C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.A ‘GGGGCC’ repeat expansion in the first intron of the The online version contains supplementary material available at 10.1007/s00401-022-02471-y. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are adult-onset neurodegenerative diseases belonging to a heterogeneous and complex disease spectrum , 71. At C9orf72 gene, which is the most common genetic cause of both disorders (C9 ALS/FTD) [C9orf72 (referred to as ‘C9’) repeat expansion [In ALS and FTD, impaired cognition often coincides with motor problems, and ubiquitinated inclusions containing transactive response DNA-binding protein 43 (TDP-43) represent a shared pathological hallmark , 69. An ALS/FTD) , 60. It ALS/FTD) , 59. PatALS/FTD) , 17. Remxpansion , indicatxpansion .post-mortem brain and spinal cord samples. A first mechanism attributes neurodegeneration to a C9orf72 loss-of-function, due to impairment of normal C9orf72 gene expression by the C9 repeat expansion [The exact mechanism underlying C9 ALS/FTD remains elusive. However, three non-mutually exclusive mechanisms have been proposed and are currently being investigated using in vitro and in vivo ALS models in addition to patient xpansion . These Dxpansion . A thirdxpansion . Of all xpansion .C9orf72 hexanucleotide repeat RNAs, are candidate modifying proteins of interest in the modulation of C9 ALS/FTD heterogeneity [C9orf72 ALS [ALYREF in a C9 Drosophila model alleviates toxicity, whereas downregulation of HNRNPA3 in primary rat hippocampal neurons and patient fibroblasts was found to aggravate the C9orf72 ALS phenotype [C9orf72 models [It is important to elucidate the contribution of RNA toxicity in C9 ALS/FTD since RBPs, shown to interact with sense and antisense ogeneity , 18, 25.rf72 ALS , 64. Forhenotype . As both2 models  and beca2 models , 78, it C9orf72 hexanucleotide repeat-associated ALS phenotype is only due to RNA toxicity, without any involvement of DPRs [C9orf72 zebrafish model, as well as patient fibroblasts, iPSC-derived motor neurons and post-mortem material, we provide evidence that HNRNPK loss-of-function contributes to disease in C9orf72 ALS. We identified the DNA damage and repair-related protein ribonucleotide reductase regulatory subunit M2 (RRM2) as a downstream target and mediator of HNRNPK dysregulation in ALS/FTD. In conclusion, we show that C9 repeat RNA toxicity is associated with an aberrant DNA damage response through disturbed RRM2 functionality. More specifically, we further emphasize the relevance of C9 repeat RNA toxicity as a disease mechanism by demonstrating the presence of DNA damage and diminished DNA repair in C9 ALS/FTD. Both hallmarks represent a novel avenue of research on C9 ALS/FTD and are exigent topics in the investigation of general ALS/FTD-related neurodegeneration.In this study, we aimed to assess the relevance of C9 RNA toxicity by identifying RBPs capable of modifying a repeat RNA-induced phenotype. Therefore, we used our previously characterized zebrafish model in which the  of DPRs . In addi of DPRs . By perfAgeI  restriction enzyme. FLAG-tagged HNRNPK  and RRM2  plasmid constructs were digested with AgeI.Plasmids, comprising 91 (91S) or 70 (70S) ‘GGGGCC’ hexanucleotide repeats under control of a T7 promoter were obtained and adapted as described earlier , 64. In XhoI and EcoRI restriction digestion of donor constructs, desired inserts were harvested using the GenElute Gel Extraction kit , according to the manufacturer’s instructions. Next, inserts were ligated  with T4 ligase into the pCMV6 vector. These constructs were then transformed in E. coli TOP10 chemically competent cells . Consequently, plasmids were linearized with AgeI restriction enzyme.HNRNPK deletion constructs were synthesized by Genscript  in a pUC57 vector and subcloned in a pCMV6-Entry vector . After ®T7 kit (Invitrogen). Antisense repeat (70AS) RNA was generated using mMESSAGE mMACHINE®T3 kit (Invitrogen). Resulting RNA was purified with MEGAclear Transcription Clean-Up Kit (Invitrogen) and quality was evaluated on a 1–2% RNA gel based on expected transcript lengths. RNA concentration was determined by spectrophotometry .Following linearization of plasmids by restriction digestion, 91S repeat RNA, RBP RNA and HNRNPK deletion construct RNA was synthesized using mMESSAGE mMACHINEOne-step RNA transcription and biotinylation of constructs containing 91 and 70 repeats was performed as previously described , using TZebrafish work was performed as previously described . All exphnRNPK splice-blocking oligo (5ʹ-AACTCAAAGACTTACTTTGCTCTGT-3ʹ), hnRPKL splice-blocking (5ʹ-ACATTTACATCACACTTACTTTGCT-3ʹ), hnRNPK translation-blocking oligo (5ʹ-GATCCATTATCTATCTACACCTAGT-3ʹ) and hnRPKL translation-blocking oligo (5ʹ-CTGCTCAATTTCTGTCTCCATTGTC-3ʹ).Antisense morpholino oligonucleotides were designed and obtained from Gene Tools : morpholino standard control oligo (sequence 5ʹ-CCTCTTACCTCAGTTACAATTTATA-3ʹ), Fixed zebrafish embryos were permeabilized with acetone for 1 h at − 20 °C, blocked with 1% bovine serum albumin (BSA)/1% dimethyl sulfoxide (DMSO)/PBS for 1 h at room temperature (RT) and immunostained with primary mouse anti-synaptic vesicle glycoprotein 2 , Supplementary table 1, online resource) for 5 h at RT. Embryos were incubated overnight at 4 °C with secondary Alexa Fluor 555 anti-mouse antibody . In-between washing steps were performed with 1%BSA/1%DMSO/PBS and following SV2 staining, samples were washed 4 times in 0.2% PBS-Triton (PBST) for 1 h and stored in PBS at 4 °C.Stained zebrafish embryos were analyzed with fluorescence microscopy  using LUCIA software . A standardized method was used to score fish on axonal length and abnormal branching of ventral root projections. Consecutively, 15 fish were analyzed per condition. For axonal length scoring, five predefined, consecutive motor axons  were measured in all 15 embryos. Data were normalized to the control condition. Correspondingly, abnormal branching was analyzed from 20 predefined and consecutive motor axons  of 15 embryos. Branching was considered to be abnormal when at least two out of 20 axons were branched at or above the ventral edge of the notochord. The number of biological replicates is indicated with “N” in the figure legends of the results section. For RNA injections, GFP mRNA-injected embryos were used as negative controls.Fixed zebrafish embryos were permeabilized with acetone for 1 h at − 20 °C, blocked with 10% NDS in 0.4% PBST for 1 h at 37 °C and immunostained with primary rabbit anti-γH2AX , Supplementary table 1, online resource) for 3 h at 37 °C. Embryos were incubated overnight at 4 °C with secondary Alexa Fluor 555 anti-rabbit antibody . In-between washing steps were performed with 0.4% PBST. Nuclei were stained using NucBlue counterstaining (Invitrogen) and embryos were mounted using ProLong Gold antifade reagent (Invitrogen). For image acquisition, a Nikon NiE microscope was used with a 40x objective, equipped with a Yokogawa CSU-X spinning-disk module and Teledyne Photometrics Prime 95B camera. The setup was controlled by NIS-Elements . For image analysis, the number of neuronal nuclei positive for γH2AX and located in the spinal cord of zebrafish embryos was counted and normalized to the length of the spinal cord section using ImageJ.C9orf72 ALS patient lines 24/4 and 24/5) were grown and maintained at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) with high glucose and L-Glutamine  supplemented with 10% fetal bovine serum , 5% non-essential amino acids  and penicillin/streptomycin (100 µg/ml). For transfection purposes, antibiotics were not added to media. Primary human fibroblasts were obtained from skin biopsies of ALS patients and controls with the approval of the Ethical Committee of the University Hospitals Leuven (S50354). Clinical information is indicated in Supplementary table 2 (online resource). Human C9orf72 ALS patient iPSC lines and their isogenic controls were obtained and were published before [Human Embryonic Kidney 293 (HEK) cells , HeLa cells  and human-derived fibroblasts  and Lipofectamine™ 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Cells of 6-well plates were lysed using RIPA buffer (Sigma) supplemented with protease inhibitor cOmplete™  and collected 48 h post-transfection.In HeLa cells, siRNA was reverse transfected using Opti-MEM™ (Gibco) and Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) following the standard protocol. SiRNAs were obtained from Dharmacon : ON-TARGETplus human non-targeting siRNA , HNRNPK SMARTpool L-011692-00-0005 targeting J-011692-05 (5ʹ-UAAACGCCCUGCAGAAGAU-3ʹ), J-011692-06 (5ʹ-GGUCGUGGCUCAUAUGGUG-3ʹ), J-011692-07 (5ʹ-UGACAGAGUUGUUCUUAUU-3ʹ) and J-011692-08 (5ʹ-GCAAGAAUAUUAAGGCUCU-3ʹ). Cells were treated, collected or fixed at 72 h post-transfection.Cultured cells were treated with 10 µM camptothecin (CPT) topoisomerase I inhibitor for 30 min. Then, medium was replaced with fresh medium (without drugs) and cells were incubated for 4 h at 37 °C before collection or cells were immediately PFA-fixed or lysed after treatment.post-mortem diagnosis of ALS and FTLD-TDP was pathologically confirmed by assessment of the pTDP-43 pathology. Clinical and neuropathological information is indicated in Supplementary table 3 (online resource).Brain tissue was collected in accordance with the applicable laws in Belgium (UZ Leuven) upon written informed consent. The study was approved by the UZ Leuven Ethical Committee . After autopsy, the right hemisphere was dissected into coronal planes and frozen at − 80 °C. The left hemisphere was fixed in 4% PFA. The post-mortem material, 50 mg of frozen brain tissue was weighed and mechanically homogenized in 0.5 ml 2% SDS in Tris-buffered saline (TBS) with Nuclease  and a cocktail of protease/phosphatase inhibitors  using a micropestle. Samples were sonicated, followed