done, for instance, by sampling peripheral blood and utilizing specific antibodies for the edited and non-edited versions of IGFBP7.Alu and IGFBP7 has a predictive value that is comparable to the expression of top heart failure markers, such as troponin subtypes, atrial natriuretic peptide (ANP), and B-type natriuretic peptide . We next wanted to assess how increased editing translated into a potentially clinically useful test. We tested this by comparing the predictive value of Alu editing and IGFBP7 editing with expression-based levels of common clinical markers of cardiac injury. We found that, in the cardiomyopathies, editing of both Advancements in diagnostics and therapeutics of cardiovascular illnesses rely on a deeper understanding of the underlying cellular processes. RNA editing is highly abundant in vascular tissues, is tissue-specific, and participates in pathways that contribute to cardiovascular diseases, such as inflammation and circular RNA regulation. Li et al. recently pointed a spotlight on RNA editing demonstrating it is a major mediator of the genotypic effects on inflammatory diseases ., includRNA editing appears to interfere with several relevant mechanisms. Jain et al.  recognizHere we present for the first time a comprehensive analysis of RNA-editing in the cardiovascular system. We found that the highest activity of editing in both coding and non-coding sequences occurs in the cardiovascular system. Patients with coronary or structural heart disease demonstrated yet higher levels of editing compared with controls. Conversely, editing decreases in patients with cerebral vascular accidents or hemorrhages but no heart disease. These opposing trends may represent differences in etiology , or else the aberrant editing may serve as an etiology.p150 expression is strongly influenced by IFN. Accordingly, we observed a close correlation between inflammation and increased ADAR1 expression and Alu editing in ischemic and dilated cardiomyopathies. Interestingly, Alu editing is most increased in ICM, where inflammation may mediate both the initial pathology and subsequent scar formation. It is possible that RNA editing represents a new inflammation-driving mechanism that is highly active in the arteries. Importantly, attempts are currently made to both activate and inhibit the editing machinery for therapeutic purposes. This may render RNA editing an actionable target.In recent years, it has become accepted that inflammation plays a central role in conditions such as atherosclerosis and the resulting ischemic cardiomyopathy –60. AlthIt is important to note that RNA editing is present and maybe upregulated even in the absence of inflammation. Alu editing was increased in the GTEx data analysis of patients with atherosclerosis and cerebrovascular disease, despite normal ISG scores. It is possible that the lack of correlation between the ISG score and Alu editing in this cohort is due to the nature of the GTEx population, which comprises mainly deceased donors, and where tissue ischemia and other perimortem changes may affect gene expression and blur an analysis that relies on differential expression. Given this potential caveat, it is noteworthy that the Alu editing in this population still differentiates patients from controls, testifying to the robustness of the measurement. Another possible mechanism by which altered editing promotes structural heart diseases and atherosclerosis is through effects on the formation of circRNA. An increase in RNA editing in intronic regions has been shown to reduce circRNA levels . This reOur results identified several specific coding sites with prominent differences between patients and controls. These recoding changes are enriched for relevant biological processes. For example, in left ventricle samples, we observed an enrichment of genes controlling actin and cytoskeleton polarity, a process that plays a crucial role in cardiomyopathy and heart failure. Similarly, we observed enrichment in atrial cell membrane repolarization regulation in the left atrial appendage -a process that may be involved in arrhythmias and thrombus formation.Finally, a primary site of interest for cardiovascular editing is within the IGFBP7 gene. This gene is highly expressed in cardiovascular tissues and contains two evolutionarily conserved editing sites. We observed altered editing in the major editing site in atherosclerosis patients. Editing in this site in cardiovascular tissues is exceptionally high, surpassing 95%, and constitutes a sizable proportion of all recoding editing in the heart and arteries. The mechanism by which the observed increase in editing of IGFBP7 influences pathogenesis