Human adenoviruses (HAdV) express either one or two virus-associated RNAs (VA RNAI or VA RNAII). The structure of VA RNA resembles human precursor microRNAs (pre-miRNA), and, like human pre-miRNA, VA RNA can be processed by DICER into small RNAs that resemble human miRNA. VA RNA-derived miRNA (mivaRNA) can mimic human miRNA post-transcriptional gene repression by binding to complementary sequences in the 3′ UTR of host mRNA. HAdV14 is a member of the B2 subspecies of species B adenovirus, and the emergent strain HAdV14p1 is associated with severe respiratory illness that can lead to acute respiratory distress syndrome. Utilizing small RNA sequencing, we identified four main mivaRNAs generated from the HAdV14/p1 VA RNA gene, two from each of the 5′ and 3′ regions of the terminal stem. There were temporal expression changes in the abundance of 5′ and 3′ mivaRNAs, with 3′ mivaRNAs more highly expressed early in infection and 5′ mivaRNAs more highly expressed later in infection. In addition, there are differences in expression between the emergent and reference strains, with HAdV14 expressing more mivaRNAs early during infection and HAdV14p1 having higher expression later during infection. HAdV14/p1 mivaRNAs were also shown to repress gene expression in a luciferase gene reporter system. Our results raise the question as to whether differential expression of mivaRNAs during HAdV14p1 infection could play a role in the increased pathogenesis associated with the emergent strain. Human adenoviruses (HAdVs) are non-enveloped viruses with a linear double-stranded DNA genome of ~36,000 bp. HAdVs are grouped into seven species (A–G) and consist of over 100 different types (consecutively numbered) based on genomic sequencing. HAdVs can infect the respiratory, gastrointestinal, and urinary tracts as well as the conjunctiva. Overall, HAdV usually results in mild, self-limited infections in immunocompetent individuals. HAdV14 is a member of the B2 subgroup of adenovirus and, as such, predominately causes kidney and urinary tract infections but can also be associated with respiratory infections. Outbreaks of emergent strains of Ad have resulted in severe and sometimes fatal infections in otherwise healthy people. In the past fifteen years, an emergent strain of HAdV14, HAdV14p1, emerged first in the U.S. and subsequently throughout the world ,6. HAdV1The HAdV genome encodes roughly 40 proteins. In addition to protein encoding RNAs, non-coding RNAs (ncRNA) are also present in HAdV-infected cells ,11,12. VVA RNA forms a complex secondary structure that is critical for its biological function ,20,21. ORecently, new roles for VA RNAI in regulating cellular miRNA processes have been elucidated. Following transcription, VA RNAI is exported from the nucleus to the cytoplasm by Exportin 5 (Exp5). Exp5 exports RNA molecules  that contain a ‘mini-helix’ from the nucleus ,34,35,36Group B2 HAdV members encode only the VA RNAI gene. Bioinformatic analysis of the HAdV14 genome showed that the VA RNAI gene of Ad14 is ~98% identical to the other group B2 members HAdV11, HAdV34, and HAdV35 . In this2 in DMEM supplemented with 5% heat-inactivated bovine calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. A549 cells were monitored for mycoplasma contamination by PCR and validated by short tandem repeat markers (STR Authentication) (ATCC). HAdV14 deWit was obtained from ATCC, and HAdV14p1 (isolate 1986T) was obtained from the United States Navy [A549 cells  were grown at 37 °C and 5% COtes Navy ,35,36. VA549 cells were infected with either HAdV14 or HAdV14p1 at a multiplicity of infection (MOI) of 10 pfu/cell in suspension for 1 h at 37 °C, after which cells were plated and allowed to adhere until collected. Adherent and non-adherent cells were collected at 6, 12, 24, 36, and 48 h post-infection. Total RNA was isolated using miRNeasy kit  with on column DNase treatment. The total RNA in each sample was quantified using the Qubit 2.0 Fluorometer , and quality was measured using the RNA6000 Nano chip on the Agilent 2100 Bioanalzyer . All samples had an RNA integrity number greater than 7.TruSeq Small RNA library prep kit  was used to create sequencing libraries. Specifically, adapters were ligated using the 5′ phosphate and 3′ hydroxyl groups common to most mature miRNAs. After adapter ligation, samples were reverse transcribed and amplified. Finally, the libraries were size selected using a 6% polyacrylamide gel and concentrated using ethanol precipitation. Purified libraries were normalized and pooled to create a double-stranded cDNA library and were sequenced on the Illumina MiSeq to render 50 base pair single end reads at the Loyola Stritch School of Medicine Genomics Facility. Adapter sequences were removed, and low-quality reads were trimmed from raw sequencing reads using Cutadapt (v. 1.11). The resulting reads were mapped to the HAdV14 deWit genome (GenBank accession number AY803294) in CLC Genomics Workbench (Qiagen).Differential expression was conducted with CLC Genomics Workbench. The VA RNA sequence was added to miRbase 22 and annotated to contain either total 5′ and 3′ reads or mivaRNA 5′A, 5′G, 3′A and 3′C seeds. Quantify miRNA 1.2 (CLC) was used with the following settings: allow for length-based isomiRs, no additional upstream bases, 5 additional downstream bases, no missing or mismatch bases, minimum sequence length = 18, maximum sequence length 25. Differential Expression for RNA-Seq 2.6 (CLC) was used for differential expression analysis with normalization set to Trimmed Mean of Means (TMM).p < 0.05 considered significant .The complement of the either the 5′ or 3′ seed sequences of Ad14 VA RNA were cloned into the multiple cloning site of pmirGLO Dual Luciferase Vector . Constructs were sequenced to confirm the correct orientation of the complimentary HAdV14 VA RNA seed sequences. Plasmids were purified using PureLink HiPure Plasmid Maxi Prep (Invitrogen) and transfected into A549 cells using Lipofectamine LTX (Invitrogen). Twenty-four hours after transfection, cells were infected with Ad14 at an MOI of 10. After an additional 24 h, luciferase activity was determined using a dual-luciferase assay (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. One-way ANOVA was followed by the Holm-Sidák test with https://www.genome.jp/kegg/, accessed on 2 May 2021) was used to predict the cellular pathways and processes potentially regulated by mivaRNA–target mRNA interaction.TargetScan and miRDB were used to predict cellular targets for mivaRNAs by seed sequences and filtered to remove duplicate targets. KEGG  on an Illumina HiSeq4000 set for pair ends 100 bp reads and demultiplexed. Reads were trimmed with Trim Reads 2.4 (CLC) before RNA-seq Analysis 2.21 (CLC) was performed, mapping to the human genome (Hg38) with a mismatch cost of 2, insertion cost of 3, deletion cost of 3, and length/similarity fractions set to 0.8. Paired reads were counted as a single read and expression value was set to reads per kilobase million (RPKM). Differential expression for RNA-Seq 2.4 (CLC) using gene expression values from RNA-seq analysis was performed and the results were filtered to the predicted mivaRNA targeted genes. Ingenuity Pathway Analysis (IPA) (Qiagen) was used for functional enrichment with the differential RNA-seq analysis from HAdV14-infected cells at 36 hpi vs. uninfected cells. FDR was set to 0.05 with absolute expression fold changes being >1.5 and expression intensity being >1.9)WG) found in the central domain [Like other HAdV group B2 members, HAdV14 and HAdV14p1 genomes encode only 1 VA RNA sequence that resembles VA RNA-I at nucleotides 10,452–10,613 . Houng al domain . HAdV14 Previous studies have shown that the majority of small RNAs produced during HAdV infection map to the VA RNA gene(s). To examine the viral small RNAs produced during HAdV14 or HAdV14p1 infection, A549 cells were infected with either HAdV14 or HAdV14p1 . Four inWhile there is diversity in the small RNAs that map to the VA RNA gene, repression of target gene expression involves the binding of miRNA to the target mRNA through the miRNA seed sequence, nucleotides 2–8 of the miRNA, and its complement on the target gene mRNA. Therefore, the seed sequences provide a better metric for both identifying and quantitating HAdV mivaRNAs. Unique miRNA seed sequences were identified for each small VA RNA molecule . The totInfection with HAdV14 or HAdV14p1 resulted in differential expression of 3′ small VA RNAs . With thDICER processes pre-miRNAs and VA RNA into mature miRNA and mivaRNA, respectively ,39. DuriSince HAdV VA RNAs are processed by DICER into mivaRNAs, we hypothesized that the mivaRNAs can repress gene expression like cellular miRNAs. To test the ability of HAdV14 mivaRNA to function as miRNA, the complement of each of the four main mivaRNA seed sequences were cloned into the 3′ UTR of luciferase in the pmirGLO Dual Luciferase Vector. Each reporter vector was transfected into A549 cells 24 h prior to infection with HAdV-14 at an MOI of 10, and luciferase activity was determined 24 hpi. As shown in Having established that mivaRNAs are expressed at levels similar to cellular miRNAs  and are Since our in vitro studies showed that the mivaRNAs can functionally repress luciferase gene expression , we souged cells B. This sed cells . CombineThe adenoviral VA RNA gene is a multifunctional non-coding RNA molecule. VA RNAs are processed by DICER into mivaRNA that resemble and function like cellular miRNAs. While all human adenoviruses encode at least one VA RNA gene, the amount of sequence similarity between VA RNA genes from all human adenoviruses is limited between all human Ad, but is somewhat conserved within Ad groups. In this study, we sought to identify the mivaRNA produced during prototype HAdV14 deWit infection, assess predicted target gene expression, and compare mivaRNA expression with that of the emergent pathogenic strain HAdV14p1, using A549 cells that are permissive for HAdV14 and HAdV14p1 infection.HAdV14, like other group B2 HAdV, only encode one VA RNA gene . In HAdVTo characterize the mivaRNA produced from the HAdV14 VA RNA gene, we infected