by a centrifugation at 14 000g for 30 min. The resulting supernatant was used. Protein quantification was done using (Micro) BCA™ Protein Assay Kit (Thermo Scientific). Indicated amounts of lysates were supplemented with Pierce™ Non-Reducing Sample Buffer (Thermo Scientific), loaded on 4–20% Mini-PROTEAN® TGX Stain-Free™ Precast Gels  and transferred to Trans-Blot® Turbo™ Mini 0.2 µm PVDF Transfer Packs (Bio-Rad) using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). After blocking with 5% non-fat dry milk in TBS supplemented with 0.1% TWEEN®20 (Sigma) (TBS-T) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibody diluted in TBS-T. Primary antibodies are listed in Supplementary table 1 (online resource). Then, three TBS-T washes and a 1 h incubation with secondary antibody  at room temperature were performed before washing two times with TBS-T and once with TBS. Blots were visualized by chemiluminescence using SuperSignal™ West ECL/Pico/Femto Chemiluminescent Substrate (Thermo Scientific) on the ImageQuant™ LAS 4000 .RIPA buffer supplemented with protease inhibitor was used to lyse 6 hpf zebrafish blastulae, HEK cells and iPSC-derived motor neurons. For ® Ribonuclease Inhibitor (Promega) by physical disruption using mortar and pestle. Magnetic streptavidin-coated beads  were prepared for RNA application, according to the manufacturer’s instructions. Next, HEK lysates were precleared for 1 h at 4 °C by incubation with magnetic beads on rotor. Non-specific binding was reduced by pre-incubating magnetic beads with precleared WT HEK lysate. Biotinylated 91S or 70AS repeat RNA was denatured at 95 °C for 10 min and incubated with 0.5 mg of precleared lysate overnight at 4 °C on rotor. The following day, 20 µl of prepared magnetic beads was added and incubated for 1 h at 4 °C on rotor. Samples were washed five times in ice-cold PBS, then denatured in Pierce™ Non-Reducing Sample Buffer (Thermo Scientific) and separated by SDS-PAGE followed by Western blotting.HEK cells were transfected with HNRNPK deletion constructs using Lipofectamine 3000 (Invitrogen), according to the manufacturer’s instructions. WT and 48 h post-transfected HEK cells were lysed in RIPA buffer supplemented with protease inhibitor and RNasinhnRNPK , hnRPKL , GAPDH .Total RNA of 30 hpf zebrafish embryos was isolated using the RNeasy kit . cDNA was generated using reverse transcription with SuperScript™ III First-Strand Synthesis SuperMix kit (Invitrogen). RT-PCR was performed using DreamTaq Green PCR Master Mix (Thermo Scientific) in a 96-well thermal cycler (Thermo Scientific) with optimized program, according to the manufacturer’s instructions. RT-PCR products were analyzed on a 2% agarose gel and visualized using MIDORI Green staining . Primers were obtained from Integrated DNA Technologies : −ΔΔct method and normalized to ZNF48 transcript levels. The following primers (IDT) were used: HNRNPK , ZNF48 .Total RNA extraction was performed with the RNeasy Mini kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using SuperScript III™ First-Strand Synthesis SuperMix (Invitrogen), according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green PCR Master Mix  on the 7500 Step OnePlus Real-Time PCR system (Applied Biosystems). Relative gene expression was determined by the 2After culturing cells  on a coverslip, cells were washed with PBS and PFA-fixed for 20 min. Three PBS wash steps were followed by 1 h blocking at 37 °C with 10% normal donkey serum (NDS) in 0.4% PBS-Triton (PBS-T). Then, cells were incubated with primary antibody diluted in 0.4% PBS-T overnight at 4 °C. Primary antibodies are listed in Supplementary table 1 (online resource). The next day, three 0.4% PBS-T wash steps were followed by 1 h incubation with secondary antibody conjugated with Alexa Fluor 555 or Alexa Fluor 488  at RT. Upon three 0.4% PBS-T washes, NucBlue™ Live ReadyProbes™ Reagent (Invitrogen) was added as a nuclear marker and after 20 min, cells were washed three times with PBS. Finally, coverslips were mounted on microscope slides using fluorescence mounting medium . Confocal images were obtained using the Leica SP8 DMI8 confocal microscope using a 40x objective. Images were processed using ImageJ.post-mortem brains, immunohistochemical analysis was performed on 7–8 µm thick sections cut from frozen tissue of motor cortex, as previously described [®  for 30 s at room temperature. After rinsing three times with PBS, slides were mounted using ProLong™ Gold Antifade reagent with DAPI (Invitrogen). Confocal images were obtained using a Leica SP8 DMI8 confocal microscope. Images were processed using ImageJ.To assess localization of HNRNPK in escribed . Frozen post-mortem brain and spinal cord, histological examination was performed on 5 µm-thick sections cut from formalin-fixed, paraffin-embedded tissue. Stainings were performed with the BOND-MAX automated IHC/ISH Stainer (Leica Biosystems). Slides were deparaffinized and epitopes were retrieved with low pH buffer. After incubation with Envision Flex Peroxidase-Blocking Reagent, slides were incubated with a primary anti-RRM2 antibody  for 30 min, followed by secondary antibody incubation. 3,3’-Diaminobenzidine (DAB) was used for visualization. Counterstaining with hematoxylin and dehydration was carried out in an autostainer, followed by mounting with an automated cover-slipper (Leica Biosystems). Images were acquired using the Leica DM2000 LED microscope using a 40x objective coupled to a Leica DFC 7000 T camera. Images were processed using ImageJ. Clinical and neuropathological information is indicated in Supplementary table 3 (online resource).To assess localization of RRM2 in post-mortem tissue were selected in the DAPI channel and corresponding regions of interest (ROIs) were placed in an unbiased manner. Cytoplasmic ROIs were captured for the selected nuclei in the HNRNPK or RRM2 channel, respectively. HNRNPK/RRM2 nuclear and cytoplasmic ROI intensity was measured as the raw integrated density and was presented as the nuclear cytoplasmic (N/C) ratio. Each data point represents the average N/C ratio per replicate.On ImageJ, nuclei of immunofluorescent-stained HeLa cells, human-derived fibroblasts, iPSC-derived motor neurons and C9orf72 ALS cases. A qualitative grading system was used in which cells were counted as positive when nuclei were stained completely and negative when nuclei were not stained for RRM2.The presence of RRM2 in the DAB-stained nuclei of neurons in motor cortex, occipital cortex and cervicothoracic spinal cord was assessed in non-neurodegenerative controls and https://microscopy.duke.edu/guides/count-nuclear-foci-ImageJ). The prominence in the ImageJ function “Find Maxima” was set to at least 40.γH2AX foci were automatically quantified using a computational algorithm based on uniform threshold per fluorescence channel in ImageJ as described by the light microscope core facility at Duke university .RNA-sequencing was performed by the Nucleomics Core Facility . RNA was isolated using the RNeasy kit (Qiagen). From extracted RNA, libraries were made using the Illumina TruSeq Stranded mRNA Library protocol. These libraries were sequenced on an Illumina NextSeq 500 paired-end 75 bp and yield an average of 37.5 million reads per sample (range 35.1–40.5). To estimate the expression of the transcript of every sample, reads were counted using Salmon (v0.14.0)  against http://www.rstudio.com)). Shapiro–Wilk normality test (GraphPad Prism) was used to assess normal distribution of data. For zebrafish experiments, axonal length and branching was analyzed with one-way ANOVA (GraphPad Prism) and Tukey’s post hoc correction was applied for multiple comparisons. Data are represented as mean ± SEM. Data from experiments with post-mortem material, iPSC-derived motor neurons, fibroblasts, HEK and HeLa cells are represented as mean ± SEM, unless indicated otherwise. Normally distributed data were analyzed using one-way ANOVA with Tukey’s post hoc correction for multiple comparisons or unpaired t tests for comparisons of two groups, unless indicated otherwise. Data that were not normally distributed were analyzed using Kruskal–Wallis non-parametric test with Dunn’s correction for multiple comparisons or Mann–Whitney U test for comparisons of two groups. Significance level was defined at 0.05. Significance levels are indicated in the figures as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Statistical analysis was performed using GraphPad Prism software  or RStudio . RStudio: Integrated Development for R. RStudio, Inc., Boston, US (C9orf72 hexanucleotide repeat in the sense direction (91S) in 1–2 cell stage zebrafish oocytes and observed motor axon abnormalities in zebrafish embryos (30 hpf) after whole-mount staining of synaptic vesicle glycoprotein 2a (SV2) , rescued the axonal phenotype in a dose-dependent manner  . These results show that HNRNPK is a strong modifier of C9 RNA toxicity in our zebrafish model.Following a dose–response experiment , we injected the highest non-toxic dose of candidate RNA in zebrafish oocytes and found that, from all tested candidates, heterogeneous nuclear ribonucleoprotein K (HNRNPK) is the most potent modifier of hnRNPK and hnRNPK-like (or hnRPKL) are the zebrafish orthologues of human HNRNPK, with 71 and 84% sequence homology, respectively. To determine whether hnRNPK dysfunction contributes to the phenotype in our C9 RNA toxicity zebrafish model, we first assessed the transcript levels of hnRNPK and hnRPKL by RT-PCR, as well as hnRNPK protein levels by Western blot. We observed that hnRNPK and hnRPKL transcript levels remained unaltered in zebrafish  using qPCR and public RNA-sequencing datasets [ish Fig. a, b, whidatasets , 57, 61.HNRNPK orthologues in zebrafish embryos using antisense morpholino oligonucleotides  [hnRNPK and hnRPKL pre-mRNA. Since exon 2 is not a