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polyclonal antibody against the ythdc2-170aa polypeptide  (GenScript corporation)

 
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    GenScript corporation polyclonal antibody against the ythdc2-170aa polypeptide
    Expression profiles and characterization of <t>circYthdc2.</t> A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments
    Polyclonal Antibody Against The Ythdc2 170aa Polypeptide, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polyclonal antibody against the ythdc2-170aa polypeptide - by Bioz Stars, 2026-02
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    1) Product Images from "CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape"

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-024-05148-9

    Expression profiles and characterization of circYthdc2. A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments
    Figure Legend Snippet: Expression profiles and characterization of circYthdc2. A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments

    Techniques Used: Expressing, Selection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Negative Control, Nucleic Acid Electrophoresis

    CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01
    Figure Legend Snippet: CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01

    Techniques Used: Activity Assay, Clone Assay, Construct, Plasmid Preparation, FLAG-tag, Expressing, Transfection, Infection, Immunofluorescence, Staining

    CircYthdc2 and Ythdc2-170aa inhibit host antiviral innate immunity. A and B The schematic diagram of siRNAs ( A ) and circYthdc2 overexpression plasmid structure ( B ). C qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MIC cells treated with siRNAs. qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MKC cells overexpressing circYthdc2. D and E qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-circYthdc2 ( D ) and transfected in MKC cells with circYthdc2 or pLC5-circ and Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG after SCRV infection ( E ). F circYthdc2 and Ythdc2-170aa promote SCRV replication. MIC cells transfected with NC or si-circYthdc2 and MKC cells were transfected with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. G MKC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MKC cells were transfected with Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG. H Effect of circYthdc2 on cell viability after SCRV infection. MIC cells were transfected with NC or si-circYthdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay was measured. I Ythdc2-170aa counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING, MAVS, TRIF, and TBK1 expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa. J Relative protein levels of STING in MIC cells after transfected with NC or si-circYthdc2 and in MKC cells with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa or Linear-FL-Ythdc2-AG. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01
    Figure Legend Snippet: CircYthdc2 and Ythdc2-170aa inhibit host antiviral innate immunity. A and B The schematic diagram of siRNAs ( A ) and circYthdc2 overexpression plasmid structure ( B ). C qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MIC cells treated with siRNAs. qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MKC cells overexpressing circYthdc2. D and E qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-circYthdc2 ( D ) and transfected in MKC cells with circYthdc2 or pLC5-circ and Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG after SCRV infection ( E ). F circYthdc2 and Ythdc2-170aa promote SCRV replication. MIC cells transfected with NC or si-circYthdc2 and MKC cells were transfected with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. G MKC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MKC cells were transfected with Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG. H Effect of circYthdc2 on cell viability after SCRV infection. MIC cells were transfected with NC or si-circYthdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay was measured. I Ythdc2-170aa counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING, MAVS, TRIF, and TBK1 expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa. J Relative protein levels of STING in MIC cells after transfected with NC or si-circYthdc2 and in MKC cells with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa or Linear-FL-Ythdc2-AG. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Techniques Used: Over Expression, Plasmid Preparation, Expressing, Transfection, Infection, RNA Expression, Staining, Viability Assay, Luciferase, Cotransfection

