Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 is upregulated in human ART-preterm placentas. ( A ) qRT-PCR detection of the expression level of YTHDC1 in placentas conceived by assisted reproductive technology (ART). Preterm: preterm placentas ( n = 12); Term: full-term placentas ( n = 17). ( B ) Western blotting detection of YTHDC1 in the placentas conceived by ART. Preterm ( n = 6), Term ( n = 6). ( C , D ) Immunohistochemistry (IHC) images and quantification of YTHDC1 positive staining areas in placentas conceived by ART. Preterm ( n = 6), Term ( n = 6). Scale bar, 200 μm and 50 μm. Data are shown as means ± SD. ** P < 0.01

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 knockdown inhibits the proliferation, migration, and invasion of trophoblastic cells. ( A ) Knockdown efficiency of YTHDC1 siRNA in HTR-8/SVneo and JAR cells as assessed by qRT-PCR. ( B ) Knockdown efficiency of YTHDC1 siRNA in HTR-8/SVneo and JAR cells as assessed by Western blotting. ( C ) Knockdown effects of YTHDC1 on the cellular viability of HTR-8/SVneo and JAR cells as detected by CCK-8. ( D ) Knockdown effects of YTHDC1 on the proliferation of HTR-8/SVneo and JAR cells as detected by colony formation assays. ( E , F ) Knockdown effects of YTHDC1 on the proliferation of HTR-8/SVneo and JAR cells as detected by EdU assay. Scale bar, 100 μm. ( G , H ) Cell apoptotic rate as analyzed by flow cytometry. ( I , J ) Knockdown effects of YTHDC1 on the migration (I) and invasion (J) of HTR-8/SVneo and JAR cells as detected by Transwell assay. Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot, CCK-8 Assay, EdU Assay, Flow Cytometry, Transwell Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: Identification of the signaling pathways regulated by YTHDC1 in trophoblastic cells. ( A ) Heatmap of differentially expressed genes (DEGs) identified by RNA-seq in HTR-8/SVneo cells. ( B ) Volcano plot of DEGs. ( C ) GO enrichment analysis of DEGs. ( D ) GSEA analysis of DEGs. ( E , F ) The knockdown (E) and overexpression (F) effects of YTHDC1 on the expression of DKK1, PIK3R3, and LIFR in HTR-8/SVneo and JAR cells as detected by Western blotting. ( G ) CCK-8 assay of JAR cells transfected with a YTHDC1 overexpression plasmid and PIK3R3 siRNA. ( H , I ) Colony formation assay of JAR cells transfected with a YTHDC1 overexpression plasmid and PIK3R3 siRNA. ( J , K ) Transwell assay about migration of JAR cells transfected with a YTHDC1 overexpression plasmid and PIK3R3 siRNA. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: RNA Sequencing Assay, Knockdown, Over Expression, Expressing, Western Blot, CCK-8 Assay, Transfection, Plasmid Preparation, Colony Assay, Transwell Assay, Migration

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 accelerates protein synthesis in trophoblastic cells. ( A ) The m 6 A motif detected by MEME algorithm analysis in identified mRNAs by MeRIP-seq in HTR-8/SVneo cells. ( B ) Metagene profiles of m 6 A enriched regions across mRNA segments in HTR-8/SVneo cells. ( C ) The distribution of m 6 A modified sites within mRNAs in HTR-8/SVneo cells. ( D ) GO enrichment analysis of the DEGs identified by RIP-seq. ( E ) Overlapping analysis of genes identified by RNA-seq, RIP-seq, and MeRIP-seq in HTR-8/SVneo and JAR cells. RNA-seq analysis was conducted on YTHDC1 knockdown HTR-8/SVneo cells. ( F ) Functional annotation of the overlapping genes. ( G ) IF staining of YTHDC1 in HTR-8/SVneo and JAR cells. Scale bar, 25 μm. ( H - J ) The effects of YTHDC1 knockout ( H , I ) or overexpression (J) on protein synthesis in HTR-8/SVneo and JAR cells detected by OP-Puro assays. Scale bar, 50 μm. ( K , L ) The effects of YTHDC1 knockdown ( K ) or overexpression (L) on de novo protein synthesis in HTR-8/SVneo and JAR cells as detected by SUnSET assays. ( M ) Polysome profiling of YTHDC1 overexpressed JAR cells