multiple of three, skipping leads to a frameshift in the subsequent amino acid sequence of hnRNPK, while retention of intron 2 (target hnRPKL) presumably results in nonsense-mediated decay. After a dose–response experiment , the effectiveness of these morpholinos was confirmed by RT-PCR  , 72. SplhnRNPK and hnRPKL compared to embryos injected with standard control morpholino . From these findings, we conclude that hnRNPK is necessary to ensure motor neuron development, that hnRNPK function is disturbed by C9 repeat expansion RNA and that C9 RNA toxicity can be rescued by HNRNPK overexpression.Similar to what was observed in the C9 RNA toxicity model, the axonal length of motor neurons was significantly decreased in embryos knocked down for ino Fig. g. Moreovino Fig. h. This pRNA Fig. g, h, indGFP mRNA, full-length HNRNPK mRNA or mRNA encoding one of the six HNRNPK deletion constructs. By comparing the rescue effect of each construct to that of the full size HNRNPK, we were able to determine which domain is essential to rescue the axonal phenotype associated with C9 RNA toxicity in our zebrafish model. Our data showed that removing the protein interaction domain (∆KI) did not alter the modifying effect on the axonal phenotype. On the contrary, the rescue effect of HNRNPK was abolished by deletion of either the nuclear shuttling domains (∆NLS/KNS) or of all RNA-binding domains at once (∆KH1-2-3) , a protein interaction domain (KI) and two nuclear shuttling domains  . As expected, HNRNPK lacking nuclear shuttling domains (∆NLS/KNS) exhibited a mainly cytoplasmic localization and was depleted from the nucleus . Interestingly, HNRNPK proteins encoded by constructs lacking all RNA-binding domains were also detected in the cytoplasm, in addition to their abundant nuclear presence . As both of these constructs did not rescue the toxicity of C9 repeat RNA, our data suggest that HNRNPK nuclear localization, as well as RNA binding function, are required for the modifying effect of HNRNPK on C9 repeat RNA toxicity in zebrafish.HNRNPK was selected as candidate modifying protein because of its known interaction with C9 repeat RNA , 25. To C9orf72 ALS, the HNRNPK localization was assessed in three patient-relevant models using immunostainings. First, two C9orf72 ALS patient fibroblast lines  were compared to two healthy control lines . We observed a > 40% reduction in HNRNPK nucleus/cytoplasm (N/C) ratio, measured as mean fluorescence intensity ratio, due to an increase in cytoplasmic localization of HNRNPK . Statistical analysis showed that three genes were significantly downregulated, while 12 genes were upregulated , of which a 0.5-fold reduced expression was detected in the C9 repeat RNA toxicity zebrafish model compared to GFP mRNA control-injected embryos  and obtained from the Gene Expression Omnibus (GSE91621) [To further investigate the HNRNPK dysfunction in the context of C9 repeat RNA toxicity, we assessed dysregulated gene expression to identify downstream HNRNPK targets. To achieve this, we performed transcriptome-wide analysis using bulk RNA-sequencing of 30 hpf zebrafish embryos injected with control ted Fig. a. The stSE91621) .Fig. 5HNC9orf72 ALS and is involved in the C9 repeat RNA toxicity mechanism.To investigate the importance of RRM2 in the modifying mechanism on C9 RNA toxicity, we tested whether overexpression of RRM2 was sufficient to rescue the axonopathy caused by 91S repeat RNA in our zebrafish model. Therefore, we injected mRNA encoding human RRM2 together with C9 repeat RNA in 1- to 2-cell stage zebrafish oocytes after confirmation of its expression  and determination of the highest non-toxic dose . Assessment of SV2-stained motor axons showed a significant increase in axonal length and a trend of a decrease in aberrant branching when compared to embryos only injected with 91S repeat RNA Fig. d, e, dempost-mortem motor cortex tissue and compared RRM2 protein levels in C9 ALS/FTD patients with those in non-neurodegenerative controls. Consistent with an expected decrease in RNA transcripts, as observed in our zebrafish model, we noted a 30% reduction in RRM2 levels in C9orf72 patients  as a downstream transcriptional target of HNRNPK, and found that its function is altered by repeat RNA-induced dysfunction of HNRNPK. We discovered that RRM2 expression is reduced in C9orf72 ALS, and found that RRM2 is mechanistically linked with HNRNPK in the DNA damage response pathway.In this study, we explored the relevance of C9 RNA toxicity as a disease mechanism in C9 ALS/FTD and investigated modifiers using a target RBP known to bind C9 repeat RNA , 25. We C9orf72 embryonic zebrafish model represents an acute toxic phenotype, rather than progressive neurodegeneration, as in C9orf72 ALS patients [C9orf72 ALS, it encompasses a consistent and reproducible motor axonopathy induced by C9orf72 repeat RNA. The purpose of including patient-relevant models  and post-mortem material in this study is to enable translation of the embryonic zebrafish screening to C9orf72 ALS in the clinic, and to assess the relevance of HNRNPK as a modifier of RNA toxicity in disease. However, investigation of DPR-induced displacement of HNRNPK, which has already been associated with other RBPs [C9orf72 ALS.The patients . In thisher RBPs , will beC9orf72 ALS [Processing of mRNAs, including alternative splicing and transcriptional as well as translational regulation, is the overall purpose of HNRNPs . HNRNPs rf72 ALS .HNRNPK gene [C9orf72 ALS patient iPSC-derived motor neurons and frontal cortex [C9orf72 repeat RNA interactome [HNRNPK is one of the most abundantly expressed HNRNP members and is widely distributed across tissues affected in ALS, such as the cerebral cortex, spinal cord and muscle , 76. TheNPK gene . MoreoveNPK gene , 49, whel cortex , 57, 61.l cortex , 63 and eractome , 25, whiC9orf72 ALS, we used two independent patient models  and post-mortem tissue to demonstrate that HNRNPK localization is mislocalized to the cytoplasm in C9orf72 ALS, as was recently shown for FTD [C9orf72-specific mechanism could be at play, as HNRNPK mislocalization in C9orf72 ALS could be instigated by the direct interaction with cytoplasmic RNA foci and/or soluble repeat RNA. This is in line with findings in the C9 RNA toxicity zebrafish model displaying solely cytoplasmic, and not nuclear, RNA foci [C9orf72 ALS patients could also be sufficient to bind and sequester HNRNPK, preventing its translocation to the nucleus where it performs its function. Crucially, evidence of pathogenicity for RNA foci remains limited, suggesting that the soluble repeat RNA might be more toxic than the RNA foci themselves, as was originally thought. Nevertheless, the exact contribution of these factors to the interplay of HNRNPK with ALS-related actors and its mislocalization remains elusive and warrants further investigation.To investigate a change in function, we used HNRNPK deletion constructs and discovered that nuclear localization and RNA binding of HNRNPK, independent from its interaction with C9 repeat RNA, are necessary for the observed modification of the phenotype associated with C9 RNA toxicity in our zebrafish model. To evaluate the overall relevance of HNRNPK localization in  for FTD . Each mo for FTD , and as  for FTD . Third,  for FTD . Aside fRNA foci . While oRNA foci , 42, 58,C9orf72 ALS. Moreover, this increases the likelihood that HNRNPK specifically regulates RRM2 transcription, in addition to its RNA binding or modification as indicated by the published CLIP-seq datasets. To confirm the mechanistic link between HNRNPK and DNA damage in our model, we found that in absence of HNRNPK, HeLa cells showed increased DNA damage. We then demonstrated that upregulation of HNRNPK expression was triggered as a response to induced DNA damage by treating the cells with CPT. These findings imply that the downstream mechanism of HNRNPK dysfunction occurs within the same cell type. However, on top of the impaired DNA damage response observed in one single cell type, we cannot exclude non-cell autonomous effects in the context of a multi-cellular model or in C9orf72 ALS patients. We found that RRM2 protein levels were reduced and that its cell distribution was altered towards an increasing nuclear localization, both in C9 ALS/FTD patient samples and upon HNRNPK depletion in HeLa cells. The use of RRM2 agonists, antagonists [Recently, HNRNPK mislocalization was reported to induce splicing dysregulation of downstream genes in the frontal cortex of FTD patients . We, theagonists  or drugsagonists , 36 coulC9orf72 repeat expansions.Over the past years, arginine-rich DPRs produced in C9 ALS/FTD have been associated with many cellular deficits leading to neurodegeneration, including a failed response to DNA damage . IncreasOverall, our working model Fig.  illustraC9orf72 ALS. We hypothesize that HNRNPK loss-of-function, either by lack of proper expression or mislocalization, contributes to pathogenicity in C9orf72 ALS. Furthermore, we demonstrated that a disrupted DNA damage response is attributable to dysfunctional HNRNPK, and that the subsequent RRM2 deficiency leads to impairments in DNA repair in the C9 RNA toxicity model. Altogether, our results highlight RNA toxicity as an undervalued mechanism that at least partially, and perhaps concomitant with DPR toxicity, contributes to neurodegeneration in C9 ALS/FTD. As related studies further illustrated that the altered DNA damage response is a recurring mechanism at play in other ALS/FTD types, such as TDP-43 ALS [In conclusion, using in vivo and in vitro models we identified HNRNPK as a novel modifying protein of RNA toxicity in P-43 ALS , 29, ourSupplementary file1 (PDF 1893 KB)Supplementary file2 (XLSX 11 KB)Supplementary file3 (XLSX 18 KB)Supplementary file4 (XLSX 25 KB)Below is the link to the electronic supplementary material.