    Ythdc2 inhibits host antiviral innate immunity. A Relative protein and mRNA levels of Ythdc2 in MIC cells after transfected with NC or si-circYthdc2. B Relative protein levels of STING in MIC cells after transfected with NC or si-Ythdc2 and in MKC cells with pcDNA3.1 or Ythdc2. C and D qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-Ythdc2 ( C ) and transfected in MKC cells with pcDNA3.1 and Ythdc2 after SCRV infection ( D ). E Ythdc2 counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1, Ythdc2. F Ythdc2 promotes SCRV replication. MIC cells transfected with NC or si-Ythdc2 and MKC cells were transfected with pcDNA3.1 or Ythdc2 for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. (G) MIC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC cells were transfected with NC or si-Ythdc2. H Effect of Ythdc2 on cell viability after SCRV infection. MIC cells was transfected with NC or si-Ythdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay were measured. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01
    Figure Legend Snippet: Ythdc2 inhibits host antiviral innate immunity. A Relative protein and mRNA levels of Ythdc2 in MIC cells after transfected with NC or si-circYthdc2. B Relative protein levels of STING in MIC cells after transfected with NC or si-Ythdc2 and in MKC cells with pcDNA3.1 or Ythdc2. C and D qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-Ythdc2 ( C ) and transfected in MKC cells with pcDNA3.1 and Ythdc2 after SCRV infection ( D ). E Ythdc2 counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1, Ythdc2. F Ythdc2 promotes SCRV replication. MIC cells transfected with NC or si-Ythdc2 and MKC cells were transfected with pcDNA3.1 or Ythdc2 for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. (G) MIC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC cells were transfected with NC or si-Ythdc2. H Effect of Ythdc2 on cell viability after SCRV infection. MIC cells was transfected with NC or si-Ythdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay were measured. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Techniques Used: Transfection, Expressing, Infection, Luciferase, Cotransfection, Plasmid Preparation, RNA Expression, Staining, Viability Assay

    Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. A MKC cells were transfected with Flag-Ythdc2 and Flag-circYthdc2 plasmids, the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed; MIC cells were silence Ythdc2 or circYthdc2, and the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed B MKC cells were transfected with Linear-Flag-Ythdc2-170aa or Flag-circYthdc2 or Flag-Ythdc2 plasmids, after 42 h, the cells were treated with DMSO or 10 μM MG132 for 6 h before immunoblot analysis was performed. C Flag-Ythdc2 and Linear-Flag-Ythdc2-170aa were cotransfected with GFP-STING into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2 and STING or Ythdc2-170aa and STING cellular localization. Original magnification is 630. D Immunoprecipitation and immunoblot analysis of Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa with Myc-STING, in EPC cells. IP, immunoprecipitation. E Upper panel: Schematic diagram of Ythdc2-△HELICc plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, Flag-Ythdc2-△HELICc or Linear-Flag-Ythdc2-170aa with Myc-STING, in MKC cells. IP, immunoprecipitation. F Schematic diagram of STING, STING-△TM, STING-△N, STING-△C plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. H Immunoprecipitation and immunoblot analysis of Linear-Flag-Ythdc2-170aa with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. I Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. J Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2, or Flag-Ythdc2-△HELICc, or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. K Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the three independent triplicated experiments
    Figure Legend Snippet: Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. A MKC cells were transfected with Flag-Ythdc2 and Flag-circYthdc2 plasmids, the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed; MIC cells were silence Ythdc2 or circYthdc2, and the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed B MKC cells were transfected with Linear-Flag-Ythdc2-170aa or Flag-circYthdc2 or Flag-Ythdc2 plasmids, after 42 h, the cells were treated with DMSO or 10 μM MG132 for 6 h before immunoblot analysis was performed. C Flag-Ythdc2 and Linear-Flag-Ythdc2-170aa were cotransfected with GFP-STING into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2 and STING or Ythdc2-170aa and STING cellular localization. Original magnification is 630. D Immunoprecipitation and immunoblot analysis of Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa with Myc-STING, in EPC cells. IP, immunoprecipitation. E Upper panel: Schematic diagram of Ythdc2-△HELICc plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, Flag-Ythdc2-△HELICc or Linear-Flag-Ythdc2-170aa with Myc-STING, in MKC cells. IP, immunoprecipitation. F Schematic diagram of STING, STING-△TM, STING-△N, STING-△C plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. H Immunoprecipitation and immunoblot analysis of Linear-Flag-Ythdc2-170aa with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. I Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. J Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2, or Flag-Ythdc2-△HELICc, or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. K Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the three independent triplicated experiments