is unclear. It is possible that editing of this highly expressed cardiovascular gene is a result of the globally increased ADAR1-mediated editing. However, some indirect evidence suggests that this site is edited by ADAR2 and is not so much susceptible to ADAR1 overexpression.IGFBP7 has already been suggested as a biomarker of atherosclerosis and heart failure ,55, and The main limitation of our study is its in-silico nature, which naturally precludes the ability to infer causality and mandates further biological experiments.Another limitation is the use of publicly available data, where complete details on the donors are sometimes lacking, along with a lack of control over the data production process. For example, no genotypic information was available in the cardiomyopathies datasets. This limitation is common in bioinformatical analysis in general, and we addressed this issue by carefully selecting datasets with high-quality and maximal annotations.We made every effort to use reproducible, meticulous analysis of the data and to use well-established and consistent statistical approaches to maximize the robustness of our findings.RNA editing in both coding and non-coding regions is increased in atherosclerosis, ischemic, and dilated cardiomyopathies. It is partially correlated with increased inflammatory signals and thus might be involved in maladaptive inflammatory remodeling. RNA editing is not fully explained by increased inflammation and may represent a discrete additional process.S1 FigEndogenous dsRNA structures are formed naturally and may activate the inflammation pathway. To prevent false activation of the immune system, ADAR1 disrupts endogenous dsRNA structures by editing adenosines to inosines. Mitochondrial Antiviral Signaling (MAVS); Melanoma Differentiation-Associated protein 5 (MDA5); Interferon Stimulated Genes (ISG); Adenosine Deaminase Acting on RNA 1 (ADAR1).(JPG)Click here for additional data file.S2 FigAlu sequences in (a) ASCVD patients, and hypo-editing in cerebrovascular patients . Increased editing levels are also observed in (b) CMP  and (c) ICM  . Note that due to differences in read length, the nominal index values cannot be compared between the two panels.The AEI demonstrates consistent increased editing levels of all (JPG)Click here for additional data file.S3 FigSpearman correlation rho = 0.48, 0.58 and 0.45; p-value = 1.5e-06, 1.2e-09 and 5.9e-06, for ADAR1 expression vs. ISG score, ADAR1 vs. Alu editing and Alu editing vs. ISG, respectively). Linear regression analysis revealed that the Alu editing is significantly correlated to both ADAR1 expression and the ISG score, with a positive interaction term (p for interaction = 0.004)(JPG)Click here for additional data file.S4 FigLevels of A-to-I editing in left ventricle DCM patients from RNA-seq (n = 37) and Ribo-seq (n = 30) datasets. Cutoff of ≥ 10 reads for each site.(JPG)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(CSV)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps – 1) immobilization of a single cell on solid substrate, 2) hypotonic lysis of immobilized single cell, and 3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression. Arguably, single cell-based approaches played the most important role in our progress in study of somatic mutation as they allow us to accurately assess random somatic mutations 4. These studies clearly demonstrated that a healthy somatic cell contains several hundred unique mutations, a number which increases due to multiple factors over the course of life and reaches thousands towards the end of life 6. However, despite evidence of increasing mutations during aging, it still remains unclear whether these changes in genome are translated to changes in cellular function. It has become apparent now that complete understanding of these mechanisms is only possible by comprehensive analysis of changes in both genome and transcriptome of a single cell, the only methodology that allows the establishment of direct links between mutations and their functional implementation.The advent and rapid development of single cell sequencing technologies permitting fast and accurate analysis of individual whole genomes resulted in an avalanche of experimental evidence on the pivotal role of somatic mutations in human aging and diseases such as cancer 9. An alternative approach was implemented by simultaneous isolation of genomic DNA and total RNA (SIDR) where RNA-containing cytoplasm is released into aqueous phase by hypotonic lysis and then separated from the immobilized nucleus by simple aspiration 10. However, practical application of SIDR is limited by availability of cell-surface specific antibodies