Moreover, mutant KRAS induces an intrinsic IFN-stimulated gene (ISG) signature that is often seen across many different cancers. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway.RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression, are preferentially released in extracellular vesicles, and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas   Many human cancers are driven by mutant KRAS, but its effects on noncoding RNA are unclear. Reggiardo et al. show that mutant KRAS regulates this RNA landscape by silencing KRAB zinc-finger genes that normally repress transposable element noncoding RNAs, which are preferentially released from mutant KRAS cells in extracellular vesicles. Most of the human genome is noncoding and transcribed into RNA . Moreovein vitro and in mutant KRAS lung adenocarcinomas in vivo. Moreover, our findings indicate that significant upregulation and extracellular secretion of TE RNAs and ISGs are transcriptomic signatures of mutant KRAS signaling.To investigate the role of mutant KRAS in reprogramming the transcriptome during early stages of cellular transformation, we characterized the composition of both intracellular and extracellular RNA, including protein-coding RNA, lncRNA, and TE RNA, using human airway epithelial cells  and humaTo determine the transcriptomic landscape of protein-coding and noncoding RNAs regulated by oncogenic RAS signaling, we performed RNA sequencing (RNA-seq) on human airway epithelial cells (AALE) that undergo malignant transformation upon the introduction of mutant KRAS . We compα and -γ responses, as well as the hallmark inflammatory response   using geresponse , along wresponse .in vivo relevance of our findings in both mutant KRAS AALE and HBEC cells, we examined ISG expression in mutant KRAS(G12D) lung adenocarcinomas (LUAD) from The Cancer Genome Atlas (TCGA), which revealed a subset of ISGs that were upregulated in KRAS(G12D) tumors when compared to lung cancer samples with wild-type (WT) KRAS  and in vivo (TCGA LUAD).To further validate the connection observed between mutant KRAS and ISG expression, we compared mutant KRAS-induced ISGs in AALE cells to those that were induced in human bronchial epithelial cells (HBECs) in response to mutant KRAS(G12V) . We obseWT) KRAS . These rTo elucidate potential mechanisms involved in inducing ISG signatures in mutant KRAS AALE cells, we performed the assay for transposase-accessible chromatin using sequencing (ATAC-seq) . In mutaThe genome-wide effects of mutant KRAS-mediated epigenomic reprogramming were further assessed with the Genome Regions Enrichment of Annotations Tool (GREAT) . GREAT aTo test whether extracellular RNAs secreted from mutant KRAS cells also exhibit differential expression relevant to intracellular reprogramming events, we isolated extracellular vesicles from the culture media of control and mutant KRAS AALEs . Extraceα and -γ signatures were enriched in both intracellular and extracellular RNA  and lncRNA   and S2A.ular RNA , indicatTo determine the effects of oncogenic KRAS on noncoding RNA secretion, we also characterized the TE RNAs that were preferentially packaged and released in extracellular vesicles. We found significant upregulation of predominantly long terminal repeat (LTR) RNAs such as LTR12, MER11C, and LTR27C, along with LINE, DNA, and Satellite repeat RNAs in mutant KRAS AALE extracellular vesicles . MoreoveGiven the prevalence of secreted TE RNAs, we investigated intracellular TE RNA dynamics in response to mutant KRAS signaling in AALE cells. Analogous to extracellular RNAs, LTR RNAs were among the most significantly upregulated TE RNAs in response to oncogenic KRAS signaling, including LTR12C RNAs . In addiBased on the functions of KZNF genes in silencing TE RNAs in other contexts , we examTo explore the mechanistic relationship between KZNFs and TE RNA expression, we examined mutant KRAS A549 lung cancer cells that overexpress ZNF257 or ZNF682 , both ofTo determine the extent to which mutant KRAS signaling epigenetically silences KZNF expression, we examined ATAC-seq data for all significantly downregulated KZNF loci. We found that mutant KRAS signaling substantially reduces chromatin accessibility at TSS regions . When weFinally, we explored the clinical significance of the mutant KRAS-induced KZNF silencing we identified in AALE and HBEC cells. Evaluation of KZNF expression in TCGA LUAD RNA-seq data revealed their significant downregulation in mutant KRAS(G12D) samples when compared to WT KRAS lung cancer or matched normal samples, respectively  and 5B. in vivo. Our conclusions are based on deeply sequencing and analyzing the intracellular and extracellular transcriptomes and epigenomes of mutant KRAS-transformed lung cells, building on previous work in which we discovered the coordinate regulation of noncoding RNAs and RAS signaling in the context of epigenomic reprogramming  that correct mutations in KRAS would reveal whether oncogenic RAS signaling is necessary for high ISG expression in tumor cells. Lastly, the biomarker potential of extracellular RNAs that are preferentially secreted from cancer cells with oncogenic RAS signaling would require validation using blood samples from patients with lung and other cancers having activating driver mutations in RAS pathway genes.daniel.kim@ucsc.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Daniel H. Kim (This study did not generate new unique reagents.Bulk RNA-seq and ATAC-seq data have been deposited at GEO (GSE120566) and are publicly available as of the date of publication. Accession numbers are also listed in the All original code has been deposited at Zenodo and is publicly available as of the date of publication. DOIs are listed in the Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.2 in SABM Basal Medium  with supplements and growth factors .Immortalized lung epithelial cells , derived at Dana-Farber and immortalized by SV40 large-T antigen  were obt2 in Keratinocyte Serum-Free Media (KSFM) with supplements .HBEC3kt cell lines  were obtained as a gift from the laboratory of Harold Varmus . The HBEC stable cell lines pLenti6/V5-GW/lacZ (control)  and pLenFor AALE cell lines, bulk RNA was isolated from cells using Quick-RNA MiniPrep kit (Zymogen) and RNA was quantified via NanoDrop-8000 Spectrophotometer. 1ug of total RNA was used as input for the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to manufacturer protocol. Library quality was determined through the High Sensitivity DNA Kit on a Bioanalyzer 2100 (Agilent Technologies). 6 multiplexed libraries, 3 biological replicates of each condition, were sequenced as HiSeq400 100PE runs.For HBEC cell lines, cells grown in 10 cm plates (n = 3 per cell line) were washed twice in cold DPBS then collected in Tri-reagent for storage at –80°C until the bulk RNA was extracted using Direct-Zol RNA Miniprep Kit (Zymo Research). Concentrations of purified RNA in nuclease-free water were determined by Nanodrop-2000 Spectrophotometer and by Qubit RNA BR Assay (ThermoFisher Scientific). Quality RIN numbers ranging from 9.4–10 were determined by TapeStation 4150 RNA ScreenTape Analysis (Agilent Technologies) before sending RNA to UC Davis DNA Technologies and Expression Analysis Core Laboratory for poly-A strand specific library preparation to obtain 60 million paired end reads by NovaSeq S4 (PE150) sequencing.100,000 AALE cells were collected and centrifuged at 500xg for 5 min at 4C. Pellets were washed with ice-cold PBS and centrifuged. Pellets were resuspended in ice-cold lysis buffer. Tagmentation reaction and purification were conducted according to manufacturer’s protocol (Active Motif). 2 Libraries, one from each condition, were sequenced on a NextSeq500 as 2 × 75 paired end reads.The exoRNeasy serum/plasma maxi kit (Qiagen) was used to isolate extracellular vesicles, which were quantified using Nanoparticle Tracking Analysis . 30 mL of AALE cell culture supernatant was filtered to remove particles larger than 0.8 um. The filtrate was precipitated with kit buffer and filtered through a column to collect extracellular vesicles. These vesicles were then lysed with QIAzol® lysis reagent. Total RNA was isolated using the indicated phase separation method and used to make 6 libraries, 3 biological replicates for each condition, for RNA-seq using the Smart Seq HT low input mRNA library prep kit (Takara). Libraries were sequenced on an Illumina NextSeq500.fastq files were trimmed with Trimmomatic 2 (0.38) (FastQC (All 2 (0.38)  using th . Input gene abundance data was first variance stabilized using DESeq2 and then filtered for genes with 0 standard deviation across the samples.PCA was performed in R using packages memes (1.1.4), universalmotif (1.10.2), BSgenome.Hsapiens.UCSC.hg38 (1.4.3), MotifDBGenomicRanges (1.44.0) and MotifDB (1.34.0)    and filtR package fgsea (1.18.0) (The (1.18.0)  was used(1.18.0)  estimateR package rGREAT (1.24.0) (Bioconductor) was used to process ATAC-seq identified peaks with GREAT and identify enriched GO terms. ATAC-seq peaks unique to either CTRL or KRAS contexts were used as input with the background set to the entire peak library comprised from both contexts.The R using the package ComplexHeatmap (2.8.0)   . Survivage (3.3) .R package bamsignals (1.24.0) (Bioconductor) using the sorted bam files produced by the nf-core pipeline.The nf-core ATAC-seq pipeline was used to process ATAC-seq reads to alignments with BWA, narrow peak calls with MACS2, and ultimately annotated peaks. Read count analysis was performed with the R – wilcox.test) unless otherwise noted and p values <0.05 were considered significant. All statistical analyses were performed with R (version 4.1.1) running from the Rocker ‘Tidyverse’ Docker container (rocker/tidyverse:4.1.1). Linear regression was carried out with the lm function.All quantitative data for functional assays have been reported as means ± standard deviation. Statistical significance for these were calculated using a Wilcox-test (https://genome.ucsc.edu/s/rreggiar/aale%2DKRAS%2DG12%2Dtransformation.UCSC Genome Browser tracks generated from ATAC-seq data: 1


This study aimed to explore possible associations between molecular subtypes and site of distant metastasis in advanced breast cancer (ABC).3577 ABC patients were selected from 21 hospitals of seven geographic regions in China from 2012-2014. A questionnaire was designed to collect medical information regarding demographic characteristics, risk factors, molecular subtype, recurrence/metastasis information, and disease-free survival (DFS). The cancers were classified into Luminal A, Luminal B, HER2-enriched and Triple Negative subtypes. Chi-square test and multivariate Cox proportional hazard models were performed to explore the associations between molecular subtypes and distant metastasis sites.A total of 2393 cases with molecular subtypes information were finally examined. Patients with Luminal A (51.1%) and Luminal B (44.7%) were most prone to bone metastasis, whereas liver metastasis was more frequently observed in HER2-enriched ABC patients (29.1%).The cumulative recurrence and metastasis rates of ABC patients at 36 months of DFS were the most significant within molecular types, of which Triple Negative was the highest (82.7%), while that of Luminal A was the lowest (58.4%). In the adjusted Cox regression analysis, Luminal B, HER2-enriched and Triple Negative subtypes increased the risk of visceral metastasis by 23%, 46% and 87% respectively. In addition, Triple Negative patients had a higher probability of brain metastasis .Molecular subtypes can predict the preferential sites of distant metastasis, emphasizing that these associations were of great help in choices for surveillance, developing appropriate screening and cancer management strategies for follow-up and personalized therapy in ABC patients. Breast cancer is the most common malignancy in women worldwide. Approximately 2,261,419 new cases and 416,371 deaths occurred in 2020 . PatientBreast cancer is widely recognized as a heterogeneous disease with a variable clinical and pathological behavior and also different prognosis and response to cancer therapies, which makes it difficult not only to define prognosis of this disease, but also predict the risk of metastasis , 8. It hThis study was approved by the Ethics Committee of Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS). Patient consent was not required as there were no risks anticipated to the participants of the study. All data were stripped of any patient identifiers.This study was a 3-year retrospective multi-center analysis in China from January 1st, 2012 to December 31st, 2014. Female ABC patients were selected randomly from 21 tertiary hospitals of seven geographic regions. Traditionally, China was divided into seven geographical regions: north, northeast, central, south, east, northwest, and southwest, three tertiary hospitals were selected from each region randomly. The aim of this method was to establish a large group of participants in ABC with complete pathological diagnosis and treatment information, which could fully represent the differences between urban and rural areas, including women of different living habits and socio-economic status in different geographical regions.The subjects of this study were pathologically confirmed ABC patients with recurrence and distant metastasis hospitalized within the specified period. The number of collected cases were determined proportionally according to the inherent population capacity of each geographical region. In each hospital, one month was randomly selected for each year according to the random number and all pathologically diagnosed