A549 cells and sampled mivaRNA expression through all phases of the Ad14 infectious cycle from very early (6 hpi) all the way through to full CPE 48 hpi). At all times, >90% of all small RNA reads that mapped to the HAdV14 genome mapped specifically to the VA RNA gene. These results are consistent with those seen during Ad5 infection of IMR-90 cells  hpi. At . The samOur results identified the core seed sequences produced during Ad14/Ad14p1 infection as coming from both the 5′ and 3′ terminal stems. Minor variants are also produced. How these arise is unclear. One possibility is that RNA pol III synthesis of VA RNAs might have some minor wobble in its exact initiation site, as these minor mivaRNA species show very slight variations in either the 5′ or 3′ ends. It is possible that this could have an effect on expression, as small differences in the 5′ start can result in a completely different mivaRNA seed sequence. We identified a group of long mivaRNAs produced from the 3′ terminal stem that start between 8 and 4 nucleotides upstream of the predominant 3′ mivaRNA. Interestingly, the longest mivaRNA produced from the 3′ terminal stem has the exact same seed sequencing as hsa-miR-584c-3p. It has been well documented that HAdV mivaRNAs can regulate gene expression, like gene-encoded miRNAs ,40,42. UThe ability of mivaRNAs to target and regulate host gene expression is an attractive theory for potential roles in HAdV replication. As a result, numerous studies have been conducted to determine potential target genes for mivaRNAs. Bioinformatic, microarray studies, and RNA-seq analysis have shown that many of the potential cellular gene targets for mivaRNAs are involved in DNA repair, cell growth, cell death, RNA metabolism, cell signaling pathways, and metabolism ,42,48. OIn this study, we characterized the HAdV14 VA RNA gene and the mivaRNAs produced from it. We identified the predominant seed sequences and the cellular genes that the mivaRNAs can target. Our bioinformatic analysis using RNA-seq data of the expression of mivaRNA target genes showed that HAdV14 mivaRNA can regulate similar pathways seen from HAdV2/5 studies despite the lack of identity of the mivaRNA seed sequences. Despite 100% sequence identity later in infection, HAdV14p1-infected cells express significantly more mivaRNA than HAdV14-infected cells. Whether this difference has an impact on viral replication or pathogenesis is unknown. Due to a lack of studies in immunocompetent animal models, the role of HAdV mivaRNA in viral pathogenesis is largely unexplored. To answer questions about viral factors, such as mivaRNAs, we have developed an immunocompetent Syrian hamster model of HAdV14p1 lung pathogenesis that displays pathology similar to what is seen in the lungs of severely infected human patients ,50. We p


Intrahepatic cholangiocarcinoma (ICC) is a malignant disease characterized by onset occult, rapid progression, high relapse rate, and high mortality. However, data on how the tumor microenvironment (TME) regulates ICC metastasis at the transcriptomic level remains unclear. This study aimed to explore the mechanisms and interactions between hepatocytes and ICC cells.6A) modification and the downstream target in vitro, in vivo experiments, and in clinical specimens.We analyzed the interplay between ICC and liver microenvironment through cytokine antibody array analysis. Then we investigated the role of N6-methyladenosine (m6A modification via vir-like m6A methyltransferase associated (VIRMA) in ICC cells. Moreover, immunohistochemical analyses showed that VIRMA correlated with poor outcomes in ICC patients. Finally, we confirmed both in vitro and in vivo that CCL3 could activate VIRMA and its critical downstream target SIRT1, which fuels tumor metastasis in ICC.Our study demonstrated that cytokine CCL3, which is secreted by hepatocytes, promotes tumor metastasis by regulating m6A-mediated regulation in ICC metastasis. These studies highlight potential targets for the diagnosis, treatment, and prognosis of ICC.In conclusion, our results enhanced our understanding of the interaction between hepatocytes and ICC cells, and revealed the molecular mechanism of the CCL3/VIRMA/SIRT1 pathway via mThe online version contains supplementary material available at 10.1186/s12967-023-03897-y. Intrahepatic cholangiocarcinoma (ICC) emanates from the epithelial lining of the intrahepatic biliary tree, and is a malignant disease characterized by onset occult, rapid progression, high relapse rate, and high mortality , 2. As tTumor metastasis is one of the most important indicators of tumor progression and a leading cause of mortality in cancer patients. Besides, the tumor microenvironment (TME) has been shown to play a key role in driving tumor development and metastasis . ICC is 6A) modification [6A dynamics which trigger cancer proliferation and metastasis [Chemokines, a family of small proteins (8–10 kDa) that interact with specific G-protein–coupled chemokine receptors, are key components of the TME . The chefication , 15. Fortastasis , 17.6A methylation occurs on the sixth N of adenylate (A) in RNA, which is the most universal and abundant epigenetic modification in eukaryotic mRNA [6A RNA methylation is a dynamic and reversible process mainly mediated by the methyltransferase complex. The process is mediated by m6A demethylases and is related to diverse cellular functions by recognizing and binding the m6A reader proteins [6A methyltransferase complex consists of METTL3, METTL14, VIRMA (vir-like m6A methyltransferase associated), and WTAP, while the m6A demethylases comprise FTO and AlkBH5. m6A methyltransferase includes YTH domain-containing family proteins  and insulin-like growth factor 2 binding proteins , which can specifically recognize m6A modification sites and recruit related proteins to regulate the stability of mRNA, nuclear output or translation efficiency. Another study suggested that the m6A modification and m6A binding proteins participate in a range of physiological and pathological processes such as DNA damage [6A methylation plays an essential role in the promotion and metastasis of many types of malignancies such as gastric cancer [6A modification of HDGF mRNA modulates the progression of gastric cancer [6A modification in ICC remain unclear.The mtic mRNA , 19. Theproteins . The m6AA damage , embryonA damage , immune A damage , tumorigA damage , 25. Besc cancer , hepatocc cancer , ovarianc cancer , breast c cancer , and colc cancer , 31, thuc cancer , while Mc cancer . To date6A modification in ICC. Our study demonstrated that CCL3, which is secreted by hepatocytes, promotes tumor metastasis by fueling m6A modification in ICC. Moreover, we showed that VIRMA was a major modulator in the m6A modification, which correlated with poor outcomes in ICC patients and promoted ICC metastasis. In addition, SIRT1 was shown to be a critical downstream target of VIRMA, which fuels tumor metastasis. Taken together, our data enhanced our understanding of the interaction between hepatocytes and ICC cells, and uncovered the molecular mechanism of VIRMA-mediated m6A modification in ICC metastasis.In this study, we analyzed the interplay between ICC and liver microenvironment and investigated the role of mEthical approval for this study was provided by the Institutional Review Board of Sun Yat-sen Memorial Hospital. A total of 110 paraffin-embedded ICC specimens and corresponding adjacent normal tissues were obtained from Sun Yat-sen Memorial Hospital, Sun Yat-sen University between January 2011 and December 2020.2 in a 1640 medium supplemented with 10% fetal bovine serum . The cells were seeded at a density of 5 × 105 cells in a T25 culture flask and passaged every 4–5 days.Immortalized normal liver epithelial cells (THLE3) and human normal bile duct epithelial cell lines (HIBEpiC) were obtained from the American Type Culture Collection . The cells were cultured as specified by respective manufacturers. Two intrahepatic cholangiocarcinoma cell lines (HuCCT1 and RBE) were acquired from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. The cell lines were grown at 37 °C under 5% CO4 cells per well in the 24-well plates, with or without Matrigel , and incubated for 24 h. Thereafter, unmigrated and uninvaded ICC cells were removed with cotton swabs. The ICC cells on the bottom surface were then fixed in methanol for 10 min, at room temperature, followed by staining with 0.5% crystal violet. The experiments were carried out three times and in triplicate.Here, we employed both the transwell migration and invasion assays. The ICC cells were inoculated in the upper transwell chamber  with an indicated density of 3–5 × 10–ΔΔCt method. The experiments were repeated at least three times.Total RNA was extracted from tissues and cells using TRIzol Reagent , following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using PrimeScript RT reagent Kit and SYBR Premix Ex Taq , and employed primer sequences as shown in Additional file Full-length VIRMA (Ensembl: ENSG00000164944) cDNA was designed and synthesized by GenePharma , and then the cloned fragments were ligated into pcDNA3.1. Lipofectamine 3000  was served as the role of transfecting the plasmids into the ICC cells, following the manufacturer’s protocol. Small hairpin RNA (shRNA) of VIRMA (shVIRMA) and negative control (shNC) were designed as shown in Additional file 6 transfected HuCCT1 cells, which were suspended in 0.2 ml of PBS, into the flank of nude mice (five mice per group). On the other hand, an orthotopic xenograft tumor model was established by inoculating 3 × 106 transfected HuCCT1 cells in 50 μl of PBS into the liver of nude mice (five mice per group). For the treated groups, HuCCT1 cells were treated with CCL3 (100 ng/ml) for 6 h before inoculation. For subcutaneous injection, the intratumoral multiple-point injection of CCL3 (20 mg/kg) diluted in 50 μl PBS was performed every 5 days. The control groups were treated with PBS. Subcutaneous tumor size was measured twice a week. After in vivo fluorescence imaging, all the study mice were sacrificed with the in vivo imaging system (IVIS) spectrum