conjugated to magnetic beads. Here we introduce a simple modification to SIDR allowing isolation and parallel analysis of DNA and RNA from a single cell using the CellRaft AIR system by Cell Microsystems as a mean to immobilize individual cells prior to lysis. We demonstrate that DNA and RNA separated with the modified approach are suitable for downstream applications and that this procedure does not introduce any significant bias to the results of DNA and RNA analysis.There are several approaches available for separation and further analysis of DNA and RNA contents of one single cell. Many of these methods are based on capture of mRNA molecules using oligo-dT probes followed by immobilization and separation of captured transcripts from genomic DNA Isolation of primary human IMR90 fibroblasts was conducted using the automated Air Raft system by Cell Microsystems. This machine allows direct visualization and identification of cells plated and adhered to the CytoSort Array, composed of thousands of microwells . The CellRafts are magnetic and can be individually selected and released from the array. When an individual cell is identified on the surface of a CellRaft, it is then picked and transferred to a PCR tube using a magnetic wand. Once deposited into tube, the individual cell is then exposed to the hypotonic lysis buffer (HLB) to disrupt the cell membrane and release cytoplasm into aqueous phase as described previously by others 10. The DNA-containing nucleus remains embedded into cytoskeleton and attached onto the CellRaft. Next, the tube is placed on a magnetic stand to immobilize the CellRaft. The RNA-containing supernatant is then aspirated and transferred to a separate tube, leaving the DNA-containing nucleus in the original tube . With thSupp. Figure 2). Finally, we assessed the total number of genes detected in single cells as well as the number of genes shared between single cells and the bulk and did not see a significant difference between separated and naïve cells . We also constructed RNA-seq libraries from intact single cells, without DNA/RNA separation, as a control. In total we analyzed 3 native cells without separation and 3 cells after DNA/RNA separation procedure. First, we tested if separation procedure affects relative representation of different transcripts in RNA-seq data. We reasoned that single-cell transcriptomes combined in one pseudo-bulk should provide a set of data similar to the bulk RNA-seq of thousands of cells and, hence, may serve as a measure for accuracy of transcriptome assessment. We composed two pseudo-bulk data sets, one containing single cell RNA-seq data after DNA/RNA separation and the other without that. Only genes with at least 3 counts in each analyzed cell were included in pseudo-bulks. We found that these two pseudo-bulks have similar levels of correlation with RNA-seq data . We performed locus drop-out test (LDO), a qPCR-based approach measuring relative representation of 8 different targets distributed across the genome 3, to assess the uniformity of the whole genome amplification.. The LDO test also did not reveal any significant differences in quality of WGA products with and without DNA/RNA separation  using our scMDA approach paration .Supp. Figure 3).We also did not observe a statistically significant difference in the rates of genome coverage in single cell genome sequencing data obtained from cells with and without DNA/RNA separation procedure  . Also, nTo further investigate a possible separation effect in more detail, we assessed the mutational spectra of sepaterated and non-separated cells. The distribution of SNVs did not change significantly in separated cells compared to non-separated cells . MoreoveHere we present a simplified method for DNA/RNA separation that allows for simultaneous investigation of gene expression profiles and mutation patterns in the same single cell. When comparing bulk RNA-seq data and pseudo-bulks compiled from single-cell RNA-seq results of cells with and without DNA/RNA separation, we found similar levels of correlation. In addition, the DNA/RNA separation procedure did not affect the number of identified genes in the single-cell RNA-seq or the proportion of identified genes overlapping with the bulk RNA-seq.Analysis of single cell WGS results revealed similar levels of observed somatic mutations, both SNV and small INDELs, in genomic DNA after separation from RNA and without separation. Spectra of identified somatic SNVs as well as distribution of insertions and deletions also remained similar between these two cohorts. While we observed more variance in genomic coverage in the separated cells than in the non-separated ones, the