    Techniques Used: Ubiquitin Proteomics, Transfection, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Plasmid Preparation, Control, Expressing

    N6-methyladenosine modification mediates circYthdc2 translation polypeptides. A Upper panel: Schematic diagram of Flag-circYthdc2-m 6 A-mut plasmid construction. Lower panel: MKC cells were transfected with Flag-circYthdc2 or Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. B Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with m 6 A modification-related genes, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. C Left panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with METTL3 or METTL14, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Middle panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with FTO or YTHDF1, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Right panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with YTHDF3 or Ythdc2, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. D Relative RNA levels of circYthdc2 in MKC cells after transfected with pcDNA3.1, METTL3, METTL14, YTHDF1, YTHDF3, FTO, and Ythdc2, respectively. E The m 6 A level alteration of circYthdc2 upon METTL3 or FTO overexpression was examined by MeRIP-qPCR. MKC cells were transfected with vector or METTL3 or FTO plasmid for 48 h. F The level of circYthdc2 upon YTHDF1 or YTHDF3 or Ythdc2 overexpression were examined by RIP-qPCR. MKC cells were transfected with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. G The protein level of YTHDF1 or YTHDF3 or Ythdc2 was examined by RNA pulldown. MKC cells were transfected MS2-GFP, MS2-circYthdc2 or MS2-circYthdc2-m 6 A-mut with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. H Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, circYthdc2, METTL3, METTL14, FTO, YTHDF1, YTHDF3. I Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-circYthdc2 or m 6 A modification-related genes expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01
    Figure Legend Snippet: N6-methyladenosine modification mediates circYthdc2 translation polypeptides. A Upper panel: Schematic diagram of Flag-circYthdc2-m 6 A-mut plasmid construction. Lower panel: MKC cells were transfected with Flag-circYthdc2 or Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. B Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with m 6 A modification-related genes, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. C Left panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with METTL3 or METTL14, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Middle panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with FTO or YTHDF1, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Right panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with YTHDF3 or Ythdc2, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. D Relative RNA levels of circYthdc2 in MKC cells after transfected with pcDNA3.1, METTL3, METTL14, YTHDF1, YTHDF3, FTO, and Ythdc2, respectively. E The m 6 A level alteration of circYthdc2 upon METTL3 or FTO overexpression was examined by MeRIP-qPCR. MKC cells were transfected with vector or METTL3 or FTO plasmid for 48 h. F The level of circYthdc2 upon YTHDF1 or YTHDF3 or Ythdc2 overexpression were examined by RIP-qPCR. MKC cells were transfected with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. G The protein level of YTHDF1 or YTHDF3 or Ythdc2 was examined by RNA pulldown. MKC cells were transfected MS2-GFP, MS2-circYthdc2 or MS2-circYthdc2-m 6 A-mut with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. H Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, circYthdc2, METTL3, METTL14, FTO, YTHDF1, YTHDF3. I Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-circYthdc2 or m 6 A modification-related genes expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Techniques Used: Modification, Plasmid Preparation, Transfection, Western Blot, Over Expression, Luciferase, Cotransfection, Expressing, Ubiquitin Proteomics