ABC cases for that month were reviewed. January and February were excluded from the random selection to decrease any confounding effects of the Chinese New Year holiday (the longest holiday period of the year). Firstly, all eligible cases were recruited in 2012, if inpatients admissions were less than predetermined numbers, more cases from the neighboring months were reviewed in that year, otherwise, the remaining cases continued to be collected in 2013 and 2014, until the total number in that year reaches the target quantity.All patients enrolled in this study must meet 3 key inclusion criteria: (1) pathology confirmed female ABC, when diagnosed as stage IV or local recurrence/distant metastasis after diagnosis; (2) inpatient admission date was within the selected month in the study hospital and (3) full information of surgical pathology results, time and location of metastasis, detailed therapeutic methods and protocols after metastasis.Medical records were reviewed by local clerks within each hospital according to the designated protocol and a questionnaire was designed by epidemiologists and breast physicians in CHCAMS to collect information from each enrolled patient’s chart regarding (1) General information; (2) Demographic characteristics and breast cancer risk factors; (3) Clinical and imaging diagnostic information at first diagnosis; (4) Molecular subtype of patients; (5) Surgical and adjuvant treatment information; (6) Recurrence and metastasis treatment information including distant metastasis sites, disease-free survival (DFS), endocrine therapy, targeted therapy and chemotherapy after metastasis.Two data-input clerks from each hospital were recruited to independently double-enter all above information from the paper to computer-based database. Then all finished double-entry databases were sent to CHCAMS for validation by running EpiData. Inconsistencies between the two databases revealed by CHCAMS were reported to the local clerks for adjudication until the databases were consistent. As a final inspection, one of the databases was chosen to undergo a final consistency check. Logical errors (e.g. the first recurrence and metastasis date were earlier than the date of the first diagnosis of breast cancer) were reported back to the local hospital, and the local collaborators reviewed the original medical chart again. After checking with the original medical record, the local staff sent the revised database back to CHCAMS for a final analysis. During the consistency check, 5% of the medical charts were randomly selected based on the study ID and sent to CHCAMS for quality control review.The cancers were classified into four molecular subtypes according to the St-gallen Guidelines, 2013 : (1) LumRecurrence and distant metastasis were diagnosed by clinical evaluations including imaging studies or biopsy. Distant metastasis was defined as metastasis of breast cancer developing beyond the ipsilateral or contralateral breast, chest wall, or regional lymph node including ipsilateral axillary, supraclavicular, or internal mammary lymph node. Five patterns of the distant metastasis were mainly classified: bone metastasis only, metastases excluding bone and brain, bone metastasis + others (excluding brain metastasis), brain metastasis only and brain metastasis + others. Cumulative frequency of bone, liver, lung, brain and visceral metastases  in this study was analyzed regarding to the molecular subtypes. Disease free survival (DFS) was defined as the time from the first diagnosis of breast cancer by surgery or puncture to recurrence and/or metastasis, whichever was the earliest.2) test or fisher for categorical variables as appropriate and analysis of variance (ANOVA) for continuous variables. The Kaplan-Meier analysis was used for cumulative recurrence and metastasis rates with a log-rank test to assess the significance among the four molecular subtypes. Cox proportional hazards regression models were used to determine hazard ratios (HRs) and 95% confidence intervals (Cis) for the associations between molecular subtypes and the risk of distant metastasis at specific sites. Potential confounders included age at diagnosis, tumor size, number of lymph node metastasis, menstrual status, education and family history. In the pairwise comparisons, Luminal A was set as the referral group for the better prognosis, all other subtypes compared with it. Statistical significance was assessed using two-tailed tests with a significant level of 0.05. Analyses were conducted using SPSS statistical software version 22.0 .Patients’ demographic and clinicopathological characteristics, pattern of the distant metastasis and cumulative frequency of the metastasis sites were compared within subtypes using chi-square , followed by HER2-enriched , Triple Negative . Clinicopathological characteristics of ABC patients according to molecular subtypes are shown in The difference of the pattern of distant metastasis was significantly different among subtypes P<0.001, Table 2.Cumulative data of distant metastasis were shown in HER2-enriched 2.27, 95% CI 1.65-3.14, HR Luminal B 1.66, 95% CI 1.24–2.23), and the HRs remained statistically significant after extended adjustment to the model . Luminal B, HER2-enriched and Triple Negative were positively correlated to lung metastasis in the univariate as well as in the multivariate analyses. Further, these types of molecular subtypes also increased the risk of visceral metastasis by 23%, 46% and 87% respectively. In terms of brain metastasis, the results showed that triple Negative was associated with a significantly increased risk of brain metastasis  and this association remained significant after full adjustment . However, it made no statistical difference in the risk of bone metastasis of the three subtypes compared with Luminal A, whether by univariate or multivariate analyses.In Cox regression analyses, crude and fully adjusted HRs and 95% CIs for the associations between molecular subtypes and distant metastasis sites are shown in The complex biological behavior of breast cancer is determined by the heterogeneity of its intrinsic molecular phenotypes. There is marked variability in the time interval between treatment of the primary tumor and the occurrence of distant metastases, in the organs involved with distant metastases and in the response to systemic treatment in patients with metastatic breast cancer . Prior sMolecular subtypes of breast cancer, as an important complement to TNM staging, have the propensity to give rise to distant metastasis at specific sites. Recent publications had shown that newer molecular subtypes which used microarrays for gene expression analysis, was the best way to perform such molecular classification –21. HowePrevious studies had demonstrated significant difference in the time of recurrence or distant metastasis within different molecular subtypes. In this study as expected, ABC patients with Luminal subtypes had relatively longer DFS and were prone to bone metastasis with good prognosis. While Triple Negative had a higher cumulative rate than other subtypes and more of them had a propensity of brain metastasis with the shortest DFS, highlighting a substantial clinical burden and unmet need for more effective treatment for these high-risk patients. These results indicated that distant metastasis of breast cancer was a non-random process, and ABC patients with different molecular subtypes have different distant metastasis mechanisms. In our study, we further observed the cumulative recurrence and metastasis rate of different subtypes on age subgroups, showing that the difference of cumulative rates were more significant in younger age group, manifested that the rates of HER2 and TNBC patients were higher than those of older age group, while rates were just opposite in Luminal types with good prognosis at the 3-year time point. Another retrospective study showed that DFS after adjuvant chemotherapy in younger patients with ER-positive tumors was significantly longer than patients with ER-negative disease . Colak eCharacterization of breast cancer metastasis to bone has been most extensively studied since bone is the most common site of distant metastasis . With thHER2 over-expression plays an important role in the proliferation, apoptosis, and angiogenesis of many solid tumors, so it is thought that the prognosis for patients with HER2 gene amplification should be poor. Some studies have shown that HER2-enriched was more likely to have multiple site and visceral metastases, among which liver metastasis was the most common . These fTo date, the association between breast cancer subtypes and lung metastasis in patients with breast cancer have been preliminarily identified . A tissuIt is obvious from studies discussed that Triple Negative is a subtype of breast cancer that is associated with high risk of developing brain metastases, with an associated subsequent poor survival outcome –45. It hAdding to the previous literature, the current study could provide important evidence for decision-makers. From the perspective of physicians, molecular subtypes can be used as an important tool for prognosis assessment of ABC patients, and it is an important supplement in assessing the time and site distribution of postoperative distant metastasis, developing targeted preventive therapeutic schedules for patients, and contributing to personalized screening during postoperative follow-up. For ABC patients, the difference in the distant metastasis pattern and overall prognosis of different molecular subtypes can provide guidance to choosing a reasonable and cost-effective approach for treatment, so as to improve the quality of life and promote rehabilitation. While for health policy maker, understanding the burden of different molecular subtypes of ABC could help in optimizing resource allocation for those who in greater need of advanced treatment.The present study exhibits several strengths. Firstly, our study was the first multi-center hospital-based clinical epidemiological investigations to explore the characteristics of ABC for women in China. All the data was collected from 21 tertiary hospitals in seven geographical regions, representing different cancer burden levels, diagnoses, and treatments. Covering different geographic regions in China would be more informative and less subject to practices specific to individual hospitals which can provided important insights about real-world clinical outcomes for ABC patients from a large nationwide sample. Secondly, convenience sampling was adopted, and the sample size was allocated according to the month to reduce the bias caused by the time of initial diagnosis. In addition, a full review of the medical records permitted a check of the detailed documented metastasis sites, thereby providing a more apparent pattern as well as cumulative frequency of distant metastasis sites.However, several potential limitations of this study should be considered inherent to hospital-based retrospective study. Firstly, the results might be subject to selection bias if confounding factors were not identified or adjusted for in the analyses. In the current study, we adjusted for confounders such as age at diagnosis, tumor size, menstrual status and family history which were thought to have the potential to affect the prognostic outcomes. In addition, molecular typing characteristics still need to be evaluated in conjunction with conventional or established gold criteria such as imaging and pathology.Secondly, the consistency of molecular typing between relapsed and metastatic tumor and primary tumor was not examined in this study, as there have been reported the rate of subtype conversion was 0% in basal-like tumors, 23.1% in HER2-enriched tumors, 30.0% in Luminal B tumors, and 55.3% in Luminal A tumors , 57. In In conclusion, this study provided data of ABC patients to clearly show that molecular subtypes were significant different in metastatic behavior with regard to the sites of distant metastasis as well as recurrence and metastasis rate, suggesting that molecular subtypes could predict the preferential site of distant metastasis in female ABC patients. Despite improving breast cancer outcomes, distant metastasis remains common and incurable, especially for the ABC patients. These observations could potentially be used in determining the appropriate schemes for follow-up of ABC patients and hopefully shed light on the development of effective surveillance strategies and targeted therapies. Therefore, future studies are warranted and can ultimately lead to the tailoring of individualized comprehensive treatment fields based on molecular subtypes combined with the conventional clinicopathologic characteristics.qiaoy@cicams.ac.cn.The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Requests to access the datasets should be directed to You-lin Qiao, The studies involving human participants were reviewed and approved by Cancer Hospital, Chinese Academy of Medical Sciences. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Y-LQ, J-YW, and B-HX had access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Y-LQ and B-HX participated in the study concept and design. SZ acquired and analyzed data. J-HF, SZ, and HY were responsible for interpreting the data. J-HF, SZ, and HY took the lead in drafting the report. The other co-authors collected the data from the corresponding study hospital. Every author participated in editing and finalization. The work reported in the paper has been performed by the authors, unless clearly specified in the text. All authors contributed to the article and approved the submitted version.