after four weeks. The mice tumors and organs were dissected, photographed, weighed, and stained.All animal experiments were approved by the Animal Research Committee of Sun Yat-sen University Cancer Center. Female Balb/c nude mice , were purchased from the Guangdong Medical Laboratory Animal Center . To generate a subcutaneous xenograft tumor model, we injected 5 × 10Additional methods and experimental details are described in Additional file This study analyzed a total of 280 cholangiocarcinoma patient records with complete follow-up and clinical data from Sun Yat-sen Memorial Hospital of Sun Yat-sen University between 2009 and 2018 . The growth of the RBE and HuCCT1 cells after co-culture with THLE3 was measured using cell counting kit-8 (CCK-8) assays. The data showed that the co-culture with THLE3 significantly promoted the proliferation of RBE and HuCCT1 cells  compared with human intrahepatic biliary epithelial cells (HIBEpiC) Fig. a. ThereaThe growth of the RBE and HuCCT1 cells after knockdown or overexpression of the VIRMA was measured using cell counting kit-8 (CCK-8) and colony formation assays. As shown in Fig. Furthermore, we performed cell cycle progression and cell apoptosis analyses. As demonstrated in Fig. We demonstrated that the downregulation of VIRMA inhibits the proliferation, invasion, and metastasis of ICC cells in vitro. Here, we analyzed the effect of VIRMA in the development of ICC in mice. Stable cells with modified VIRMA expression were subcutaneously injected into the BABL/c nude mice. First, we investigated the effect of VIRMA on the proliferation of ICC. As shown in Fig. P < 0.05). Taken together, these findings suggested that the higher expression of VIRMA was associated with adverse prognosis in ICC patients.In parallel, we analyzed the expression of VIRMA in ICC patients using the TCGA dataset and showed that the VIRMA expression was remarkably increased in CCA tissues Fig. g. Moreov6A binding sites were located in the protein-coding region (CDS region) and were enriched in the 5′ untranslated region (5′ UTR) as well as 3′ untranslated region (3′ UTR). Further analysis showed that the proportion of specific m6A binding sites in the 3′UTR was reduced in the siVIRMA group as compared with that in the siNC group and this was consistent with findings that VIRMA was mainly involved in regulating m6A modification in the 3′UTR region of mRNA  consensus sequence, results of MeRIP-seq in the present study showed that an m6A peak was detected around the stop codon of SIRT1 mRNA in non-target control siRNA cells and were all decreased upon VIRMA knockdown  protein level was decreased after SIRT1 knockdown in both RBE and HuCCT1 cells Fig. a. Cell vIt was evident that the CCL3 can abolish the effect of VIRMA knockdown in SIRT1 expression  subcutaneously into nude mice. As shown in Fig. We further showed that CCL3 promoted the metastasis of intrahepatic bile duct cancer cells by regulating VIRMA/SIRT1 pathway in the orthotopic ICC tumor model. The liver metastasis was significantly enhanced with CCL3 stimulation, whereas the progression was significantly inhibited by VIRMA knockdown Fig. g–j. FurtIn conclusion, the results of the current study confirmed that CCL3/VIRMA/SIRT1 pathway promotes ICC proliferation and tumor metastasis in vivo.Metastasis is the main clinical challenge in ICC. Tumor metastasis is affected by the TME, a unique environment that arises because of tumor progression. Most studies on the mechanism of ICC metastasis have focused on the analysis of the ICC cells, but only a few have assessed the role of other cells in TME. The liver cancer microenvironment is composed of cells such as immune cells, fibroblasts, and liver parenchymal cells as well as extracellular matrix components . Previou. Moreover, to clarify the molecular interaction between hepatocytes and ICC cells, we performed a cytokine microarray analysis and showed that CCL3 is the main chemokine that is involved in the interaction. CCL3, also referred to as macrophage inflammatory protein-1 (MIP-1α), belongs to the C–C chemokine family. Studies have shown that CCL3 contributes to the development and progression of various malignancies, such as esophageal cancer, breast cancer, osteosarcoma, leukemia, and multiple myeloma [In this study, we showed that the co-culture of the ICC cells with hepatocytes significantly enhanced the migration and invasion ability of the cells myeloma . As expe6A modification [6A RNA modification has been identified as a source of epigenetic regulation in DNA modification. The m6A modification accounts for more than 80% of RNA modification and is a reversible and dynamic process regulated by m6A methyltransferase, demethylases, and readers [6A methylation of mRNA has been reported to play significant roles in many RNA functions, such as RNA splicing, RNA processing, translation regulation, and RNA decay [6A modification in ICC remains limited. In this study, there was an increased m6A modification in ICC cells co-cultured with hepatocytes, which was potentially affecting cancer metastasis. We showed that CCL3 was the key factor mediating the interaction between the liver microenvironment and ICC cells. Subsequently, we characterized methyltransferase VIRMA and showed that it is the aberrant m6A modification in ICC cells co-cultured with hepatocytes.Thereafter, we analyzed the mechanisms of CCL3 activity in the communication between hepatocytes and ICC. Functional enrichment analysis showed that the genes that interacted with CCL3 were enriched for functions related to RNA metabolism. We further demonstrated that chemical modification occurs at the RNA level, which affects gene-regulatory networks at the epigenetic level. Previous studies demonstrated an association between extracellular signals such as cytokines or chemokines and mfication , 15. The readers . m6A metNA decay , 38, 39.6A methyltransferase complex and is mainly located in the nucleosome and cytoplasm. As a key regulator of m6A methylation, VIRMA was shown to form a complex with WTAP/METTL3/METTL14 heterodimer to catalyze m6A formation [6A modification in ICC. In this study, we showed that VIRMA was significantly upregulated in ICC tissues, which correlated with poor prognosis and thus regarded as a prognostic factor for tumor recurrence in ICC. Moreover, our in vitro and in vivo assays demonstrated that VIRMA promotes malignant development and progression of ICC, and its expression showcased a significant correlation with various metastatic, angiogenic and proliferation gene markers. These findings suggested that VIRMA may function as a tumor-promoting gene and might be a potential clinical prognostic biomarker and therapeutic target for ICC.VIRMA is an important member of the mormation , 40. Recormation , lung caormation , germ ceormation , and livormation , and is 6A methylation. Thereafter, we conducted GO and KEGG enrichment analyses to explore the major biological functions and downstream signaling pathways of the genes from the RNA-Seq and MeRIP-Seq analyses, respectively. SIRTI, a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, plays important roles in oncogenesis via transcription, translation, and post-translational modifications [Analysis of the RNA-Seq and MeRIP-Seq data identified SIRT1 (Sirtuin type 1), as a crucial downstream target of VIRMA. Our RNA pull-down and RIP assays demonstrated that SIRT1 was positively regulated by VIRMA and modified by VIRMA-mediated mications . SIRT1 wications , 46. In ications , 48 and ications  to contr6A dot blot assay to quantify the m6A levels of ICC, and showed that SIRT1 is a downstream target of VIRMA. Thus, there is a need for further studies which would take the following variables into account: (1) examine the m6A levels of ICC using a colorimetric strategy or liquid chromatography-mass spectrometry (LC–MS), (2) detect whether VIRMA functions independently of its m6A catalytic activity in cancer progression, and (3) develop a peptide inhibitor to target VIRMA domain and explore whether it may be beneficial in the treatment of ICC.In our study, we only employed the m6A modification in ICC progression and metastasis. Mechanistically, we demonstrated that CCL3 is secreted by hepatocytes and may promote metastasis of ICC cells by regulating m6A methylation. The regulation of m6A methylation is mediated by VIRMA, which epigenetically promotes SIRT1 expression through an m6A methylation-dependent mechanism. Our results suggested that the interaction between hepatocytes and ICC cells might offer a possible interventional target for ICC. Besides, the m6A modification on tumor metastasis will contribute to further studies that would explore molecular mechanisms and identify efficient treatment strategies against ICC.In conclusion, our study highlights the critical role of VIRMA-mediated mAdditional file 1: Table S1. Primers used in qRT-PCR analysis.Additional file 2: Table S2. siRNA sequences.Additional file 3: Figure S1. Functional enrichment analysis of cytokine antibody array. Figure S2. Validation of VIRMA knockdown and overexpression in ICC cells. (a) qRT-PCR (up) and western blot (down) analysis of VIRMA expression in VIRMA-knockdown ICC cells. siVIRMA-1 and siVIRMA-2 indicated VIRMA knockdown in both RBE and HuCCT1 cells. (b) qRT-PCR (up) and western blot (down) analysis of VIRMA expression in VIRMA-overexpression ICC cells. oeVIRMA indicated RBE cells and HuCCT1 cells transfected with an overexpressing plasmid. The relative quantification was calculated using the 2−ΔΔCt method and normalized based on GAPDH. Figure S3. MeRIP-seq and RNA-seq analysis. (a) GO analysis of down-regulated genes identified for MeRIP-seq. (b) Motif analysis. (c) The peak annotation for m6A signal enrichment in MeRIP-seq. (d) GO analysis of down-regulated genes identified for RNA-seq. (e) KEGG analysis of down-regulated genes identified for RNA-seq. (f) GO analysis of up-regulated genes identified for RNA-seq. (g) KEGG analysis of up-regulated genes identified for RNA-seq. Figure S4. Quantification of rescue experiments.Additional file 4: Additional methods and experimental details.