    Ythdc2-170aa is highly conserved in structure and function in vertebrates. A CircYthdc2 exists in Miichthys miiuy , Nibea albiflora , Sciaenops ocellatus , Larimichthys ocellatus , Xenopus tropicalis (GenBank accession no. XM_031893156.1), Bufo gargarizans (GenBank accession no. XM_044275926.1), Podarcis muralis (GenBank accession no. XM_028748342.1), Numida meleagris (GenBank accession no. XM_021380759.1), Mus musculus (GenBank accession no. NM_001163013.1), Homo sapiens (GenBank accession no. NM_022828.5), and is composed of exon 13 to exon 18, with a length of 634nt. We confirmed the head-to-tail splicing of hsa -circYthdc2 in the hsa -circYthdc2 RT-PCR product by Sanger sequencing. B Sequence alignment of circYthdc2 from teleost fish to mammals. C Amino acid sequence alignment of circYthdc2 translated polypeptides from teleost fish to mammals. D HEK293 cells were transfected with vector or hsa -Flag-circYthdc2, hsa -Flag-circYthdc2-ATG-mut, hsa -Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. E HEK293 cells were transfected STING and hsa -Flag-circYthdc2 and hsa -Flag-circYthdc2-m 6 A-mut with METTL3 or METTL14 or YTHDF1 or FTO plasmids, after 48 h, the immunoblot analysis was performed. F HEK293 cells were transfected STING and hsa -Flag-circYthdc2 with si- has -METTL3 or si- has -METTL14 or si- has -YTHDF1 or si- has -FTO, after 48 h, the immunoblot analysis was performed. All data represented the three independent triplicated experiments
    Figure Legend Snippet: Ythdc2-170aa is highly conserved in structure and function in vertebrates. A CircYthdc2 exists in Miichthys miiuy , Nibea albiflora , Sciaenops ocellatus , Larimichthys ocellatus , Xenopus tropicalis (GenBank accession no. XM_031893156.1), Bufo gargarizans (GenBank accession no. XM_044275926.1), Podarcis muralis (GenBank accession no. XM_028748342.1), Numida meleagris (GenBank accession no. XM_021380759.1), Mus musculus (GenBank accession no. NM_001163013.1), Homo sapiens (GenBank accession no. NM_022828.5), and is composed of exon 13 to exon 18, with a length of 634nt. We confirmed the head-to-tail splicing of hsa -circYthdc2 in the hsa -circYthdc2 RT-PCR product by Sanger sequencing. B Sequence alignment of circYthdc2 from teleost fish to mammals. C Amino acid sequence alignment of circYthdc2 translated polypeptides from teleost fish to mammals. D HEK293 cells were transfected with vector or hsa -Flag-circYthdc2, hsa -Flag-circYthdc2-ATG-mut, hsa -Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. E HEK293 cells were transfected STING and hsa -Flag-circYthdc2 and hsa -Flag-circYthdc2-m 6 A-mut with METTL3 or METTL14 or YTHDF1 or FTO plasmids, after 48 h, the immunoblot analysis was performed. F HEK293 cells were transfected STING and hsa -Flag-circYthdc2 with si- has -METTL3 or si- has -METTL14 or si- has -YTHDF1 or si- has -FTO, after 48 h, the immunoblot analysis was performed. All data represented the three independent triplicated experiments

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Plasmid Preparation, Western Blot

    Schematic diagram of the mechanism underlying Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. Under normal circumstances, circYthdc2 does not translate to produce polypeptides. When SCRV virus infects the host, the pathway of circYthdc2 translating polypeptides is activated. There are two pathways for circYthdc2 to be translated into polypeptides, the one is IRES-mediated translation pathway and another m 6 A modification mediated translation pathway. In addition, Ythdc2 will preferentially promote the RNA degradation of circYthdc2 when circYthdc2 is produced in large quantities. Ythdc2-170aa and Ythdc2 both could promote the STING protein degradation and represses STING-mediated antiviral responses, thereby regulating viral replication. Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING, thereby inhibited the antiviral responses and help the virus escape
    Figure Legend Snippet: Schematic diagram of the mechanism underlying Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. Under normal circumstances, circYthdc2 does not translate to produce polypeptides. When SCRV virus infects the host, the pathway of circYthdc2 translating polypeptides is activated. There are two pathways for circYthdc2 to be translated into polypeptides, the one is IRES-mediated translation pathway and another m 6 A modification mediated translation pathway. In addition, Ythdc2 will preferentially promote the RNA degradation of circYthdc2 when circYthdc2 is produced in large quantities. Ythdc2-170aa and Ythdc2 both could promote the STING protein degradation and represses STING-mediated antiviral responses, thereby regulating viral replication. Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING, thereby inhibited the antiviral responses and help the virus escape

