Journal: Frontiers in Immunology

Article Title: Transcriptome-wide N6-methyladenosinem modifications analysis of chicken cecum in responding to Campylobacter jejuni inoculation

doi: 10.3389/fimmu.2025.1630008

Figure Lengend Snippet: Validation of m 6 A peaks and mRNA levels of differentially expressed genes containing differential m 6 A peaks in the C. jejuni -susceptible (S) and resistant (R) groups. (A) qRT-PCR validation of gene expression in the resistant and susceptible groups. (B) The expression levels of m 6 A modification related genes METTL3 , METTL14 , YTHDC2 , ALKBH5 , and FTO in the resistant and susceptible groups of chicken cecum. (C) qRT-PCR results of SUSD5 , WDR41 , EPG5 , FOS , STAB2 , and IFT74 in the resistant and susceptible groups of chicken cecum. (D) meRIP-qPCR results of SUSD5 , WDR41 , EPG5 , FOS , STAB2 , and IFT74 in the resistant and susceptible groups of chicken cecum. The data are pooled from 2 independent experiments with 6 replicates per group (n = 6) and presented as the mean ± SEM; * and ** represent P < 0.05, P < 0.01, respectively.

Article Snippet: The small interfering RNA (si-YTHDC2) for YTHDC2 (Sense: 5’-CAGCUUUAAUUGUGAGAAATT-3’; Anti-sense: 3’-UUUCUCACAAUUAAAGCUGTT-5’) and negative control si-NC were obtained from Sangon Biotech (Shanghai, China).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Gene Expression, Expressing, Modification

Journal: Frontiers in Immunology

Article Title: Transcriptome-wide N6-methyladenosinem modifications analysis of chicken cecum in responding to Campylobacter jejuni inoculation

doi: 10.3389/fimmu.2025.1630008

Figure Lengend Snippet: The regulatory role of YTHDC2 for chicken HD11 cell line responding to C. jejuni LPS stimulation. (A) Flow cytometric analysis of apoptosis in HD11 cells after YTHDC2 knock down followed by C. jejuni LPS stimulation. (B) Effects of interference YTHDC2 on the mRNA expression of apoptosis and autophagy related genes involved in responding to C. jejuni LPS stimulation. The data are pooled from 4 independent experiments with 4 replicates per group (n = 4) and presented as the mean ± SEM; * and ** represent P < 0.05, P < 0.01, respectively.

Article Snippet: The small interfering RNA (si-YTHDC2) for YTHDC2 (Sense: 5’-CAGCUUUAAUUGUGAGAAATT-3’; Anti-sense: 3’-UUUCUCACAAUUAAAGCUGTT-5’) and negative control si-NC were obtained from Sangon Biotech (Shanghai, China).

Techniques: Knockdown, Expressing

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet: Negative correlation between YTHDC2 and RUNX2 in BMSCs osteogenesis. A-B. The mRNA and protein expression of YTHDC2 in BMSCs quantified by qRT-PCR and Western blot. C-D. The mRNA and protein expression of RUNX2 in BMSCs quantified by qRT-PCR and Western blot. (*P < 0.05).

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet: Knockdown of YTHDC2 promoted BMSCs osteogenic differentiation and RUNX2 expression. BMSCs were transfected with YTHDC2-siRNA (YTHDC2-si) or negative control-siRNA (NC), after which they were cultured in OM for 7 days. A-B . The qRT-PCR and Western blot results of YTHDC2 expression in BMSCs. C-D . ALP staining and alizarin red staining of BMSCs. E-F . The qRT-PCR and Western blot results of YTHDC2 expression in BMSCs. (*P < 0.05).

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Knockdown, Expressing, Transfection, Negative Control, Cell Culture, Quantitative RT-PCR, Western Blot, Staining

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet: YTHDC2 regulated RUNX2 expression through m6A modification. BMSCs were cultured in osteogenic medium (OM) or growth medium (GM) for 7 days. A. m6A-IP showed OM-induced BMSCs had more m6A modification on RUNX2 mRNA. B. YTHDC2-IP showed YTHDC2 tightly bound to RUNX2 m6A. C. Immunofluorescence staining showed negative correlation between YTHDC2 and RUNX2. D. Fluorescence intensity of OM-induced and GM-induced BMSCs. E. The mechanism of YTHDC2 potential effects on BMSCs osteogenesis through m6A modification on RUNX2 mRNA. (*P < 0.05).