Pairing of splice sites across an intron or exon is the central point of intron or exon definition in pre-mRNA splicing with the latter mode proposed for most mammalian exons. However, transcriptome-wide pairing within endogenous transcripts has not been examined for the prevalence of each mode in mammalian cells. Here we report such pairings in rat GH3 pituitary cells by measuring the relative abundance of nuclear RNA-Seq reads at the intron start or end (RISE). Interestingly, RISE indexes are positively correlated between 5′ and 3′ splice sites specifically across introns or exons but inversely correlated with the usage of adjacent exons. Moreover, the ratios between the paired indexes were globally modulated by depolarization, which was disruptible by 5-aza-Cytidine. The nucleotide matrices of the RISE-positive splice sites deviate significantly from the rat consensus, and short introns or exons are enriched with the cross-intron or -exon RISE pairs, respectively. Functionally, the RISE-positive genes cluster for basic cellular processes including RNA binding/splicing, or more specifically, hormone production if regulated by depolarization. Together, the RISE analysis identified the transcriptome-wide regulation of either intron or exon definition between weak splice sites of short introns/exons in mammalian cells. The analysis also provides a way to further track the splicing intermediates and intron/exon definition during the dynamic regulation of alternative splicing by extracellular factors. Pre-mRNA splicing occurs between pairing splice sites across an intron (intron definition) or exon (exon definition). The former is likely the preferred way of splicing in species such as drosophila with mostly short introns, and the latter in vertebrates with relatively short exons among long introns ,2,3,4. BOur interest in the splice site pairing of the intermediates was prompted by the depolarization-regulated splicing of the STREX (stress hormone axis-regulated exon) in pituitary cells, a model exon for studying activity-dependent splicing ,14,15,16Here, we use RNA-Seq of nuclear RNA , to measure the ratio of reads at the intron start or end over exon reads (index), the global relationship between the indexes of splice sites across introns or exons and their regulation by membrane depolarization. This detected the transcriptome-wide pairing of indexes of 5′ and 3′ splice sites specifically across introns or exons including STREX in the transcriptome, and regulation of the pairing by depolarization. To track the nuclear pre-mRNA intermediates in the splicing regulation of STREX and other alternative exons by membrane depolarization, we made use of the RNA-Seq samples from nuclear, cytoplasmic, or whole cell lysates of rat GH3 pituitary cells . We analyzed the uniquely mapped read pairs using DEXSeq or edgeR, for the Ensembl-annotated (spliced) RNA transcripts or their intron regions.U4 and U1 snRNAs, Small Cajal body-specific RNAs ScaRNA2, 6, 7, 13, 17, and 21, RNase-P nuclear RNA, Xist-intron RNA, and 7SK RNA were about 100 to 10,000 times enriched, consistent with their nuclear localization [Hnrnp, U2af, and Gapdh genes all had an N/C ratio of less than 1, consistent with their mainly cytoplasmic localization. This verified the specificity of the nuclear RNA samples by our nucleo-cytoplasmic RNA fractionation method.The nuclear RNA fraction was highly enriched with known nuclear RNA transcripts by edgeR analysis of their relative levels over those in the cytoplasmic fractions . In partlization ,23,24,25p < 0.001, hypergeometric density test) with little increase at the downstream 5′SS. The prominent increase of the upstream 3′SS reads is consistent with its increase in RT-PCR from the pre-mRNA at about five times over that of the downstream 5′SS  similarly as in previous reports [Manual examination of the STREX exon and its flanking introns in the integrated genomics viewer (IGV ) identif reports ,27,28. T reports ,14,15 anPrmt5 gene, which is inhibited by G-tracts between the polypyrimidine tract and the 3′ AG (REPAG) [Similar splice site-specific accumulation of reads was also observed for other alternative exons such as the 86nt-long exon 3 of the  (REPAG) ,30. TogeTherefore, the accumulated reads at the intron start or end (RISE) likely reflect pre-mRNA intermediates of alternative and/or depolarization-regulated splicing in the presence of spliced introns of the same pre-mRNA transcripts.hnRNP L gene [Further manual examination of a group of RISE-containing splice sites of cassette exons in the IGV identified three types of such reads A: in theP L gene , it has p < 0.05, KCl vs. NT) that had adjacent RISE, from DEXSeq analysis of poly(A) RNA. Exons adjacent to either type I or II RISE were enriched of changed ones by about 2 folds , consistent with the overall 5′ to 3′ direction of intron removal from nascent pre-mRNA transcripts [To determine if the RISE abundance is globally correlated between the 5′SS and 3′SS pairs across introns or exons, as predicted by the SS pairing in intron or exon definition , we obtanscripts . In totar = −0.18). Furthermore, similar correlation patterns were also observed in the KCl-treated samples . The stronger correlation between paired RISE indexes supports the existence of a constraint beyond their mere presence in the same genes/transcripts, which is likely the driving force for splice site pairing in intron or exon definition.To make sure that the global positive correlation is not simply due to that the paired RISE are in the same gene transcripts, we further examined the Pearson coefficients with 218 RISE pairs of 25 genes. The mean t-test [To determine the global effect by depolarization, we examined the index net changes and the ratios of the 5′SS to 3′SS RISE indexes (t-test ). Globalt-test ,34,35,36Lars1 exon E022 was increased by depolarization and further by 5-azaC; as is the ratio between the competing 3′SS . However, they deviate significantly from the genome-wide consensus  E3 and STREX are targets of splicing regulation by hnRNP L/LL upon depolarization and calcium signaling [Further analysis of the splice sites of the RISE-adjacent, depolarization-regulated exons using multiple Em for motif elicitation (MEME) , showed ignaling ,15. r = −0.005 on average), but the type II inversely correlated with exon lengths , consistent with a higher index for delayed exon definition of shorter exons. More interestingly, the type I and II groups showed significant differences. Type I is associated with more of the introns less than 1000 nt, compared to type II  or exons (type II) in the rat genome, consistent with their inverse correlation with exon usage  and moduAlthough the RISE indexes are inversely correlated with exon usage and globally modulated by depolarization , after sp > 0.05 by DEXSeq) gave rise to clusters for basic cellular processes such as signaling (ATP-binding or transferases) or RNA splicing (p < 0.05 by DEXSeq) did not give rise to the same clusters. Instead, it gave rise to clusters for more specific pituitary functions: genes with alternative splicing, prenylation, nuclear functions, and protein interactions , essential for luteinizing hormone production [cullin-associated and neddylation-dissociated 2 (Cand2) with a homolog Cand1 regulating vasopressin secretion [DAVID functional clustering analysis of genes whose RISE-associated exons were not changed upon depolarization , even among those less than 400 nt (r = 0.02 on average). Alternatively, the across-intron RISE may pair with those further away rather than in the same introns only (type III), thereby adding to more variation, but the same could also be applicable to the RISE of the across-exon pairs. Therefore, the observation likely supports inherently stronger exon than intron definition in the mammalian cells.We detected more instances of across-intron than -exon RISE pairs, which might be just for this cell line under the experimental conditions since they represent only the delayed splice sites at the 6h time point. However, the correlation coefficients for the across-exon pairs are much higher than the across-intron ones . One couThe RISE index analysis has allowed a glimpse of a group of splicing intermediates in the transcriptome. For a more complete and dynamic view of transcriptome-wide RISE pairing and its regulation by depolarization, however, many more intermediates have to be detected at additional time points and/or with more in-depth nuclear RNA-Seq.cis-acting pre-mRNA sequence motifs as demonstrated previously in other systems [Potential molecular mechanisms for the regulation of splice site pairings in these cells by KCl depolarization could involve not only the previously identified hnRNP L/LL at the 3′SS of STREX ,18 but a systems ,6,7. For systems . However systems ,36, whic systems , or hist systems . Particu systems ,50. Alte systems ,52.In summary, the nuclear RNA-Seq analysis of intron RISE has allowed us to see for the first time the coordinated transcriptome-wide splice site pairing among endogenous pre-mRNA transcripts that is consistent with delayed intron and/or exon definition. The global view suggests that there is an overall much stronger exon than intron definition in the mammalian transcriptome. The detected intermediates of the weak splice sites and short introns/exons likely impact genes in basic as well as pituitary-specific cellular functions depending on their regulation by depolarization.Cell culture: We cultured rat GH3 pituitary cells in Ham’s F10 nutrient mixture with 10% horse serum plus 5% fetal bovine serum with 5% CO2 at 37 °C.KCl treatments, total or cytoplasmic RNA extraction, and RNA-Seq analysis: For treatment, KCl (50 mM) was added and incubated for 6h before RNA extraction. We extracted total RNA with the GenElute™ Mammalian Total RNA Miniprep Kit . We extracted cytoplasmic RNA using our previous nucleo-cytoplasmic fractionation protocol [protocol ,53, the RNA-Seq was carried out after purification of poly(A) RNA using oligo d(T) and random-primed for cDNA synthesis following the protocol of Illumina Truseq stranded mRNA sample preparation. Illumina HiSeq4000 paired-end 100-bp sequencing was used for the total RNA of non-treated (NT) and KCl-treated (6 h) samples, and the Illumina NovaSeq 6000 S2 paired-end 100-bp sequencing was used for the cytoplasmic RNA samples. RNA-Seq analyses by edgeR or DEXSeq were performed as in our previous report .Nuclear RNA preparation and RNA-Seq analysis: The nuclear RNA samples were obtained from a test intended for lariat and RNA secondary structures. Briefly, GH3 cells were untreated, treated with KCl (50 mM), or KCl (50 mM) plus 5-azaC (50 μM), respectively in triplicate (three wells of cells). Upon 6h treatment, cells were collected and nuclear RNA extracted in a procedure modified from the nucleo-cytoplasmic fractionation protocol [protocol ,53: cellprotocol ,55) withprotocol , chilledn = 12) were obtained for each sample. Approximately 1 μg of nuclear RNA from each sample of the three groups was reverse transcribed using random primers for NEB rRNA-depleted (HMR) stranded library and NovaSeq 6000 sequencing. On average, 42 million uniquely mapped reads (paired, ±6.6, For the RISE analysis, the following criteria were empirically chosen after referring to a similar method in the previous study of 3′SS , and tesStatistical tests: All samples were in triplicate. Two-tailed Student’s t-test was used unless otherwise noted. The DEXSeq and edgeR analysis use Fisher’s exact test [act test ,60.


Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesized that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed, this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV), and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses. Positive strand RNA viruses infect all forms of life and impose significant environmental, economic and social hardship due to the diseases that they cause. For those viruses that infect higher eukaryotic organisms, viral genome replication occurs in membrane rich compartments within the cytosol often referred to as replication complexes (RCs) . These cThe basic principles of viral genome replication were first determined by pulse chase experiments that allowed RNA synthesis to be followed over time . The curcis-acting replication elements (CREs); RNA elements that play a more direct role in genome replication itself. The function of these CRE elements ranges from controlling the switch between translation to replication  as a substitute for a 5′ cap to ensure translation of their viral proteins . Use of lication , acting lication  and servlication .The single-stranded nature of the viral genome provides the CREs within it the relative freedom to fold. However, once [−] RNA synthesis has occurred, constraints are encountered. This is because the complementary nature of the [−] and [+] RNAs promotes dsRNA formation. Despite this several viruses harbor structured CREs in their [−] RNA; CREs that act as promoters for genomic and subgenomic RNA production . Thus, aRibozymes are self-cleaving RNAs whose activity depend on both RNA secondary structure and other higher ordered RNA interactions . There acis, we initially chose to focus on HCV. The basis for this decision was because of evidence that HCV allows folding of relatively complex native structures in its [−] RNA , when the luciferase activity arises from translation of the input RNAs alone, the signal was twofold lower for both the HdV constructs compared to JFH1DVR-mono and replication-defective control constructs. Given that no replication would have occurred at this point, as illustrated by the signal being the same between the replication-competent and -defective controls, this difference likely comes from partial disruption of luciferase activity arising from the insertion of the reverse complemented HdV Rbz sequence into the Renilla-FMDV2A fusion protein. After this 4 h time point, HCVgt2a_HdV(wt), HCVgt2a_HdV(ko), and JFH1DVR-mono all produced an initial increase in luciferase over time compared to a decrease seen for the polymerase-defective control construct, demonstrating that all three former RNAs were replication-competent. However, the luciferase signal from the replicon with the active Rbz significantly diverged from its inactive Rbz counterpart as the latter produced a more rapid rise in luciferase activity, with luciferase levels peaking at 48 versus 96 hpt. This coincided with the observation that replication of HCVgt2a_HdV(wt) was suppressed, with luciferase levels at 32% and 44% of those produced by its HdV(ko) counterpart at 24 and 48 hpt. While at 96 hpt this trend was reversed, the likely explanation is that this was due to HCVgt2a_HdV(ko) transfected cells exhibiting earlier cytopathic effects and luciferase levels in this experimental group starting to drop.If Rbz activity was suppressing replication, then cleavage of the [−] RNA would be anticipated. To establish whether this was the case, RNA was recovered from these same experiments and assessed by northern blot C. StrandS. mansoni that had been further engineered for enhanced cleavage activity    and anot; 83 nt) . The fou; 83 nt) A,B, more; 83 nt) C. The reIn order to confirm the availability of the [−] RNA strand to form functional RNA structure in other regions of the virus genome during HCV replication, the positional independence of the Rbz was investigated. To achieve this, a second reverse complemented active N79 or inactive N79 sequence was introduced into the NS5A/5B boundary of both HCVgt2a_N79(wt) and HCVgt2a_N79(ko) A. This wHaving established that the N79 Rbz effectively suppressed replication of the HCV gt2a replicon, we assessed its impact on replication when placed in the [−] RNA of other positive strand RNA virus constructs. A variety of HCV genotype replicons exist although they display reduced levels of replication compared to JFH-1 gt2a constructs. Introduction of a functional N79 reverse complemented sequence into a HCV gt1b replicon abolished replication whereas an inactive N79 Rbz did not A.Caliciviridae family; viruses which harbor relatively large stem–loops in this strand that function as a subgenomic promoter . We also introduced an RNA polymerase promoter immediately downstream from the 3′ end of the genome in the plasmids encoding five of the N79-containing virus replicons to allow in vitro production of full length  or near full length (YFV) [−] RNA. Analysis of these RNAs showed that they experienced no more than a twofold difference in rates of cleavage, with cleavage ranging from 30% to 60%  and N79(ko) transcripts appeared uncleaved and were equally abundant. The relative abundance of full length HRV [−] RNA N79(wt) and N79(ko) transcripts was also broadly similar, although unlike YFV a low level of cleavage of the N79(wt) transcript was also observed, consistent with the transient inhibition of replication observed at early time points in the replication assay. Importantly, the addition of Mg2+ resulted in an increase in cleavage of the HRV N79(wt) [−] transcript and cleavage of a proportion of the YFV N79(wt) [−] transcript not seen for the equivalent RNAs carrying the N79(ko) Rbz. We conclude a functional Rbz sequence is maintained in the [−] RNA strand of HRV and YFV during the time that genome amplification is occurring, but its activity is suppressed by dsRNA formation.Another scenario that might have accounted for poor cleavage is mutation of the Rbz sequence such that activity is rapidly lost. To exclude this possibility, cells were transfected with N79-containing HRV and YFV replicons and cellular RNA was recovered 48 h later. Samples were heat denatured to melt all dsRNA, then diluted and cooled to promote RNA folding. Half of each sample was supplemented with 1 mM Mgthis RNA B. In theSupplemental Fig. S3). Importantly, about half of the N79(wt) RNA produced by T7 was cleaved into products of a size consistent with that expected from N79 activity (444 and 100 nt). Indeed, this was specifically due to Rbz activity as the N79(ko) transcript produced by T7 remained uncleaved. In contrast, no obvious difference was observed between RNA products generated in the NS5B reactions when comparing between N79(wt) and N79(ko) experimental groups irrespective of the temperature of the transcription reaction; the RNAs present consisting of an approximately unit length (∼500 nt) product identical in size to the uncleaved RNA produced in the T7 reactions, as well as several smaller RNAs mostly ranging in size between 300–400 nt and likely to represent internal initiation products. To assess the double-stranded nature of the various polymerase reactions, all products were incubated with RNase A under high salt conditions. No degradation of the NS5B products was seen, indicating that they were duplexed with their template and therefore double-stranded. In contrast, the T7-derived products were highly sensitive to RNase A treatment. Finally, to confirm that dsRNA formation was preventing Rbz cleavage, the surviving RdRp product from the RNase treatment was subjected to heat denaturation and renaturation. In the N79(wt) experimental group, this resulted in a decrease in size of all RNAs by ∼100 nt as well as the appearance of an additional ∼90 nt band. In contrast, there was no change in appearance of the N79(ko) containing RNAs. Taken together these data show that in the absence of any other host or viral encoded activity, RNA produced by HCV NS5B is unable to even transiently access secondary structures that might be encoded within it.The extent that Rbz induced cleavage of the [−] RNA of HCV was much greater than most other constructs tested. To investigate whether RNA synthesized by the HCV viral polymerase in vitro was equally prone to Rbz cleavage and thus able to sample secondary structure, we performed a series of RNA polymerase reactions. HCV NS5B RNA-dependent RNA polymerase reactions were set up to produce a 544 nt RNA transcript containing either an active (wt) or inactive N79 (ko) Rbz A. ParallLeviviridae family which infect prokaryotes, and for members of Narnoviridae which infect fungi and insects, the [+] and [−] RNAs are kept in a predominately single-stranded state  has been suggested to depend on host cell proteins TIA-1 and TIAR binding to a 75 nt stem–loop found within the 3′ end of the [−] strand . The helOur observations of Rbz activity within the [−] RNA of HCV is consistent with the virus relying on a large structured promoter at the 3′ end of this same RNA species. Even so it was noticeable how much more active Rbzs were in the HCV constructs compared to many of the other viral constructs. This begs the question, why does HCV expend what is likely to be a considerable amount of resources and energy driving duplex separation within its replication complex when it does not produce subgenomic RNAs? Indeed, it seems strange that any virus would evolve a large structured promoter within its [−] RNA unless there was already preexisting evolutionary pressure driving stand separation. Perhaps by keeping its [+] and [−] strands in a more separated state, HCV reduces the chances of it being detected by dsRNA antiviral sensors. While the replication complex membranes protect the RF and RI from antiviral sensors in the cytosol, turnover/destruction of these organelles does expose them to endosomal TLR3, something that HCV already seeks to minimize by promoting RC secretion . By bein2. Huh7.5 cells were maintained under similar conditions but with an additional supplementation of nonessential amino acids made to the media.HeLa (Ohio), CRFK and BHK21 cells were maintained in DMEM (Invitrogen) supplemented with 50 units penicillin, 50 mg streptomycin and 10% (v/v) fetal calf serum at 37°C and 5% COSupplemental Table S1 for primer and synthetic DNA sequences) with template pMNV* , have been described  and servte pMNV* , and priShistosoma mansoni N79 Rbz  or HCVgt2a_N79(ko). All three products were combined in a second round PCR using primer pairs 17 + 22 and the resultant DNA cloned into both the HCVgt2a_N79(wt) or HCVgt2a_N79(ko), containing plasmids via SacII and SalI restriction sites to generate plasmids containing HCVgt2a_2xN79(wt/wt), HCVgt2a_2xN79(ko/ko), HCVgt2a_2xN79(wt/ko) and HCVgt2a_2xN79(ko/wt).Insertion of the N79 Rbz into other replicon constructs was achieved as follows. For the HRV replicons, PCRs were undertaken using primer pairs 23 + 24 in combination with either HCVgt2a_N79(wt) or HCVgt2a_N79(ko) as a template, as well as primer pairs 25 + 26 in combination with pR16.11  as a template. The resultant sets of DNAs were combined in a second round PCR with primers 23 + 26 and the resultant product cloned into an in-house pR16.11 derived replicon (sequence available on request) via AscI and ClaI restriction sites to generate plasmids containing HRV_N79(wt) and HRV_N79(ko). A control replication-defective version of HRV_N79(ko) was generated by cutting the associated vector with NsiI, polishing with Phusion polymerase (NEB) and religating. For the YFV replicons, PCRs were undertaken using primer pairs 27 + 28 and 29 + 30 using pACNR-FLYF-17D  a gift  via ApaIFor the HEV replicons, the GFP coding region from pSK-E2-GFP   or HCVgt2a_N79(ko) and using primer pairs 47 + 48 with template pFKI341PVIlucUbiNS3-3′dgET  . A pair of