    Techniques Used: Ubiquitin Proteomics, Virus, Modification, Produced



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    GenScript corporation polyclonal antibody against the ythdc2-170aa polypeptide
    Expression profiles and characterization of <t>circYthdc2.</t> A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments
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    Expression profiles and characterization of circYthdc2. A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: Expression profiles and characterization of circYthdc2. A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Expressing, Selection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Negative Control, Nucleic Acid Electrophoresis

    CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Activity Assay, Clone Assay, Construct, Plasmid Preparation, FLAG-tag, Expressing, Transfection, Infection, Immunofluorescence, Staining

    CircYthdc2 and Ythdc2-170aa inhibit host antiviral innate immunity. A and B The schematic diagram of siRNAs ( A ) and circYthdc2 overexpression plasmid structure ( B ). C qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MIC cells treated with siRNAs. qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MKC cells overexpressing circYthdc2. D and E qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-circYthdc2 ( D ) and transfected in MKC cells with circYthdc2 or pLC5-circ and Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG after SCRV infection ( E ). F circYthdc2 and Ythdc2-170aa promote SCRV replication. MIC cells transfected with NC or si-circYthdc2 and MKC cells were transfected with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. G MKC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MKC cells were transfected with Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG. H Effect of circYthdc2 on cell viability after SCRV infection. MIC cells were transfected with NC or si-circYthdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay was measured. I Ythdc2-170aa counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING, MAVS, TRIF, and TBK1 expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa. J Relative protein levels of STING in MIC cells after transfected with NC or si-circYthdc2 and in MKC cells with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa or Linear-FL-Ythdc2-AG. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: CircYthdc2 and Ythdc2-170aa inhibit host antiviral innate immunity. A and B The schematic diagram of siRNAs ( A ) and circYthdc2 overexpression plasmid structure ( B ). C qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MIC cells treated with siRNAs. qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MKC cells overexpressing circYthdc2. D and E qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-circYthdc2 ( D ) and transfected in MKC cells with circYthdc2 or pLC5-circ and Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG after SCRV infection ( E ). F circYthdc2 and Ythdc2-170aa promote SCRV replication. MIC cells transfected with NC or si-circYthdc2 and MKC cells were transfected with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. G MKC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MKC cells were transfected with Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG. H Effect of circYthdc2 on cell viability after SCRV infection. MIC cells were transfected with NC or si-circYthdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay was measured. I Ythdc2-170aa counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING, MAVS, TRIF, and TBK1 expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa. J Relative protein levels of STING in MIC cells after transfected with NC or si-circYthdc2 and in MKC cells with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa or Linear-FL-Ythdc2-AG. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Over Expression, Plasmid Preparation, Expressing, Transfection, Infection, RNA Expression, Staining, Viability Assay, Luciferase, Cotransfection