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Expressing, Modification, Cell Culture, Immunofluorescence, Staining, Fluorescence

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet:

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Sequencing

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet:

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Sequencing

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

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

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: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Ubiquitin Proteomics, Virus, Modification, Produced

Journal: Science Advances

Article Title: RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

doi: 10.1126/sciadv.abq2945

Figure Lengend Snippet: ( A ) Identification of YTHDC2-associated partners by immunoprecipitation (IP) experiments with either YTHDC2 or normal immunoglobulin antibodies from P12 mouse testes. Immunoprecipitated YTHDC2 protein complexes were separated by SDS-PAGE, and the gel was stained with silver before mass spectrometry (MS). Testis lysates were prepared from mouse at P12 ( n = 8). ( B ) Selective YTHDC2-associated protein candidates identified by MS analysis from P12 mouse testes. ( C ) Immunoprecipitation of YTHDC2 from mouse testis lysates at P21 and Western blot with anti-YTHDC2, anti-MEIOC, and anti-RBM46 antibodies (top). RBM46 protein was immunoprecipitated with anti-RBM46 antibodies with or without RNases, followed by Western blot analysis with indicated antibodies (bottom). ( D ) Full-length GFP- Rbm46 and FLAG- Ythdc2 or HA- Meioc expression constructs were cotransfected in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG or anti-HA antibodies and Western blot analysis with indicated antibodies. IB, immunoblotting. ( E ) GFP-tagged RBM46 mutants with deleted fragments were coexpressed with FLAG-YTHDC2 in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibodies before Western blot analysis with GFP antibodies. ( F ) GST pull-down assay showing that in vitro translated YTHDC2 does not bind to GST-RBM46. GST-RBM46 was expressed in E. coli and purified with glutathione beads. YTHDC2 with N-terminal His tag was in vitro translated. ( G and H ) Immunofluorescence analysis of Rbm46 -HA knock-in mice with anti-HA antibodies and the acrosome marker PNA that defines different stages of spermatogenesis. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. Lower panels show magnification image of single cell in the upper panels. Spg, spermatogonia; PL, pre-leptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene; RS, round spermatids; ES, elongating spermatids. Scale bars, 5 μm.

Article Snippet: GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega).

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Western Blot, Expressing, Construct, Pull Down Assay, In Vitro, Purification, Immunofluorescence, Knock-In, Marker

Journal: Science Advances

Article Title: RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

doi: 10.1126/sciadv.abq2945

Figure Lengend Snippet: ( A ) RBM46 and YTHDC2 exhibit a similar distribution pattern along the length of mRNA. ( B ) RBM46 eCLIP mRNA targets substantially overlapped with YTHDC2 mRNA targets by CLIP-seq in P10 testis. In total, 53 and 52% of P10 YTHDC2–binding sites were co-occupied by RBM46 within a 100- and 70-nt window, respectively. ( C ) Top motif at best-scored YTHDC-binding sites from P10 and adult YTHDC2 CLIP-seq using HOMER. Sequences within ±100 nt relative to YTHDC2-binding sites were used for de novo motif analysis. ( D ) RBM46-binding motif AAUCAUGU is the top motif within a 100-nt window centered on YTHDC2 U-rich motifs in both P10 and adult testis. Motif analysis by HOMER was performed with best-scored P10 and adult YTHDC2 CLIP targets. ( E ) Position and distance between RBM46 and YTHDC2 binding on their shared targets. ( F ) Schematic illustrations for the function of RBM46-MEIOC-YTHDC2 complex during spermatogenesis. RNA binding protein RBM46 forms an ancient posttranscriptional network with MEIOC and YTHDC2 to recognize and destabilize mitotic transcripts for a successful meiotic entry.

Article Snippet: GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega).

Techniques: Binding Assay, RNA Binding Assay