Long noncoding RNAs (lncRNAs) play important regulatory roles in a variety of pathological processes involving cancer. However, the exact molecular mechanisms of lncRNA regulation in endometrial carcinoma (EC) remain poorly defined. The aim of this study was to illustrate the mechanism of LINC00958 in regulating the function of IGF2BP3, an RNA binding protein involved in mRNA stability, and their clinical implications in EC. First, we investigated the clinical role of IGF2BP3 in EC and demonstrated its prognostic value. Loss-of-function and gain-of-function studies showed that IGF2BP3 promoted EC cell proliferation, migration and invasion. Then, we carried out RNA immunoprecipitation sequencing (RIP-seq) analysis, RNA pulldown and immunofluorescence-RNA fluorescence in situ hybridization to identify LINC00958 that interacted with IGF2BP3 in the cytoplasm of EC cells. Rescue experiments indicated that knockdown of LINC00958 partially offset the EC cell progression mediated by IGF2BP3. After that, RNA sequencing was used to screen out the downstream genes of IGF2BP3 and LINC00958. The results revealed that IGF2BP3 upregulated E2F3 expression by interacting with LINC00958. Furthermore, RNA stability assays demonstrated that silencing LINC00958 partially rescued the IGF2BP3-mediated promoting effect on the mRNA stability of E2F3. Collectively, this study suggests that LINC00958, as an oncogene, assists IGF2BP3 in stabilizing E2F3 mRNA and ultimately promotes EC progression, providing a promising therapeutic target for patients with EC. Endometrial carcinoma is the most common malignant tumor of the female reproductive system in developed countries , 2. StanLong noncoding RNAs (lncRNAs), referred to as transcripts of noncoding RNAs over 200 nucleotides in length, are involved in various tumorigenesis processes –9. We prThe insulin-like growth factor mRNA-binding protein family (IGF2BPs) consists of three proteins (IGF2BP1-3) . IGF2BP3In this research, we demonstrated that IGF2BP3 facilitated EC development and subsequently screened out LINC00958 interacting with IGF2BP3 by RIP-seq. We further revealed that LINC00958 could promote the ability of IGF2BP3 to enhance the mRNA stability of E2F3. The results of our study illustrate novel insights into the regulation of EC progression mediated by lncRNAs and IGF2BP3, which provide new ideas for therapeutic strategies in EC.To investigate the role of IGF2BP3 in EC, we first used data retrieved from the UALCAN platform . and fouTo investigate the oncogenic functions of IGF2BP3 in EC, we stably silenced the expression of IGF2BP3 in Ishikawa and HEC-1-A cell lines and confirmed their knockdown efficiency Fig. . The res2FC > 2.0 and FDR < 0.01. The results indicated that ten differentially expressed lncRNAs possessed the potential capacity to bind with IGF2BP3 in EC cells  assays to identify the lncRNAs involved in the regulation of IGF2BP3 function. One hundred ninety-one candidates as IGF2BP3-interacting lncRNAs were identified in the RIP-seq results. Then, we exploited RNA-seq web tools in Lnc2Cancer 3.0  to obtaills Fig. .Fig. 3IGTo verify the lncRNAs that could actually bind with IGF2BP3, we overexpressed IGF2BP3 in EC cells and found that only the interaction between IGF2BP3 and LINC00958 was increased by RIP-qPCR, which meant that LINC00958 specifically bound to the IGF2BP3 protein Fig. . MoreoveIt has been reported that LINC00958 has oncogenic properties in EC and predicts poor overall survival , 22. As Functional assays demonstrated that the increased ability to proliferate, migrate and invade in IGF2BP3-overexpressing EC cells was partially rescued by the knockdown of LINC00958 Fig. . CollectP < 0.05)  to identify the differentially expressed genes when LINC00958 was silenced in Ishikawa cells. A total of 305 mRNAs were regulated after LINC00958 knockdown  Fig. . Subsequ05) Fig. . We furt05) Fig. . Consist05) Fig. . Finally05) Fig. . Taken tSeveral studies have demonstrated that E2F3 is a cancer-promoting gene in EC , 24. To To confirm the role of IGF2BP3 in EC proliferation in vivo, we established subcutaneous xenograft nude mouse models. Consistent with the in vitro results, knockdown or overexpression of IGF2BP3 in HEC-1-A cells contributed to the corresponding decrease or increase in the growth of subcutaneous xenograft tumors Fig. . ImmunohMounting evidence indicates that IGF2BPs, as RNA binding proteins, improve the stability and storage of many target mRNAs, such as MYC, and thus influence gene expression , 25, 26.The expression of IGF2BP3 in EC tissues has been shown to be elevated compared to that in normal endometrial tissues, especially in endometrial serous carcinoma and clear cell carcinoma , 31. HowAs an RNA binding protein, IGF2BP3 modulates the stability of multiple mRNAs to facilitate cancer development. In triple-negative breast cancer, IGF2BP3 and IGF2BP2 synergistically recruited CNOT1 complex, reduced the stability of progesterone receptor and ultimately promoted the migration and metastasis of breast cancer . High IGIn this study, we applied the RIP-seq assay to identify LINC00958 that interacted with IGF2BP3. LINC00958 was originally recognized as a cancer-promoting gene in bladder cancer . SeveralTo further explore the downstream genes of the LINC00958/IGF2BP3 axis in EC, we combined RNA sequencing results and genes coexpressed with IGF2BP3 from the TCGA database and discovered that E2F3 was the downstream effector. E2F3, as a member of a small family of transcription factors, is involved in the tumorigenesis of many malignancies –44. Somehttp://www.cuilab.cn/sramp/) [A prominent study reported that IGF2BP1/2/3 targeted many mRNA transcripts by recognizing the consensus sequence of GG (m6A) C, thus enhancing the stability of target mRNA and regulating gene expression . RNA sta/sramp/) . Two sitIn general, this study revealed that IGF2BP3 could promote the proliferation, migration, and invasion of endometrial carcinoma. Mechanistically, IGF2BP3 played a moderating role by altering the stability of E2F3 mRNA. Moreover, LINC00958 helped IGF2BP3 complete its function through binding with it without changing its expression level. The results of our study illustrated novel insights into the regulation of endometrial carcinoma progression mediated by lncRNAs and IGF2BP3, which highlighted the value of IGF2BP3 combined with LINC00958 as predictors for prognosis and therapeutic candidates in EC.All tissue specimens were taken from patients who underwent curettage or hysterectomy at Shengjing Hospital Affiliated with China Medical University  from January 2011 to December 2019. They included 65 fresh endometrial carcinomas and 52 normal endometrial tissues, 58 paraffin-embedded endometrial carcinomas and 20 normal endometrial tissues. All of the patients received no preoperative radiation or chemotherapy treatments. All the samples were diagnosed by at least two qualified histopathologists according to the staging criteria of the International Federation of Obstetrics and Gynecology (2009). The study was approved by the relevant ethics committee of our institution, and each patient gave informed consent to our research.Serial 4 μm thick paraffin sections were used for immunohistochemistry with antibodies specific for IGF2BP3 , E2F3 , and Ki67 . A streptavidin peroxidase (SP) immunohistochemical kit  was used according to the instructions. Images were taken with a Nikon microscope . Image-Pro Plus 6.0 was employed to analyse the levels of protein expression by calculating the values of mean integrated optical density  for statistical analysis .Lentiviruses overexpressing IGF2BP3 or LINC00958 and hairpin-derived small RNAs (shRNAs) targeting IGF2BP3 or LINC00958 were purchased from Shanghai GeneChem of China. Seventy-two hours after lentivirus infection, 2.5 µg/ml puromycin was applied to screen for stably infected cells. Small interfering RNAs (siRNAs)  targeting E2F3 were introduced into EC cells by JetPRIME transfection reagent . The target sequences of shRNAs and siRNAs are listed in Table The cells were harvested and lysed on ice with RIPA lysis buffer . The BCA protein concentration determination kit  was used to determine the protein concentration. After boiling for 10 min, proteins were separated by 10% SDS–PAGE and then transferred to PVDF membranes . The membrane was subsequently blocked for 1 h in 5% fat-free milk, incubated with the primary antibodies for 1.5 h at 25 °C, and then incubated with the horseradish peroxidase-labeled secondary antibody  for 1 h at 25 °C. After repeated washing with TBST, the membranes were developed by an enhanced chemiluminescence kit . The primary antibodies used were as follows: GAPDH , IGF2BP3 , and E2F3 .We performed an RIP assay with an RNA Immunoprecipitation (RIP) Kit  according to the manufacturer’s protocol. Cells were lysed and incubated with 4 µg antibodies against IGF2BP3  overnight at 4 °C with constant rotation. IgG was used as a control. Input and coimmunoprecipitated RNAs were extracted, reverse-transcribed and analysed by qPCR.In human Ishikawa and HEC-1-A cells, RNA pulldown assays were conducted according to the instructions of an RNA–protein pulldown kit . Biotin-labeled LINC00958 was synthesized by GenePharma  and interacted with cell lysates for 4 hours. LacZ was employed as the negative control. Streptavidin magnetic beads pulled down the proteins bonded with biotin-labeled LINC00958 or LacZ following overnight incubation. The generated protein-RNA-bead compounds were collected, and then the proteins were eluted. Finally, western blot assays were used to test the retrieved proteins. The primary antibodies used were as follows: GAPDH , IGF2BP3 .We conducted fluorescence in situ hybridization of RNA with a Ribo Fluorescent in Situ Hybridiazation Kit  according to the manufacturer’s protocols. Specific FISH probes for LINC00958 were designed and synthesized by RiboBio. We captured all images with a confocal laser scanning microscope .EC cells on glass coverslips were incubated with specific antibodies against IGF2BP3  overnight at 4 °C and then incubated with Alexa Fluor 488 goat anti-rabbit IgG . To detect colocalization, we treated the cells with FISH probes and the anti-IGF2BP3 primary antibody in hybridization buffer at the same time. Images were captured by a confocal laser scanning microscope .We seeded EC cells into 12-well plates until 70–80% confluency after 24 h. Then, we added 5 µg/ml actinomycin D  to the growth medium and collected them at 0 h, 2 h, 4 h, and 6 h. Total RNA was extracted, reverse-transcribed to cDNA and amplified by qPCR. The half-life of mRNA was calculated according to a previously published article .6 HEC-1-A cells were subcutaneously injected into the right axilla of 4–6-week-old female BALB/c nude mice (n = 5 per group), which were purchased from HFK Bioscience . Tumor growth rates were monitored every week. The calculation formula used for the tumor volume was V = π/6 × length×width2. Three weeks after injection, the mice were sacrificed. Then, the tumor samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned for immunohistochemical staining.The animal experiments were approved by the ethics committee of Shengjing Hospital affiliated with China Medical University  and performed according to the relevant regulatory standards. A total of 1 × 10t tests were used to compare statistical significance between two groups. When more than two groups were compared, we used one-way analysis of variance. P < 0.05 were defined as statistically significant.GraphPad Prism 8.0  and SPSS 23.0  were applied for all statistical analyses. The results are presented as the mean ± standard deviation. We adopted the chi-squared test to evaluate relationships between IGF2BP3 expression and the clinicopathologic features of patients. The Kaplan–Meier method with log-rank tests was employed to analyse overall survival. The linear relationships between two variables were analysed by Pearson’s correlation coefficient. Independent sample Student’s Original picturesOriginal western blot picturesSupplementary figure and table legendsSupplementary MethodsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Table 1Supplementary Table 2