    Ythdc2 inhibits host antiviral innate immunity. A Relative protein and mRNA levels of Ythdc2 in MIC cells after transfected with NC or si-circYthdc2. B Relative protein levels of STING in MIC cells after transfected with NC or si-Ythdc2 and in MKC cells with pcDNA3.1 or Ythdc2. C and D qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-Ythdc2 ( C ) and transfected in MKC cells with pcDNA3.1 and Ythdc2 after SCRV infection ( D ). E Ythdc2 counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1, Ythdc2. F Ythdc2 promotes SCRV replication. MIC cells transfected with NC or si-Ythdc2 and MKC cells were transfected with pcDNA3.1 or Ythdc2 for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. (G) MIC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC cells were transfected with NC or si-Ythdc2. H Effect of Ythdc2 on cell viability after SCRV infection. MIC cells was transfected with NC or si-Ythdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay were measured. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: Ythdc2 inhibits host antiviral innate immunity. A Relative protein and mRNA levels of Ythdc2 in MIC cells after transfected with NC or si-circYthdc2. B Relative protein levels of STING in MIC cells after transfected with NC or si-Ythdc2 and in MKC cells with pcDNA3.1 or Ythdc2. C and D qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-Ythdc2 ( C ) and transfected in MKC cells with pcDNA3.1 and Ythdc2 after SCRV infection ( D ). E Ythdc2 counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1, Ythdc2. F Ythdc2 promotes SCRV replication. MIC cells transfected with NC or si-Ythdc2 and MKC cells were transfected with pcDNA3.1 or Ythdc2 for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. (G) MIC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC cells were transfected with NC or si-Ythdc2. H Effect of Ythdc2 on cell viability after SCRV infection. MIC cells was transfected with NC or si-Ythdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay were measured. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Transfection, Expressing, Infection, Luciferase, Cotransfection, Plasmid Preparation, RNA Expression, Staining, Viability Assay

    Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. A MKC cells were transfected with Flag-Ythdc2 and Flag-circYthdc2 plasmids, the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed; MIC cells were silence Ythdc2 or circYthdc2, and the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed B MKC cells were transfected with Linear-Flag-Ythdc2-170aa or Flag-circYthdc2 or Flag-Ythdc2 plasmids, after 42 h, the cells were treated with DMSO or 10 μM MG132 for 6 h before immunoblot analysis was performed. C Flag-Ythdc2 and Linear-Flag-Ythdc2-170aa were cotransfected with GFP-STING into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2 and STING or Ythdc2-170aa and STING cellular localization. Original magnification is 630. D Immunoprecipitation and immunoblot analysis of Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa with Myc-STING, in EPC cells. IP, immunoprecipitation. E Upper panel: Schematic diagram of Ythdc2-△HELICc plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, Flag-Ythdc2-△HELICc or Linear-Flag-Ythdc2-170aa with Myc-STING, in MKC cells. IP, immunoprecipitation. F Schematic diagram of STING, STING-△TM, STING-△N, STING-△C plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. H Immunoprecipitation and immunoblot analysis of Linear-Flag-Ythdc2-170aa with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. I Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. J Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2, or Flag-Ythdc2-△HELICc, or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. K Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the three independent triplicated experiments

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. A MKC cells were transfected with Flag-Ythdc2 and Flag-circYthdc2 plasmids, the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed; MIC cells were silence Ythdc2 or circYthdc2, and the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed B MKC cells were transfected with Linear-Flag-Ythdc2-170aa or Flag-circYthdc2 or Flag-Ythdc2 plasmids, after 42 h, the cells were treated with DMSO or 10 μM MG132 for 6 h before immunoblot analysis was performed. C Flag-Ythdc2 and Linear-Flag-Ythdc2-170aa were cotransfected with GFP-STING into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2 and STING or Ythdc2-170aa and STING cellular localization. Original magnification is 630. D Immunoprecipitation and immunoblot analysis of Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa with Myc-STING, in EPC cells. IP, immunoprecipitation. E Upper panel: Schematic diagram of Ythdc2-△HELICc plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, Flag-Ythdc2-△HELICc or Linear-Flag-Ythdc2-170aa with Myc-STING, in MKC cells. IP, immunoprecipitation. F Schematic diagram of STING, STING-△TM, STING-△N, STING-△C plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. H Immunoprecipitation and immunoblot analysis of Linear-Flag-Ythdc2-170aa with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. I Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. J Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2, or Flag-Ythdc2-△HELICc, or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. K Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the three independent triplicated experiments

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Plasmid Preparation, Control, Expressing