complementary oligos containing either a SP6  or T7 (CHIKV) promoter and with overhanging ends compatible with those of the digested plasmid DNA were ligated into these same DNAs. The presence and orientation of the inserted DNA was confirmed by PCR and sequencing. For the purpose of identification, the names of the constructs produced bear the parental constructs name from which they derive but contain an additional “as” (antisense) prefix .wt and pT7HCV(3′-)341asN79ko. Constructs were linearized with MfeI and polished with Mung Bean nuclease prior to use in T7 transcription reactions that generated RNA template for subsequent NS5B RdRp reactions.To generate constructs containing a reverse complemented N79 sequence to be used for transcribing template RNAs for in vitro NS5B RNA-dependent RNA polymerase (RdRp) assays, a two-step PCR was performed. Primer pairs 53 + 54 and 55 + 56 generated amplified product from HCVgt2a_N79(wt) and HCVgt2a_N79(ko) templates. These two reaction products were then combined in a second PCR reaction with primer pairs 53 + 56 and the resultant DNA cloned into pCR-Blunt (Invitrogen) before being excised using XbaI + EcoRI and cloned into XbaI + EcoRI cut pSGRJFH1luc, generating pT7HCV(3′-)341asN79wt and pT7HCV(5′+)341N79ko.To generate control constructs for producing the same RNA species to be synthesized in the NS5B RdRp reactions, but via T7 polymerase, PCRs were performed using the primer pair 57 + 54 in combination with pSGRJFH1luc as template DNA, and the primer pair 55 + 58 in combination with either HCVgt2a_N79(wt) or HCVgt2a_N79(ko) as template DNAs. First round products were combined in a second PCR using primers 57 and 58. Products were first cloned into pCR-Blunt, then excised with XbaI and EcoRI and cloned into XbaI + EcoRI cut pSGRJFH1luc to produce pT7HCV(5′+)341N79secure (Thermo Fisher) and used in a 50 µL transcription reaction containing 5× transcription buffer (Thermo Fisher), 8 mM rNTPs, 1.25 units RiboLock RNase Inhibitor (Thermo Fisher), and 60 units of either T7 or SP6 RNA Polymerase (Thermo Fisher). After an overnight incubation at 30°C, 2 units RNA-free DNase (NEB) was added, and reactions left at 37°C for 30 min. RNA transcripts were precipitated by addition of LiCl and the resultant pellet washed in 70% ethanol before resuspension in RNase-free water. Capping of YFV and CHIKV replicon RNA transcripts was performed using the Vaccinia Capping System (NEB) according to manufacturer's recommendations. HEV and FCV transcripts were generated using the HiScribe T7 ARCA mRNA kit (NEB) using 1 µg of linearized plasmid template following manufacturer's instructions. The integrity of all RNAs was verified by running RNAs on a MOPS-formaldehyde agarose gel and visualizing product by SYBR Gold Staining (Thermo Fisher). When required, relative quantification of RNA species on the gels was achieved using a ChemiDoc XRS+ Imager (Bio-Rad).All RNAs other than HEV, FCV and radiolabeled transcripts (see RdRp assay protocol) were generated as follows. Five micrograms of linearized plasmid template were pretreated with RNA7 cells/mL in ice-cold PBS. Four hundred microliters of the cell suspension was mixed with 2 µg replicon transcript, transferred to a 0.4 cm cuvette, electroporated at 270 V, 960 µF (BioRad GenePulse II) and resuspended in 6 mL growth medium. Cells were plated out in 12-well plates and maintained at 37°C  or at 33°C (HRV) until harvested by washing in PBS and lysis in Passive Lysis Buffer (Promega). Luciferase activity was determined using either the Renilla Luciferase Assay Kit (Biotium), Nano-Glo Luciferase Assay System (Promega) or Dual Luciferase Reporter Assay (Promega). Live cell imaging was performed with an IncuCyte Dual Colour Zoom FLR (Essen BioScience) within a 37°C humidified CO2 incubator scanning hourly up to 120 h post-transfection, collecting multiple images per well. The .mp4 files generated had individual stills extracted from them using VLC media player.Huh7.5 cells were used to monitor replication of HCV, HEV, and YFV replicons, BHK21 used to monitor replication of CHIKV replicons, HeLa (Ohio) used to monitor replication of HRV replicons and CRFK cells used to monitor replication of FCV. Once cells had reached confluence they were detached, washed 2× in ice-cold RNase-free phosphate buffered saline (PBS) and resuspended at 1 × 10Supplemental Fig. S1).To generate probes, the HCVgt2a_HdV(wt) vector was used as a template in an asymmetric PCR reaction  containiwt and HCV(3′-)341asN79ko RNA transcripts were heated to 90°C for 2 min in a final volume of 16.2 µL before slowly cooling to room temperature and placing on ice. To these RNA templates the following was added; 2.5 µL 10× NS5B transcription buffer , 0.6 µL RNaseOUT (Thermo Fisher), 1 µL 10 mM rATP, 1 µL 10 mM rCTP, 1 µL 10 mM rGTP, 1 µL 0.1 mM rUTP, 1 µL [α-32P]rUTP  and 0.7 µL JFH1 NS5BΔC21 or JFH1 NS5BΔC21(GAA) at 0.3 mg/mL (wt and pT7HCV(5′+)341N79ko as templates in a 25 µL final reaction volume containing 2.5 µL 10× T7 transcription buffer (NEB), 0.6 µL RNaseOUT, 1 µL 10 mM rATP, 1 µL 10 mM rGTP, 1 µL 10 mM rCTP, 1 µL 1 mM rUTP, 0.5 µL [α-32P]rUTP and 1 µL T7 polymerase (NEB). Experiments were performed where transcription was allowed to occur for 2 h at room temperature or at 37°C. Afterwards the NS5B RdRp reactions were placed on ice while the T7 reactions were supplemented with 1 unit RNase-free DNase and incubated at 37°C for 20 min. All samples were centrifuged through MicroSpin G-25 columns (Amersham) preequilibrated with 10 mM Tris (pH 7.5) 1 mM EDTA (TE) buffer before further addition of TE buffer to a final volume of 100 µL, phenol/chloroform extraction and ethanol precipitation in the presence of 20 µg glycogen. Half of each RNA was kept back for subsequent analysis while the other half was subjected to digestion with RNase A in a 100 µL high salt buffer  for 60 min at 30°C before addition of 4 µL 20 mg/mL proteinase K and 6.6 µL 10%SDS and incubating at 37°C for 30 min. Reaction products were then phenol/chloroform extracted, ethanol precipitated, resuspended in 8 µL H2O and again half the recovered material was kept back for subsequent analysis. The remaining 4 µL of samples were mixed with 4 µL of formamide and the RNAs heated to 85°C for 2 min before being rapidly diluted by the addition of 196 µL Rbz cleavage buffer preheated to 85°C. Samples were allowed to cool slowly to room temperature before being subjected to ethanol precipitation in the presence of 20 µg glycogen as a carrier. RNAs from both treated and untreated arms of the experiment were separated on a 5% denaturing acrylamide:bisacrylamide (19:1) gel alongside a RiboRuler Low Range RNA Ladder (Thermo Fisher) and the gel fixed, stained with Methylene Blue and dried onto Whatman 3 MM filter paper. Gel images were captured by exposure to both CL-Xposure X-ray film (Thermo Fisher) or BAS-MP IP phosphoimager plate (Fujifilm) and scanned at 50 µm resolution at 635 nm in a FujiFilm FLA-5100 fluorescent imager analyzer.Five hundred nanograms of HCV(3′-)341asN79.3 mg/mL . ParalleSupplemental Table S1 and Supplemental Figures S1–S3 are provided as Supplemental Files. Raw luciferase data as well as original .jpg, .tiff and .mp4 files produced during this study are available at https://doi.org/10.5281/zenodo.5873054.Supplemental material is available for this article.


Specificity of RNA-binding proteins for target sequences varies considerably. Yet, it is not understood how certain few proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two distinct binding modes. The first exclusively accommodates cognate and closely related RNAs with high affinity. The second mode accommodates all other RNAs with reduced affinity by imposing large thermodynamic penalties on non-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA-binding site. Distinct binding modes by a single RNA-binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation in RNA-protein interactions. Here the authors show that the RRM of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes - one for its cognate RNA and one for all non-cognate RNAs. A pivotal aspect in this choreography of regulation is the specificity of RBPs for their target sites7; that is, the degree to which an RBP discriminates between cognate and non-cognate RNAs4. Specificity of RBPs varies considerably; some RBPs are highly selective for certain RNA sites, others bind degenerate sites, and yet other RBPs interact with RNAs broadly, with little sequence discrimination14. Defining the molecular mechanisms through which RBPs acquire their specificity is critical for understanding the rules of RNA biology and for devising therapeutic strategies against diseases associated with RBP- and RNA-related processes15.In eukaryotic cells, the expression of tens of thousands of RNAs is regulated by thousands of diverse RNA-binding proteins (RBPs) that interact with RNA in a carefully orchestrated manner16. The three RbFox proteins contain divergent N- and C-terminal regions that are likely unstructured but all share a highly conserved RNA-binding domain that belongs to the RNA Recognition Motif (RRM) superfamily17. The RbFox RRM binds RNAs with a consensus 5’-GCAUG motif with low nanomolar affinity in vitro21. Even minor changes in the consensus motif decrease the RNA affinity by orders of magnitude21, indicating an inherent specificity of RbFox proteins for their consensus sequence that is markedly higher than for most other RBPs11. In the cell, however, biological effects by RbFox proteins are also exerted by binding to non-consensus sites characterized by lower affinity, when expression levels of RbFox increase22.Among RBPs with the greatest specificity are the three closely related human RbFox proteins (RbFox 1-3), which function in pre-mRNA splicing, miRNA processing and other RNA metabolic steps20. Therefore, we set out to systematically examine the basis for the high specificity of the RbFox proteins through a combination of high throughput biochemical techniques followed by NMR structure determination. We first determined the affinity of the RRM of RbFox for all possible 16,384 7-mer RNA sequence variants. The resulting affinity distribution reveals two distinct binding modes, one associated with binding to the consensus 5’-GCAUG and a second mode for binding to all other sequences. Mutations in RbFox only have a small effect on the binding of the consensus sequence, but markedly increase affinity for all other variants, thereby diminishing specificity. The results indicate that the binding mode for the non-consensus sequences imposes a large thermodynamic penalty on these sequence variants, thereby enabling exquisite discrimination between consensus and non-consensus RNAs. Comparison of the NMR structures with a non-consensus and the consensus sequence reveals that the two binding modes are associated with substantial structural differences within the RRM. Remarkably, the structural rearrangement extends to protein regions distant from the RNA-binding interface, suggesting that the distinct binding modes can transmit RNA sequence information to potential protein binding partners of RbFox.How RbFox proteins accomplish higher specificity than most other RBPs is not understood. Even the existing NMR structures, which provide detailed views of the RNA-binding interface, fail to provide a cogent reason for this extraordinary specificityThus, our data show that RbFox employs a previously undescribed mechanism to accomplish its extraordinary specificity - structurally distinct binding modes that enable the imposition of large thermodynamic penalties on non-consensus sequences and transmit RNA sequence information to distant regions of the RRM. The results reveal another layer of biological complexity in RNA-protein interactions