resulting WGS data was well qualified for further analysis and accuracy of variant calling was not affected.11 allowing to directly investigate the regulation of gene expression in a particular cell in a comprehensive manner. Taken together we present a powerful tool for studying single cells for a refined insight on processes in any given cell.Thus, we described a simplified method for single cell DNA/RNA separation allowing their concurrent analysis and demonstrated that the separation procedure per se does not influence observed gene expression patterns, mutation frequency and spectra. Of note, the manual version of the CellRaft method is also available that makes this approach applicable in virtually any laboratory. These open up new possibilities to study the direct relation between mutations and their effect on gene expression in a wide range of cells. Moreover, we also successfully used the DNA fraction as input for bisulfite treatment for the assessment of DNA methylation pattern after a previously published protocol for single cells Commercially available IMR90 human lung fibroblasts (ATCC) were cultured in EMEM (ATCC) supplemented with 10% FBS (Gibco) and 1% Pen/Strep (Gibco) at 37°C, 3% O2 and 10% CO2. Cell viability, apoptosis rate and cell number were assessed with the Guava EasyCyte cytometer. The CellRaft array was prepared following the manufacturer’s instruction and washed three times for 3 minutes with warm PBS before addition of cell suspension. 1000 cells were seeded in 1ml for one CellRaft array. Cells were allowed to settle for 3–4 hours at 37°C before isolation. Before isolation the raft was carefully washed with warm medium to remove potential debris and dead cells.10. Tubes containing cells and HLB were briefly spun and incubated for 10 minutes at room temperature. 2 minutes before incubation time was over, tubes were briefly spun again and placed in magnetic stand. The supernatant was transferred to a new tube and placed immediately on dry ice. 2.5 μl PBS were added to the remaining cell raft and then also placed on dry ice. Tubes with DNA and RNA respectively were stored at −80°C until further processing.Single cells were isolated using the CellRaft AIR system (Cell Microsystems) and individual rafts containing one cell were deposited in PCR tubes with 2.5ul PBS. After isolation, tubes were quickly checked under a microscope to ensure proper deposition of the raft into the tube. Then tubes were put on wet-ice and 9 μl freshly prepared hypotonic lysis buffer (HLB) was added. The HLB composition was 0.2% Triton X-100 (Sigma-Aldrich) and 0.5% RNAse Inhibitor (Takara) in nuclease-free water 4. For quality control purposes, a locus drop out test (LDO) was performed using primers and SYBR reagent 12. Library size was assessed with a TapeStation (Agilent) using HS5000 screen tape for quality control of cDNA and HS1000 screen tape for all final libraries. All RNA-Seq and WGS libraries were sequenced on an Illumina HiSeq platform by Novogene Corp Inc., CA.For RNA sequencing, cDNA was prepared using the Smart seq 4 kit (Takara) using manufacturer recommended protocol. Briefly, the RNA of the cytosolic fraction(11μl) was used as input for conversion. The product was quantified via Qubit (ThermoFisher Scientific) and quality was assessed using a HS5000 screen tape on the TapeStation (Agilent). Following this, sequencing libraries were prepared utilizing the Nextera XT kit (Illumina) with a 4minute tagmentation step and 13 cycles of amplification followed by a purification using AMPure beads (Beckman Coulter). Libraries for whole genome sequencing (WGS) were prepared according to the previously described scMDA protocol using a NEBNext Ultra II FS kit (NEB) 13 and quality control performed using FastQC 14, STAR 15 was used for alignment, duplicates were removed using Picard (http://broadinstitute.github.io/picard/) and RSEM 16 was used for quantification. Gene level counts and effective lengths were extracted using tximport 17 into the R environment. Genes with evidence of consistent expression, those with at least 3 counts in all bulk samples or single cells of the same condition, were used for downstream analysis. Pseudo bulk samples were created by summing counts across cells of the same condition. Transcripts per million (TPM) were calculated for bulk and pseudo bulk samples after normalization using effective gene lengths.For RNA-Seq processing, paired-end 150 bp raw reads were first trimmed using Trim Galore 18 and variants were called using SCcaller 4.For WGS processing, paired-end reads were trimmed using Trim Galore and quality control performed using FastQC. Alignment was performed with BWA MEM