    N6-methyladenosine modification mediates circYthdc2 translation polypeptides. A Upper panel: Schematic diagram of Flag-circYthdc2-m 6 A-mut plasmid construction. Lower panel: MKC cells were transfected with Flag-circYthdc2 or Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. B Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with m 6 A modification-related genes, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. C Left panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with METTL3 or METTL14, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Middle panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with FTO or YTHDF1, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Right panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with YTHDF3 or Ythdc2, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. D Relative RNA levels of circYthdc2 in MKC cells after transfected with pcDNA3.1, METTL3, METTL14, YTHDF1, YTHDF3, FTO, and Ythdc2, respectively. E The m 6 A level alteration of circYthdc2 upon METTL3 or FTO overexpression was examined by MeRIP-qPCR. MKC cells were transfected with vector or METTL3 or FTO plasmid for 48 h. F The level of circYthdc2 upon YTHDF1 or YTHDF3 or Ythdc2 overexpression were examined by RIP-qPCR. MKC cells were transfected with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. G The protein level of YTHDF1 or YTHDF3 or Ythdc2 was examined by RNA pulldown. MKC cells were transfected MS2-GFP, MS2-circYthdc2 or MS2-circYthdc2-m 6 A-mut with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. H Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, circYthdc2, METTL3, METTL14, FTO, YTHDF1, YTHDF3. I Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-circYthdc2 or m 6 A modification-related genes expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: N6-methyladenosine modification mediates circYthdc2 translation polypeptides. A Upper panel: Schematic diagram of Flag-circYthdc2-m 6 A-mut plasmid construction. Lower panel: MKC cells were transfected with Flag-circYthdc2 or Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. B Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with m 6 A modification-related genes, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. C Left panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with METTL3 or METTL14, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Middle panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with FTO or YTHDF1, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Right panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with YTHDF3 or Ythdc2, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. D Relative RNA levels of circYthdc2 in MKC cells after transfected with pcDNA3.1, METTL3, METTL14, YTHDF1, YTHDF3, FTO, and Ythdc2, respectively. E The m 6 A level alteration of circYthdc2 upon METTL3 or FTO overexpression was examined by MeRIP-qPCR. MKC cells were transfected with vector or METTL3 or FTO plasmid for 48 h. F The level of circYthdc2 upon YTHDF1 or YTHDF3 or Ythdc2 overexpression were examined by RIP-qPCR. MKC cells were transfected with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. G The protein level of YTHDF1 or YTHDF3 or Ythdc2 was examined by RNA pulldown. MKC cells were transfected MS2-GFP, MS2-circYthdc2 or MS2-circYthdc2-m 6 A-mut with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. H Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, circYthdc2, METTL3, METTL14, FTO, YTHDF1, YTHDF3. I Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-circYthdc2 or m 6 A modification-related genes expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Modification, Plasmid Preparation, Transfection, Western Blot, Over Expression, Luciferase, Cotransfection, Expressing, Ubiquitin Proteomics

    Ythdc2-170aa is highly conserved in structure and function in vertebrates. A CircYthdc2 exists in Miichthys miiuy , Nibea albiflora , Sciaenops ocellatus , Larimichthys ocellatus , Xenopus tropicalis (GenBank accession no. XM_031893156.1), Bufo gargarizans (GenBank accession no. XM_044275926.1), Podarcis muralis (GenBank accession no. XM_028748342.1), Numida meleagris (GenBank accession no. XM_021380759.1), Mus musculus (GenBank accession no. NM_001163013.1), Homo sapiens (GenBank accession no. NM_022828.5), and is composed of exon 13 to exon 18, with a length of 634nt. We confirmed the head-to-tail splicing of hsa -circYthdc2 in the hsa -circYthdc2 RT-PCR product by Sanger sequencing. B Sequence alignment of circYthdc2 from teleost fish to mammals. C Amino acid sequence alignment of circYthdc2 translated polypeptides from teleost fish to mammals. D HEK293 cells were transfected with vector or hsa -Flag-circYthdc2, hsa -Flag-circYthdc2-ATG-mut, hsa -Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. E HEK293 cells were transfected STING and hsa -Flag-circYthdc2 and hsa -Flag-circYthdc2-m 6 A-mut with METTL3 or METTL14 or YTHDF1 or FTO plasmids, after 48 h, the immunoblot analysis was performed. F HEK293 cells were transfected STING and hsa -Flag-circYthdc2 with si- has -METTL3 or si- has -METTL14 or si- has -YTHDF1 or si- has -FTO, after 48 h, the immunoblot analysis was performed. All data represented the three independent triplicated experiments