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Modification, RNA Sequencing Assay, Knockdown, Functional Assay, Staining, Knock-Out, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 regulates RPL37 translation in an m 6 A-dependent manner. ( A ) Relative mRNA level of RPL37 and eIF4G in human ART-preterm and ART-term placentas as detected by qRT-PCR. Preterm: preterm placentas ( n = 12); Term: full-term placentas ( n = 17). ( B , C ) Relative mRNA level of RPL37 and eIF4G in HTR-8/SVneo and JAR cells upon YTHDC1 knockdown ( B ) or overexpression ( C ) as detected by qRT-PCR. ( D , E ) Relative protein levels of RPL37 and eIF4G in HTR-8/SVneo and JAR cells upon YTHDC1 knockdown ( D ) or overexpression ( E ) as detected by Western blotting. ( F ) Integrative genomics viewer (IGV) tracks of m 6 A peaks and YTHDC1 binding peaks across RPL37 transcript. ( G ) qRT-PCR analysis of RPL37 in the MeRIP products. MeRIP carried out in HTR-8/SVneo and JAR cells with antibody against m 6 A, or the control unimmunized IgG. ( H ) qRT-PCR analysis of RPL37 in the RIP products. RIP carried out in HTR-8/SVneo and JAR cells with antibody against YTHDC1, or the control unimmunized IgG. ( I , J ) Schematic representation of wild-type (RPL37-WT) and mutant (RPL37-MUT) RPL37 luciferase reporters. ( K ) Luciferase activities of RPL37-WT or RPL37-MUT measured in 293T cells with or without YTHDC1 knockout. ( L ) Western blotting detection of YTHDC1 and RPL37. HTR-8/SVneo and JAR cells were co-transfected with YTHDC1 siRNA and RPL37 overexpression plasmid. ( M , N ) Rescue assay of cellular viability as detected with CCK-8. HTR-8/SVneo (M) and JAR cells ( N ) were co-transfected with YTHDC1 siRNA and RPL37 overexpression plasmid. ( O , P ) Rescue assay of colony formation. ( Q ) Rescue assay of SUnSET experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Binding Assay, Control, Mutagenesis, Luciferase, Knock-Out, Transfection, Plasmid Preparation, Rescue Assay, CCK-8 Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: Estradiol (E2) promotes YTHDC1 transcription through RXRA. ( A ) Luciferase assay of the YTHDC1 promoter. HEK-293T cells were transfected with reporter plasmids containing a series of YTHDC1 promoter truncations. ( B ) qRT-PCR quantification of potential transcription factors, RXRA and GTF2I , in human placental tissues. The potential transcription factors about the human YTHDC1 promoter between − 1000 to -900 bp are predicted by the ALGGEN-PROMO version 8.3 online tool of TRANSFAC. ( C ) Relative mRNA level of YTHDC1 as detected by qRT-PCR in HTR-8/SVneo and JAR cells upon knockdown of RXRA or GTF2I. ( D ) Western blotting analysis of YTHDC1 in HTR-8/SVneo and JAR cells upon knockdown of RXRA or GTF2I . ( E ) Luciferase assay about the effect of RXRA on the transcription of YTHDC1 . HEK-293T cells were transfected with RXRA siRNA or overexpression plasmid. ( F ) Alignment of ChIP-seq data from HTR-8/SVneo to the hg38 genome. The box showed the enrichment of RXRA and H3K27ac in the promoter of YTHDC1 . ( G ) ChIP-qPCR assay about the binding of RXRA to the promoter of YTHDC1 in HTR-8/SVneo and JAR cells. ( H ) Western blotting analysis of RXRA, YTHDC1, RPL37. HTR-8/SVneo and JAR cells were treated with E2 at the indicated concentrations for 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Luciferase, Transfection, Quantitative RT-PCR, Knockdown, Western Blot, Over Expression, Plasmid Preparation, ChIP-sequencing, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: High E2 exposure elevates YTHDC1 expression and knockdown of YTHDC1 postpones murine preterm delivery in vivo. ( A - D ) Murine model of high E2 exposure. C57BL/6J mice were pretreated with E2 or corn oil, and preterm delivery was induced at E15.5d by RU486, while controls were treated with PBS (Oil-MF, n = 6; Oil-PBS, n = 6, E2-MF, n = 5; E2-PBS, n = 6). ( A ) The schematic diagram. ( B ) Relative mRNA level of YTHDC1 in the placentas as detected by qRT-PCR. ( C ) Western blotting detection of the expression of YTHDC1, RPL37, and PIK3R3 in the placentas. ( D ) Representative IHC staining images of YTHDC1, RPL37 and PIK3R3 in the placentas. Scale bar, 200 μm and 50 μm. ( E-H ) The schematic diagram for murine model of YTHDC1 siRNA treatment. Pregnant C57BL/6J mice were was intravenously administered with siYTHDC1 or siNC via the tail vein, and preterm delivery was induced at E15.5d by RU486. ( E ) The schematic diagram. ( F ) The initiation time of preterm labor (siYTHDC1, n = 9; siNC, n = 7). ( G ) Western blotting detection of YTHDC1, RPL37, and PIK3R3 in the placentas. ( H ) Representative IHC staining images of YTHDC1, RPL37, Ki-67, 11-β-HSD2 and CGB in the placentas. Scale bar, 50 μm. ** P < 0.01