Oxidative stress represents one of the principal causes of inherited retinal dystrophies, with many related molecular mechanisms still unknown. We investigated the posttranscriptional RNA editing landscape of human retinal pigment epithelium cells (RPE) exposed to the oxidant agent N-retinylidene-N-retinyl ethanolamine (A2E) for 1 h, 2 h, 3 h and 6 h. Using a transcriptomic approach, refined with a specific multialgorithm pipeline, 62,880 already annotated and de novo RNA editing sites within about 3000 genes were identified among all samples. Approximately 19% of these RNA editing sites were found within 3′ UTR, including sites common to all time points that were predicted to change the binding capacity of 359 miRNAs towards 9654 target genes. A2E exposure also determined significant gene expression differences in deaminase family ADAR, APOBEC and ADAT members, involved in canonical and tRNA editing events. On GO and KEGG enrichment analyses, genes that showed different RNA editing levels are mainly involved in pathways strongly linked to a possible neovascularization of retinal tissue, with induced apoptosis mediated by the ECM and surface protein altered signaling. Collectively, this work demonstrated dynamic RNA editome profiles in RPE cells for the first time and shed more light on new mechanisms at the basis of retinal degeneration. In addition to transcription, posttranscriptional RNA modifications (PTMs) contribute to extending function and diversity of transcripts. One of the most interesting PTMs is RNA editing, which increases biologically relevant diversity of transcripts or protein isoforms . RNA ediPresently, two major canonical RNA editing types in mammals are known, including adenosine-to-inosine (A-to-I) editing (the most frequent in vertebrates) under the catalysis of adenosine deaminase acting on RNA (ADAR) proteins , and cytRNA editing can also occur in the form of U-to-C, G-to-A and others, collectively called “alternative RNA editing”. U-to-C was initially identified in the mRNA of Wilm’s tumor 1 (WT1) human transcript , while GFurthermore, primary transcripts of tRNA genes, tRNA precursors, are heavily processed  before they fold into functional mature tRNAs. Thus, it is not surprising that editing events can also occur in tRNA, and such modifications are catalyzed by the ADAT (adenosine deaminase acting on tRNA) family enzyme. For example, tRNA genes with defectively paired or incomplete acceptor stems can be transcribed into tRNA precursors whose acceptor stem is reconstructed in a template dependent editing pathway. Other examples of editing involve the insertion or deletion of single bases or short sequences in the tRNA precursor . MicroRNAs (miRNAs) and their binding sites may also be processed by ADARs to control miRNA biogenesis and regulation activity .Such a complex scenario has highlighted the need for novel technologies to investigate the effects of RNA editing events, and the advancement of computational methods has enabled the elucidation of many RNA editing sites in humans . Today, cis-retinaldehyde (11-cis-RAL) or all-trans-retinal retinaldehyde  [In the present work, for the first time we describe the RNA editing event landscape of retinal pigment epithelium (RPE) cells in a physiological condition versus exposition to the oxidant agent N-retinylidene-N-retinyl ethanolamine (A2E). A2E is a toxic bis-retinoid deriving from the condensation and oxidation of 11-ans RAL) ,24, capaans RAL) ,26. Evenans RAL) ,28. Humaans RAL) ,30. An a®  with 2% v/v fetal bovine serum (FBS), 1% penicillin/streptomycin, and then incubated at 37 °C with 5% CO2. H-RPE cells were then plated into 96-well plates (4 × 104 cells/well) and cultivated for 24h to reach confluence before A2E  addition, in a final concentration of 20 μM for 6h and following rinsing with medium. A2E was synthesized by incubating ethanolamine  and all-trans-retinal  in ethanol (3 mL) at room temperature for 2 days in the dark. A2E was then purified by silica gel chromatography, and purity was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). HPLC-purified A2E was exposed to room light for varying time periods to provide mixtures of A2E-related photoisomers. Control cell groups were incubated without A2E. Confluent cultures were transferred to PBS supplemented with Mg2+, Ca2+ and glucose, before exposure to blue light emitted by a tungsten-halogen source  for 30 min to induce phototoxicity of A2E and subsequent incubation at 37 °C, as already described by confirmed protocols [Human RPE-derived cells  were cultivated following previously described protocols . Cells arotocols ,33,34. ACell viability was determined by mitochondrial-dependent reduction of methylthiazolyldiphenyl-tetrazolium bromide (MTT)  to formazan-insoluble crystals, following a previously described protocol . ResultsTM Reagent  following manufacturer’s protocol, and quantified at Qubit 2.0 fluorimeter. The RNA-Seq samples consisted of 3 factor groups, represented by H-RPE cells, before treatment with A2E and at 4 following time points of 1 h, 2 h, 3 h and 6 h, chosen on the basis of our precedent experiences [Whole RNA was isolated by TRIzoleriences . These rhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 17 August 2022) and QualiMap  [Obtained raw sequences were filtered to remove low-quality reads (average per base Phred score <30) and adaptor sequences. The quality of analyzed data was checked using FastQC (v.0.11.9)  , while thttps://digitalinsights.qiagen.com/products-overview/analysis-and-visualization/qiagen-clc-genomics-workbench/, accessed on 17 August 2022), BWA_MEM v.2.2.1 (accessed on 17 August 2022) [In the first phase, filtered reads were aligned using a combination of mappers; in detail, CLC Genomics Workbench v.22.0 (st 2022) , HISAT vst 2022) , TopHat st 2022) , STAR 2.st 2022)  and RASEst 2022)  against After the previous individual mappings, the best record for each read from among the six available was chosen according to the following rules: (1) mapped reads preferred to unmapped; (2) among mapped reads, those characterized by more matches are always better than those with fewer; (3) among mapped reads with the same number of matches, records presenting fewer indels were preferred.10 scale, with integer granularity) were considered better; (3) pairs in forward-reverse orientation were preferred; (4) pairs from the same aligner and database combination were ultimately preferred.In the presence of multiple candidates, the best-read pair was chosen based on the following criteria: (1) pairs on the same chromosome are preferred to pairs mapping on different chromosomes; (2) pairs that are closer (on a loghttps://software.broadinstitute.org/gatk/ (accessed on 17 August 2022)).Finally, the individual aligned BAM files were sorted and merged, duplicates were removed, and the files were indexed using Picard tools SortSam, MergeSamFiles and Mark-Duplicates. Recalibration of the aligned reads was then performed by the Genome Analysis Toolkit (GATK) (v.4.1.3.0)  tool of the Ensembl Genome Browser  was used for annotation of the de novo editing sites as well as for classification, according to functional class [Once the alignment phase was concluded, the RNA editing detection step was performed. The analytic pipeline started with variant calling using the fixed ploidy variant detection in the CLC Genomics Workbench suite, reporting variants with >95% probability. The algorithm behind the Fixed Ploidy Variant Caller combines a Bayesian model (examining posterior probabilities) with a maximum likelihood approach. Found variants were then annotated by the ANNOVAR v.20211019 (accessed on 17 August 2022) tool and included databases, limiting analysis to single nucleotide variants (SNVs) and removing false editing events due to germline variants, paralogous mapping and homopolymer regions. Furthermore, to rescue low-confidence editing events for which editing was not detected de novo, we compared identified editing sites with ones annotated in the RADAR (accessed on 17 August 2022)  and REDIal class . In ordehttps://github.com/BGI-shenzhen/BamDeal, accessed on 17 August 2022), identified RNA editing events were filtered on the basis of high-confidence, defined by the following criteria: (1) an editing site showing read coverage of >100 required at least 3 mutant reads to be considered edited with high confidence; (2) sites with 20–99 reads of coverage needed at least 2 mutant reads; (3) for sites with <20 reads of coverage, only 1 mutant read was required to consider them as high confidence. Such thresholds were based on analysis of adjacent control sites that were within 2 base pairs upstream or downstream of the identified RNA editing sites in all RNA-Seq sample data, determining the rate of true-positive editing, quantifying the background error mutation rate. The editing level of each site was calculated as Gs/As + Gs. The overall editing level of each sample was calculated as the number of Gs divided by the total number of As + Gs at all editing sites.Using the BamDeal toolkit  genes edited in both 3 h and 6 h treated samples but showing different editing events and frequency; (2) genes only edited in 3 h treated samples; (3) genes only edited in 6 h treated samples. For each editing site, editing ratio between treated samples and/or control was calculated. Frequencies of editing events were calculated by IBM SPSS Statistics 26.0 software (accessed on 17 August 2022)) .p-value of <0.05 were considered. In order to explore the effect of RNA editing on miRNA targeting, the flanking 13 bp-long sequence of the editing site (6 bp each side) and the seed of known miRNA were compared. A site was regarded as a candidate binding site if any 7 bp sequence could be completely complementary to a known miRNA seed . Subsequp-values of <0.05 filtered and evaluated. Furthermore, as the FCs were linear, they were replaced by log2 values to make the variation more noticeable .From previous mappings, the paired end reads were categorized and assigned to the genes, according to abundances, using the expectation-maximization (EM) algorithm . Shortly−ΔΔCt method [p-values of < 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS 26.0 software (accessed on 17 August 2022) [The different deaminase dysregulated mRNAs previously identified were validated by quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) to confirm RNA-Seq data results, following an already described protocol . Each ret method . Statistst 2022) . The lisThe MTT cell viability assay highlighted a significant, different trend in RPE-treated cells versus control. The addition of A2E to cultures led to a dose-dependent increase in cell death percentage .ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/, accessed on 17 August 2022). All previous mapping statistics were based on average values calculated for all three replicates in each time point. RNA sequencing carried out on Illumina NovaSeq 6000  yielded a total of approximately 96 million quality reads  with a percentage of 68% uniquely mapped. About 16,200 genes and 70,000 transcripts were identified out of 60,609 and 227,462 reference counterparts, respectively, considering the whole human transcriptome. The annotated reference assembly v.40 (GRCh38.p13) was downloaded from GeneCode FTP server  . We nextMEAF6, CCDC69, OGDH, MLF2, MYO1E, MYO9A, TUBB6, CTD-2369P.2.5, COPE, FBX017, TMEM91, EWSR1 and MRMR3 , especially ALU elements. Interestingly, the number of editing modifications in ALU repeats drastically reduced during time exposure, especially if directly compared to controls B. The ednd MRMR3 .One of the most interesting aspects related to RNA editing deals with its role in coding regions as a cause of non-synonymous shifts. The most non-synonymous events involved the same sites for all considered time points, with the highest percentage reached after the first 3 h of treatment C.As shown in The known editing site characterization for events common to the whole treatment is summarized in the graphs in The characterization of de novo RNA editing events foresaw the detection of 12 different types already described for known events, with 35.1% belonging to the canonical A-to-I and C-to-U . This tiMAN2C1 (AluSx) and MLF2 (AluSc)  . Intrigu (AluSc) B.Interestingly, also for de novo analysis, most non-synonymous editing involved the same sites for all considered time points but showed a constant decrease from start to end of treatment, with the lowest percentage reached after 6 h of A2E exposure C. The VEP prediction permitted us to better detail the number of differences of RNA editing sites among the various genomic regions. Most edited sites common to all considered time points harbored 3′UTR (25.1%) and synonymous (22.6%) variants, followed by missense (13.8%) and downstream (12.3%) ones. The lowest number of differentially edited sites was detected at regulatory region and stop retained level, with only one editing site changed D.The de novo editing site characterization for events common to the whole treatment is summarized in the graphs in common = 1059, n° edited genes3h = 194, n° edited genes6h = 107), and involved in signal transduction . Interestingly, the editing events that occurred during A2E exposure seemed to alter the catalytic activity of many enzymes , as well as transcription regulation of specific genes after 3 h (n° edited genes3h = 19) or 6 h from the treatment (n° edited genes6h = 14). More differences among considered time points were evidenced regarding biological pathways. Common edited genes were mainly involved in integrin family cell surface interactions (n° edited genescommon = 285), while the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) signaling pathway and proteoglycan syndecan-mediated signaling involved the highest number of editing events after 3 and 6 h from treatment start, respectively. Details on edited gene pathway enrichment are available in Most editing events that occurred during the whole treatment, especially after 3 h and 6 h from treatment, were carried by genes encoding protein products acting mainly within cytoplasm . According to enrichment analysis, the impacted target genes were mainly involved in signal transduction (22.5%), cell communication (21%), regulation of nucleic acid metabolism (18%), transport (7.5%) and apoptosis (2%) (Like miRNA binding site single nucleotide polymorphisms (SNPs), RNA editing sites within miRNA binding sites may impact interactions between miRNA and target RNA. Approximately 19% between known and de novo RNA editing sites were located within 3′ UTR, which included sites common to all time points that were predicted to change the binding capacity of 359 miRNAs towards 9654 target genes. In detail, a global decrease in edited miRNA binding sites was seen, as well as their related targets (n° miRNA edited binding sitessis (2%) . Interes2FC3hvsCTRL = 1.18, p-value = 0.01), while the lowest was represented by another member of the ADAT family, adenosine deaminase tRNA specific 3 (ADAT3), but during the late stages of treatment . The canonical A-to-I editors, instead, showed a more gradual variation, with the highest expression changes reached by adenosine deaminase RNA specific (ADAR) during the last 6 h of exposure, and with an oscillatory trend manifested by ADAR2 . Finally, the cytidine deaminases within the activation induced cytidine deaminase/apolipoprotein B editing complex (AID/APOBEC) family, mainly responsible for C-to-U mRNA editing, were all overexpressed during the whole treatment, with the highest peak reached by APOBEC3C after 6 h of A2E exposure . The deaminases selected mRNAs were validated by qRT-PCR analysis and the obtained expression profiles were similar to the results of transcriptome analysis (data not shown). Moreover, ANOVA statistics, conducted to compare means among multiple groups, highlighted high significance .The transcriptome experiment carried out permitted the analysis of expression levels of the main enzyme families involved in editing events. Overall, an increased expression of all editing enzymes was seen during each step of analysis, with the lowest growth from 3 h to 6 h of treatment . The higEukaryotic organisms are characterized by complex transcriptomes whose regulation is critical for all cellular processes and homeostasis . The dynWith the growing adoption of genome re-sequencing and RNA-Seq technologies, a considerable number of RNA editing sites in the genome of several animal species, Homo sapiens included, have been identified . CurrentTo our knowledge, this is the first systematic study of RNA editing in human retinal cells. We detected a total of 62,880 already annotated and de novo RNA editing sites throughout all time-related samples. Approximately 20,000 RNA editing sites were shared by control and both 3 h and 6 h treated RPE cells, and a global decrease in editing events was seen from start to end of A2E exposure. Then, 31,324 RNA editing sites were found only in the control samples, 6637 were found only in RPE cell samples after 3 h of treatment, and 4562 were detected only in RPE cells after 6h from A2E exposure, which suggested that the induced oxidative stress could heavily impair RNA editing activity. Twelve types of RNA editing were identified, among which A-to-I and C-to-U were identified as being the canonical RNA editing events, accounting for about 36% of all identified sites, whereas the remaining RNA editing types were the non-canonical ones . A-to-I MEAF6, CCDC69, OGDH, MLF2, MYO1E, TUBB6, CTD-2369P.2.5, COPE, FBX017, TMEM91, EWSR1, MTMR3, MAN2C1) involved in the “intracellular anatomical structure” molecular pathways (GO:0005622), especially in the cytoplasm and nucleus. Our data revealed that a significant number of RNA editing events occurred in the coding regions, followed by intron and regulative ones, also in repetitive elements, suggesting that RNA editing may control nuclear retention and alternative splicing. In mammals, canonical editing frequently occurs within Alu repeat elements, which are mammal-specific SINEs . It is aAnnotation of the edited genes with missense mutations permitted us to speculate on a possible neovascularization of retinal tissue, with induced apoptosis mediated by the extracellular matrix (ECM) and surface protein impaired signaling. Such pathways were already shown as possibly involved in RPE oxidative stressed cells, as already evidenced after the toxicity induced by A2E ,75. Commcis-regulatory impact on gene expression by influencing mRNA degradation and stabilization. In particular, the detection of RNA editing sites potentially related to miRNA activity on LRAT gene, fundamental for the esterification of all-trans-retinol to all-trans-retinyl esters and already associated in carcinomas [In addition to missense editing sites, several RNA editing sites detected in our study were identified in UTRs of different transcripts, especially 3′UTR, and might alter miRNA-mediated posttranscriptional gene silencing and expression. The role of miRNAs in oxidative stress induced RPE cells is well described in literature , but no rcinomas  was intercinomas . These eRecent advances in RNA-editing research have led to the creation of several RNA editing resources for humans. However, a database of RNA editing for the human retina is currently lacking. Thus, we created a comprehensive and dynamic atlas of the RNA editome in RPE cells across four different A2E exposure time points  after the basal one. To our knowledge, this is the first genome-wide atlas of the RNA editome of the human retina. We identified different clusters of RNA editing sites associated with ECM and vascularization alterations, with consequent induction of RPE cell apoptosis. However, it has to be underlined that obtained results were produced with the condition of huge cell death at 6h from treatment. Thus, further experiments will be performed with a less dramatic stress stimulus and then progress towards an irreversible situation, to propose a combined functional assessment of the most relevant RNA editing which can eventually be made reversible upon intervention. At the same time, it will be useful to improve knowledge on the specific role of editing regulation on identified molecular pathways.In conclusion, our present work provides information regarding the dynamic landscape of RNA editing during an experiment of induced oxidative stress in RPE cells, which might be useful for biomedical researchers who study retinal degeneration mechanisms to find new molecular markers.