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: Ythdc2-170aa is highly conserved in structure and function in vertebrates. A CircYthdc2 exists in Miichthys miiuy , Nibea albiflora , Sciaenops ocellatus , Larimichthys ocellatus , Xenopus tropicalis (GenBank accession no. XM_031893156.1), Bufo gargarizans (GenBank accession no. XM_044275926.1), Podarcis muralis (GenBank accession no. XM_028748342.1), Numida meleagris (GenBank accession no. XM_021380759.1), Mus musculus (GenBank accession no. NM_001163013.1), Homo sapiens (GenBank accession no. NM_022828.5), and is composed of exon 13 to exon 18, with a length of 634nt. We confirmed the head-to-tail splicing of hsa -circYthdc2 in the hsa -circYthdc2 RT-PCR product by Sanger sequencing. B Sequence alignment of circYthdc2 from teleost fish to mammals. C Amino acid sequence alignment of circYthdc2 translated polypeptides from teleost fish to mammals. D HEK293 cells were transfected with vector or hsa -Flag-circYthdc2, hsa -Flag-circYthdc2-ATG-mut, hsa -Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. E HEK293 cells were transfected STING and hsa -Flag-circYthdc2 and hsa -Flag-circYthdc2-m 6 A-mut with METTL3 or METTL14 or YTHDF1 or FTO plasmids, after 48 h, the immunoblot analysis was performed. F HEK293 cells were transfected STING and hsa -Flag-circYthdc2 with si- has -METTL3 or si- has -METTL14 or si- has -YTHDF1 or si- has -FTO, after 48 h, the immunoblot analysis was performed. All data represented the three independent triplicated experiments

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Plasmid Preparation, Western Blot

    Schematic diagram of the mechanism underlying Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. Under normal circumstances, circYthdc2 does not translate to produce polypeptides. When SCRV virus infects the host, the pathway of circYthdc2 translating polypeptides is activated. There are two pathways for circYthdc2 to be translated into polypeptides, the one is IRES-mediated translation pathway and another m 6 A modification mediated translation pathway. In addition, Ythdc2 will preferentially promote the RNA degradation of circYthdc2 when circYthdc2 is produced in large quantities. Ythdc2-170aa and Ythdc2 both could promote the STING protein degradation and represses STING-mediated antiviral responses, thereby regulating viral replication. Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING, thereby inhibited the antiviral responses and help the virus escape

    Journal: Cellular and Molecular Life Sciences

    Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

    doi: 10.1007/s00018-024-05148-9

    Figure Lengend Snippet: Schematic diagram of the mechanism underlying Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. Under normal circumstances, circYthdc2 does not translate to produce polypeptides. When SCRV virus infects the host, the pathway of circYthdc2 translating polypeptides is activated. There are two pathways for circYthdc2 to be translated into polypeptides, the one is IRES-mediated translation pathway and another m 6 A modification mediated translation pathway. In addition, Ythdc2 will preferentially promote the RNA degradation of circYthdc2 when circYthdc2 is produced in large quantities. Ythdc2-170aa and Ythdc2 both could promote the STING protein degradation and represses STING-mediated antiviral responses, thereby regulating viral replication. Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING, thereby inhibited the antiviral responses and help the virus escape

    Article Snippet: A polyclonal antibody against the Ythdc2-170aa polypeptide produced by circYthdc2 was obtained by inoculating rabbits (GenScript).

    Techniques: Ubiquitin Proteomics, Virus, Modification, Produced