and post-transcriptional regulation of gene expression.K1/2) for the RbFox cognate sequence (5’-GCAUG) was reduced by orders of magnitude by this single nucleotide substitution  of RbFox for two RNAs that differ by a single nucleotide Fig. . The appion Fig. , consistHigh Throughput Sequencing Equilibrium Binding (HiTS-Eq)24. This approach allows the simultaneous determination of apparent affinities for large numbers of sequence variants within an RNA pool23. We constructed a pool of 16,384 unique RNAs encoding all possible 7-mer sequences in a segment with 7 randomized nucleotides  for all substrate variants from binding reactions with increasing RbFox concentrations, as previously described21. Experiments were performed in independent duplicates, which provided highly correlated sequencing read values , RNACompete and HTR-SELEX approaches18 G is lower by orders of magnitude, compared to its cognate 5-mer. To our knowledge, this extraordinary ability to discriminate against all but two closely related sequence variants has not been reported for other RBPs. Second, the bimodal affinity distribution of RbFox differs from the unimodal distributions seen for other RBPs24. To account for variations in the register of each possible 5-mers in our RNA pool with 7 randomized nucleotides, we plotted affinity values for all 48 5-mers for each of the 1,024 5-mer variants and determined the median relative affinity for each 5-mer  model Fig. , the simers Fig. . Howeverers Fig. . Most iners Fig. . These o24. Considering coupled contributions from multiple positions within the binding sites often improves the description of the experimental variance2. We analyzed the binding profiles to RbFox for 5’-GCNUG sequences  between all nucleotidesant Fig. . Collectmut, Fig. 20. We hypothesized that mutations in the RRM should affect the two distinct binding modes differently. We first measured the binding of RbFoxmut to the individual RNAs examined above with wild type (wt) RbFox  or (5’-UUCAUGU), designated as RNA1 and RNA2. RNA1 and RNA2 both represent examples for non-cognate sequence binding modes, and rank in the 17.4 and 37.6 percentile, respectively, among the 16,384 sequence variants. We titrated each RNA into the wt RbFox RRM until saturation, while monitoring 1H-15N HSQC  and two non-cognate RNAs, which differ from the cognate variant at a single nucleotide  between the RbFox complexes bound to the consensus RNA and to RNA1 and RNA2 Fig. . For RNA1H-15N HSQC and NOESY spectra changed during data collection, suggesting multiple binding orientations that are most likely due to the weak binding of the non-cognate RNA. These characteristics made unambiguous NOE identification difficult. To overcome this problem, when collecting spectra for the protein, we oversaturated the protein with RNA and changed the sample frequently during data collection to ensure RbFox was consistently in the fully bound conformation. For the RNA, we used some NOEs for the GCA sequence from our previous NMR dataset within the pre-miR20b stem loop in complex with RbFox25. Since chemical shifts are very similar for both proteins and RNAs, we obtained a dataset that converged to a reliable structural ensemble . However, the bimodal affinity distribution of RbFox cannot be described by a single binding model.The notion of distinct binding modes for the RRM of RbFox is based on three converging, independent lines of evidence. First, the affinity distribution of RbFox for all 16,384 7-mer RNA sequence variants is bimodal 34.Third, structural differences between complexes of RbFox with the cognate and a non-cognate sequence are observed Figs. , 6. Beyo37. However, these scenarios differ fundamentally from the multiple binding modes of a single RNA-binding module shown here. The two binding modes of the RbFox RRM enable exceptionally high sequence selectivity by enabling the imposition of a high thermodynamic penalty on the non-cognate sequences. Mutations in the RRM diminish the ability to maintain the high thermodynamic penalty because the affinity for non-cognate variants increases and specificity decreases  competent cells. Transformants were grown in LB media at 37 °C to OD600nm = 0.6. The recombinant proteins were overexpressed in E. coli at 18 °C overnight upon induction of 0.5 mM IPTG. The cells were harvested by centrifugation and resuspended in lysis buffer  and lysed by sonication at 4 °C. The crude extracts were centrifuged at 27,000 × g for 60 min to remove the cell debris. To purify the recombinant proteins, the supernatant was applied onto a HisTrap column  and a Heparin column  in succession. Protein concentrations were measured by UV absorbance at 280 nm and confirmed by the Bradford assay.Expression and purification of RbFox RRM and RbFoxProtein preparation for NMR experiments required complete removal of traces of RNase contaminants, due to the long incubation and weak binding of wt RbFox to the non-cognate RNA. To accomplish this, RbFox was double tagged with both Hisx6 and GST at its N-terminus and purified with HisTrap and GSTrap columns in succession, followed by a Heparin column to remove residual non-specifically bound RNAs. Both affinity tags were then cleaved with TEV protease and removed by Nickel affinity chromatography. The purified protein eluent was concentrated and loaded onto a Superdex 75 10/300 GL column equilibrated with NMR buffer (10 mM sodium phosphate at pH 6.0 with 30 mM NaCl). Aliquoted protein samples were flash-frozen for storage at −80 °C.32P-ATP and T4 Polynucleotide Kinase 40. Uniformly radiolabeled RNA substrates were purified with 20% denaturing PAGE  and concentrations were quantified by scintillation counter.RNA substrates were purchased from Dharmacon  and Sigma-Aldrich . RNA substrates were 5’ end labeled using γ40.For the HiTS-Eq randomized RNA pool, 5’ end radiolabeled randomized RNA substrates containing a central segment of 7 randomized (NNNNNNN) nucleotides flanked by illumina sequencing primers (underlined sequences) were annealed with two complementary DNA oligonucleotides (underlined sequences) Table . The RNAmut with all RNAs were performed at 30 °C  and protein concentrations as indicated in the Figures . was incubated with RbFox for 10 min. Competitor RNAs at increasing concentrations were added and incubated for 10 min. Longer incubation times did not change observed fractions of bound and free RNA. Samples were loaded on 8% non-denaturing PAGE, as above. Gels were dried and radioactivity in bound and free bands were quantified using a Phosphorimager (GE) and ImageQuant 5.2 software . Plots of the fraction bound RNA vs. chase RNA concentrations were fitted against the binding equation for competitive inhibition using KaleidaGraphA: reaction amplitude, R1: Substrate concentration, R2: Competitor concentration, [K1/2]1: apparent dissociation constant for substrate, [K1/2]2: apparent dissociation constant for competitor)(40.HiTS-Eq reactions (50 µL) were performed with 1 nM of 5’ end radiolabeled RNA pool with increasing protein concentrations, as indicated in the figures. RNA and protein were equilibrated for 10 min. Longer incubation times did not change the fraction of bound and free RNA. Bound and free RNAs were separated by 8% non-denaturing PAGE. The gel was exposed for 30 min to autoradiography film, free RNA species were located, cut out, and eluted from the gel and RNA was extracted and recovered as previously described23. The RNA was reverse transcribed into cDNA with Superscript III Reverse Transcriptase . The cDNA was PCR amplified into HiTS-Eq libraries using index barcode primers . Subsequently, the HiTS-Eq libraries for different protein concentrations were pooled (equimolar) and sequenced using 50 bp single-end reads on Illumina HiSeq 2500.The HiTS-Eq libraries for Illumina next generation sequencing (NGS) were generated from the eluted RNA as previously describedhttp://novocraft.com) or the Fastx-Toolkit v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/). The sequencing reads were then aligned to the sequence of nucleotides 6–29 of the single strand randomized substrate, allowing one mismatch but no gaps using Perl scripts. The counts of the individual 7-mer sequences and two degenerative random nucleotides in the index barcode primers were added and exported into excel tables using Perl scripts .Raw sequencing reads were quality checked with FastQC v0.11.5 and de-multiplexed based on their corresponding index barcode primers using Novocraft v3.0.8  were calculated from concentration dependent changes of the 7-mer sequence variants analogous to the procedure described previously23. The ratios of the two RNA substrates S1 and S2 at a given protein concentration (E) was described using a competitive binding scheme according to: S1,0 and S2,0 are initial concentrations of the RNA substrates S1 and S2 at HiTS-Eq control reaction, and equilibrium constants are described as K1 and K2.Relative equilibrium constants (K2) for S2 is:The association constant  is the value for the given RNA variant (S2), normalized by the value for substrate S1 [UGCAUGU], according to:The relative association constant for a given RNA variant  value is determined by a set of linear coefficients at individual nucleotide positions, according to:The relative association constants were fitted with the Position Weight Matrix (PWM) model as previously describedNi  are based on the nucleotide identity at position i . Ni was assigned to value 1 for matched nucleotide at position i, and 0 otherwise. The cognate sequence motif UGCAUGU or GCAUG for 5-mers were used as baseline and excluded from the linear regression for the PWM models. Linear coefficients for the PWM model correspond to the parameters αi, βi, γi and δi, i = 1 − n.The numbers for 12. Interaction coefficients between individual nucleotide positions were added to the PWM model to fit the KA,rel data from HiTS-Eq in equation, as previously described23. Interaction coefficient values (In) with a T value larger than 3.5 were considered statistically significant. Obtained interaction coefficients values were plotted as heatmaps.The Pairwise Coupling (PWC) model considers all pairwise interactions between two nucleotides13C, 15N labeled RRM of RbFox (residues 109-208) was prepared in minimal M9 medium supplemented with 1 g/L 15NH4Cl and 2 g/L 13C-glucose. The complex was formed by titrating unlabeled RNA into 13C, 15N labeled protein, and monitored by recording the 15N HSQC of the protein. NMR experiments were performed on Bruker Avance 600 and Avance 800 spectrometers equipped with HCN cryo-probes and pulsed field gradients. NMR data were processed with NMRPipe42 and analyzed with CCPNMR43. 2D F1-, F2-filtered NOESYs44 (mixing times 100 and 300 ms) and 2D TOCSY (mixing time 80 ms) were collected on unlabeled RNA in complex with 13C, 15N labeled protein in D2O at 298 K to assign the aromatic and sugar protons of the RNA. 2D 15N/13C HSQCs, 3D HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HBHA(CO)NH and HCCH-TOCSY spectra were collected on 15N, 13C labeled protein in complex with unlabeled RNA to assign the backbone and the non-aromatic side-chains of the protein. 2D 13C HSQC with carbon centered at ~125 ppm and 3D 13C NOESY-HSQC (mixing time 150 ms) were also recorded to assign aromatic side chains of the protein.RNAs for NMR measurements were purchased from Integrated DNA Technologies. Oligonucleotides were dissolved in RNase-free water, desalted using a PD MiniTrap G-10 and re-suspended in NMR buffer after overnight lyophilization. 