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Expressing, Knockdown, In Vivo, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: The schematic diagram about working model of YTHDC1. Estradiol (E2) promoted YTHDC1 expression through RXRA upregulation. Elevated YTHDC1 upregulated the expression of RPL37 by promoting the binding of YTHDC1 to m 6 A-modified RPL37 mRNA, thereby augmenting total mRNA translation and indirectly modulated the JAK/STAT/PIK3R3 signaling pathway in trophoblastic cells

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Expressing, Binding Assay, Modification

Journal: bioRxiv

Article Title: FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

doi: 10.1101/2025.02.01.633781

Figure Lengend Snippet: a Experimental pipeline for HyPro-MS analysis of FUSint6&7-RNA condensates. b Efficient labelling of the endogenous FUSint6&7-RNA condensates using HyPro probes. Stellaris FUSint6-specific probe was used for co-staining. Arrows indicate the foci labelled by both Stellaris and HyPro probes. Scale bar, 10 µm. c Principal component analysis (PCA) demonstrating clustering of triplicated HyPro-MS samples for both probes and the no-probe control. d Volcano plot for FUSint6&7-RNA condensates versus no-probe control (Ctrl). Proteins with padj<0.05 are labelled in black. Proteins with padj<0.05 and involved in RNA metabolism-related pathways and/or implicated in neurodegeneration, are labelled in red. e Volcano plot for FUSint6&7-RNA condensates versus ACTB probe. Proteins with padj<0.1 are labelled in black and the top 15 hits are labelled in red. f Dot plot of GO Biological Process term enrichment analysis for nuclear proteins identified in FUSint6&7-RNA condensates and significantly enriched as compared to no-probe control. g Dot plot of GO Biological Process term enrichment analysis for nuclear proteins identified in FUSint6&7-RNA condensates and significantly enriched as compared to ACTB probe control. h Validation of HyPro-MS proteins enriched in FUSint6&7-RNA condensates, as well as FUS and other proteins previously shown to bind FUSint6&7-RNA. Cells expressing exoFUSint7 were analysed by RNA-FISH and immunofluorescence with appropriate antibodies. Representative images are shown. Top graph, 28-35 transfected cells with condensates were analysed per protein; bottom graph, 41 and 27 individual condensates were analysed for YTHDC1 and FUS enrichment, respectively, ****p<0.0001, two-tailed unpaired t test.

Article Snippet: The following commercial primary antibodies were used: FUS (mouse monoclonal, Santa Cruz, sc-47711 and rabbit polyclonal, Proteintech, 11570-1-AP); SMN (mouse monoclonal, BD Biosciences, 610646); coilin p80 (mouse monoclonal, BD Biosciences, 612074); TDP-43 (rabbit polyclonal, C-terminal, Sigma); YTHDC1 (rabbit polyclonal, Proteintech, 14392-1-AP); HDGFL2 (rabbit polyclonal, Proteintech, 15134-1-AP); hnRNPC (rabbit polyclonal, Proteintech, 11760-1-AP); PNN/pinin (rabbit polyclonal, Proteintech, 18266-1-AP); ANP32B (rabbit polyclonal, Proteintech, 10843-1-AP); FUBP1 (rabbit polyclonal, Proteintech, 24864-1-AP).

Techniques: Staining, Control, Expressing, Immunofluorescence, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: FUS post-transcriptional splicing is autoregulated via RNA condensation with therapeutic potential for ALS-FUS