25. Manually assigned intermolecular NOE distance restraints derived from 3D NOESYs at 100 ms mixing time were separated into three ranges based on the cross-peak intensities, strong (1.8–3.5 Å), medium (1.8–4.5 Å) and weak (1.8–5.5 Å). Additional NOEs observed only in 2D NOESY with 300 ms of mixing time were assigned as very weak (1.8–6.5 Å).Protein assignments followed the regular protocol using triple resonance spectra listed above. RNA resonance assignments were obtained from 2D NOESY and TOCSY by comparison with chemical shifts of GCAUG motif of pre-miR20b stem loop complexed with RbFox and those reported for UGCAUGU-RbFox complex15N and 13C NOESY-HSQCs obtained by CYANA; intra-RNA NOEs manually assigned from 2D F1-, F2-filtered NOESYs spectra; and intermolecular NOEs manually assigned from 2D F1-filtered, F2-edited NOESY and 3D 13C F1-filtered, F3-edited NOESY-HSQC.The NOE distance restraints for the complex were obtained from the combination of: intra-protein NOEs automatically assigned from 45, and the 20 structures with the lowest target function were further regularized in an implicit solvent model using the SANDER module of Amber 14.046. We have used ff99bsc0xOL3 force field for RNA and ff14SB force field for protein. The script for the restrained simulated annealing protocol was modified from Tolbert et al.47. Protein torsion angles were obtained by TALOS+48. For the complex, we heated the system to 1500 K during Amber simulated annealing refinement. The 20 lowest-energy structures were analyzed with PROCHECK-NMR49.Dihedral angle restraints for the conformation of sugar rings (C2’-endo or C3’-endo) were added, based on H1’-H2’ cross-peak intensities in 2D TOCSY spectrum. With all of the restraints above, 100 initial structures were generated in CYANAR) scores for the selected RBPs were acquired from ENCODE data portal (https://www.encodeproject.org). The R scores were then normalized to a scale of 1-to-Euler’s constant (e), followed by natural log-normalization to fit R scores to a scale of 0 to 1. The normalized affinities for 5-mer sequence variants were then used to generate the affinity distributions for the six selected RBPs. While there is no agreed-upon method to quantitatively define the inherent specificity, we used the ratio between the highest affinity value to the median affinity value.We utilized RbFox HiTS-Eq data as well as RNA Bind-n-Seq (RBNS) datasets from the ENCODE consortium to generate affinity distributions. First, we normalized HiTS-Eq RbFox global binding affinity data to a scale of 0 to 1. In addition, for RBNS datasets, we sorted through the RBPs analyzed by the ENCODE consortium to select only the RBPs that had a single RNA binding domain in their structures or; if the RBP had multiple RBDs, then a single RBD was purified and examined. Specifically, we selected 5 RBPs  to generate the affinity distributions. Next, the 5-mer RBNS enrichment (Further information on research design is available in the Supplemental InformationPeer Review FileReporting Summary


Metastasis is the leading cause of treatment failure and tumor-related death in colorectal cancer (CRC). Our previous studies report that CEMIP functionally promotes CRC metastasis and is closely related to poor outcomes. However, the molecular network of CEMIP promoting CRC metastasis is still not fully understood. In the current study, we identify CEMIP interacting with GRAF1, and the combination of high-CEMIP and low-GRAF1 predicts poor survival of patients. Mechanistically, we elucidate that CEMIP interacts with the SH3 domain of GRAF1 through the 295–819aa domain, and negatively regulates the stability of GRAF1. Moreover, we identify MIB1 to be an E3 ubiquitin ligase for GRAF1. Importantly, we uncover that CEMIP acts as a scaffold protein in bridging MIB1 and GRAF1, which is critical to GRAF1 degradation and CEMIP-mediated CRC metastasis. Furthermore, we found that CEMIP activates CDC42/MAPK pathway-regulated EMT by enhancing the degradation of GRAF1, which is indispensable to CEMIP-mediated migration and invasion of CRC cells. Subsequently, we prove that CDC42 inhibitor suppresses CEMIP-mediated CRC metastasis in vitro and in vivo. Collectively, our results reveal that CEMIP promotes CRC metastasis through GRAF1/CDC42/MAPK pathway-regulated EMT, and suggest that CDC42 inhibitor could be a novel therapeutic strategy for CEMIP-mediated CRC metastasis. Althouged death –7. Unfor2+ release and activate PKCα signaling, then accelerated metastasis of breast cancer [Recently, it was reported that CEMIP  was associated with migration, invasion and drug resistance in various tumors –14, partt cancer . Rodrigut cancer . In termt cancer –17, and t cancer . CollectGRAF1  was reported to be a tumor suppressor that was functionally or genetically inactivated in several tumors, including gastric cancer, ovarian cancer, and metastatic brain cancer –22. GRAFIn this study, we identify CEMIP interacting with GRAF1, and the combination of high-CEMIP and low-GRAF1 predicts poor survival of patients. Mechanistically, we elucidated that the 295–819aa domain of CEMIP interacted with the SH3 domain of GRAF1, and negatively regulates the stability of GRAF1. Moreover, we uncovered that CEMIP acted as a scaffold protein in bridging MIB1 (an E3 ubiquitin ligase) and GRAF1, which was critical to GRAF1 degradation. Furthermore, we found that CEMIP promoted CRC metastasis by enhancing the degradation of GRAF1 and activating CDC42/MAPK pathway-regulated EMT. Subsequently, we proved that CDC42 inhibitor suppressed CEMIP-mediated CRC metastasis in vitro and in vivo. Collectively, our results suggested that CDC42 inhibitor could be a novel therapeutic strategy for CEMIP-mediated CRC metastasis.Our previous studies demonstrated that the elevated expression of CEMIP was closely related to poor outcomes and promoted CRC metastasis –18. To eTo further validate the interaction between CEMIP and GRAF1, we performed a series of Co-IP experiments. As showed in Fig. To further investigate which domain of CEMIP was required for binding to GRAF1, we constructed six truncated variants of CEMIP with a C-terminal myc tag , as indicated in Supplementary Fig. It was reported that, the SH3 domain of GRAF1 was conserved in different species  and screened the potential E3 ubiquitin ligases of GRAF1  and tumor metastasis, we deduced that CEMIP promoted CRC metastasis via MAPK signaling-induced EMT , 31. We S10S10Then we validated the correlation of CEMIP-mediated CRC metastasis and CDC42/MAPK pathway-regulated EMT. Our results indicated that up-regulation of CEMIP increased the migration and invasion of CRC cells, which was attenuated by CDC42 inhibitor Fig. . MeanwhiS10S10S10Metastasis is one of the main causes of death in patients with advanced colon cancer, and it is an obstacle in the clinical treatment. Our previous research proved that CEMIP functionally promoted CRC metastasis and was closely related to poor outcomes. And CDC42/MAPK pathway was proved to play a key role in the regulation of tumor growth, metastasis and drug resistance , 32, 33.Accumulating evidence showed that CEMIP was potential to serve as a therapeutic target for tumor metastasis. To further explore the underlying molecular mechanisms of CEMIP-mediated CRC metastasis, we identified CEMIP interacting with GRAF1, and CEMIP was negative correlated with GRAF1 in CRC. GRAF1 was reported to be a tumor suppressor that was genetically or functionally inactivated in several tumors, including gastric cancer, ovarian cancer, and metastatic brain cancer –22. InteAlthough CEMIP promoted the ubiquitination level and degradation of GRAF1, CEMIP had no canonical catalytic structure of E3 ubiquitin ligase. Hence, we hypothesized that CEMIP regulated the stability of GRAF1 by relying on an E3 ubiquitin ligase. Then we identified MIB1 as a potential E3 ubiquitin ligase of GRAF1. MIB1 was reported as an E3 ubiquitin ligase that promoted the ubiquitination and degradation of Notch ligands , and plaand in vivo. Altogether, our results revealed that CEMIP promoted CRC metastasis through GRAF1/CDC42/MAPK pathway-regulated EMT.Previous studies indicated that GRAF1, as a GTPase activating protein, inhibited the function of CDC42 through GTP hydrolysis via protein-protein interaction , 39. CDCIn conclusion, we identified CEMIP interacting with GRAF1, and the combination of high-CEMIP and low-GRAF1 predicted poor survival of patients. Moreover, we uncovered that CEMIP acted as a scaffold protein in bridging MIB1 and GRAF1, which was critical to GRAF1 degradation and CEMIP-mediated CRC metastasis. Furthermore, we found that CEMIP activated CDC42/MAPK pathway-regulated EMT by enhancing the degradation of GRAF1, which was indispensable to CEMIP-mediated migration and invasion of CRC cells. Importantly, we proved that CDC42 inhibitor suppressed CEMIP-mediated CRC metastasis in vitro and in vivo. Therefore, our research suggested that CDC42 inhibitor could be a novel therapeutic strategy for CEMIP-mediated CRC metastasis.Human CRC cells  and HEK293T cells were purchased from Cell Bank, Type Culture Collection, Chinese Academy of Sciences , and were cultured in DMEM or RPMI 1640  supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 μg/mL streptomycin. All human cell lines have been authenticated using STR profiling within the last 3 years. All experiments were performed with mycoplasma-free cells.For immunoprecipitation, cells were harvested and resuspended in NETN buffer . Cell lysate was centrifuged for 20 min at 13200 rpm at 4 °C. The supernatant was incubated with Pierce Protein G agarose beads  which had been incubated with the primary antibody or IgG, shaking overnight at 4 °C. Then the precipitates were washed five times with NETN buffer and analyzed by NanoLC-Ultra 1D plus system mass spectrometer , and analyzed by the Chemiluminescent Western Blot Detection Kit .4 per well for migration, 10*104 per well for invasion) were seeded in the upper well of the transwell chamber  coated with or without Matrigel  with serum-free medium and allowed to migrate or invade towards the medium containing 10% FBS in the lower compartment for 24 h or 48 h. Cells reaching the lower surface of each chamber were fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 30 min and counted in five randomly selected microscopic fields.For transwell assays, cells . Then, the primary antibodies, GRAF1  and CEMIP , GRAF1  and MIB1 , CEMIP  and MIB1  or IgG  and IgG  were incubated with the cells for 2 h at 37 °C. Then, cells were washed with wash buffer and incubated with PLA probe for 1 h at 37 °C. The ligation-ligase was added to cells at 37 °C for half an hour, cells were next incubated with amplification-polymerase solution for 100 min. The Duolink In Situ Mounting Medium with DAPI was applied to take photos under the confocal microscope.n = 10). Intrasplenic injection with 3*106 HCT116 cells  was conducted in mice. Three days after cells inoculation, mice were treated with ZCL278(50 mg/kg) or DMSO every 2 days by intraperitoneal injection. Four weeks later, five randomly selected mice from each group were sacrificed, and liver metastasis was analyzed. The rest five mice in each group were observed for other 4 weeks for survival analysis. Mice were excluded if died from other causes (such as fighting and infection). Researchers were not blinded to the group allocation during the experiment and outcome assessment.All animal experiments were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. We established stable HCT116 lines which respectively overexpressed CEMIP, GRAF1 and CEMIP + GRAF1. Female BALB/c nude mice (4–6 weeks old) were allocated randomly into four groups (The other Methods and materials used in this research are described in Supplementary Methods and Materials.original WBSupplementary Figure S1–10 and Table S1–3Supplementary Table.S4Supplementary methods and materialsE3 ligase from ubibrowser databaseSTR reportAnimal Ethics ProvementEthics ProvementSupplementary sequencing and other datacheck list