doi: 10.1101/2025.02.01.633781

Figure Lengend Snippet: a,b YTHDC1 depletion or m6A downregulation leads to a loss of FUSint6&7-RNA condensate integrity. Representative images (general plane) and quantification of condensates (a) as well as high-resolution images (b) are shown. 107 and 65 cells (4 or 5 FoV) were analysed for scrambled and YTHDC1 siRNA, respectively, from a representative experiment. **p<0.01, Mann-Whitney U test. HeLa cells were used in a and SH-SY5Y cells were used in b . Representative images are shown. STM2457-treated cells are also shown. In b , PNN was used as a speckle marker. Scale bar, 20 μm in a and 5 μm in b . c YTHDC1 depletion does not affect FUSint6&7-RNA level but downregulates FUS mRNA. qRT-PCR analysis with FUS intron 6- and 7-specific primers was performed in HeLa cells. N=5-7, *p<0.05, Mann-Whitney U test. d High-confidence DRACH motif in FUS intron 7 shown in a structural context, as predicted by SRAMP. e,f FUS introns 6 and 7 are extensively methylated. m6A-Atlas 2.0 database was used for mapping methylation sites (e) and calculating the m6A mark density (f). Also see Supplementary Table S3. g,h Pharmacological inhibition of a m6A writer METTL3 affects FUSint6&7-RNA condensate integrity without changing levels of this RNA. Representative images and quantification of the condensate number in STM2457-treated cells (g) and qRT-PCR analysis of RNA level (h) are shown. In g , cells were treated for 16 h, and >200 cells (5 FoV) were analysed per condition from a representative experiment, **p<0.01, Mann-Whitney U test. Scale bar, 20 μm. In h , qRT-PCR analysis was done using FUS intron 6- and -7 specific primers. N=4. i Pharmacological inhibition of a m6A eraser FTO promotes FUSint6&7-RNA condensate assembly. Representative images and quantification of the condensate number in FB23-2 treated cells are shown. Cells were treated with the compound for either 4 or 16 h. 79, 106 and 117 cells (5 FoV) were analysed for DMSO, 4-h FB23-2 and 16-h FB23-2 treatments, respectively, from a representative experiment. *p<0.05, Kruskal-Wallis with Dunn’s test. Scale bar, 20 μm. j FUS intron 6 RNAscope-ISH reveals partial cytoplasmic redistribution of FUSint6&7-RNA in STM2457-treated cells. Arrows indicate cytoplasmic accumulations of this RNA; nucleus is circled in the insets. Scale bar, 20 μm. k FUSΔNLS lines have preserved or enhanced ability to form FUSint6&7-RNA condensates. Representative images and quantification are shown. 234, 239, 234, 141 and 62 cells were analysed for WT, ΔNLS4, ΔNLS7, ΔNLS10 and ΔNLS11 lines, respectively (from 4-9 FoV). *p<0.05, **p<0.01, Kruskal-Wallis with Dunn’s test. l MeRIP demonstrates increased FUSint6&7-RNA methylation in FUSΔNLS lines. Total RNA purified from ΔNLS7 (homozygous) and ΔNLS10 (heterozygous) lines was subjected to pulldown using m6A antibody-coated beads and used for qRT-PCR analysis with FUSint6-specific primers. Methylated FUSint6&7-RNA level was normalised to GAPDH mRNA (extensively methylated transcript), then to total FUSint6&7-RNA level in the respective cell line, and finally, to no-antibody beads control. N=4, *p<0.05, Kruskal-Wallis with Dunn’s test. m Enhanced association of FUSint6&7-RNA with YTHDC1 in FUSΔNLS lines. RIP was performed using Flag-Trap agarose in YTHDC1-Flag expressing WT and FUSΔNLS cells, followed by qRT-PCR analysis with FUSint6-specific primers. Venus-Flag was used as a control (“vector”). FUSint6&7-RNA level was normalised to GAPDH and then to total FUSint6&7-RNA level in the respective cell line. Results for ΔNLS4, ΔNLS7 and ΔNLS10 lines were combined. N=2. *p<0.05, Mann-Whitney U test (WT vs . ΔNLS).

Article Snippet: The following commercial primary antibodies were used: FUS (mouse monoclonal, Santa Cruz, sc-47711 and rabbit polyclonal, Proteintech, 11570-1-AP); SMN (mouse monoclonal, BD Biosciences, 610646); coilin p80 (mouse monoclonal, BD Biosciences, 612074); TDP-43 (rabbit polyclonal, C-terminal, Sigma); YTHDC1 (rabbit polyclonal, Proteintech, 14392-1-AP); HDGFL2 (rabbit polyclonal, Proteintech, 15134-1-AP); hnRNPC (rabbit polyclonal, Proteintech, 11760-1-AP); PNN/pinin (rabbit polyclonal, Proteintech, 18266-1-AP); ANP32B (rabbit polyclonal, Proteintech, 10843-1-AP); FUBP1 (rabbit polyclonal, Proteintech, 24864-1-AP).

Techniques: MANN-WHITNEY, Marker, Quantitative RT-PCR, Methylation, Inhibition, RNAscope, Purification, Control, Expressing, Plasmid Preparation

Journal: Stem Cells International

Article Title: YTHDC1 Promotes Stemness Maintenance and Malignant Progression in Head and Neck Squamous Cell Carcinoma

doi: 10.1155/2022/7494354

Figure Lengend Snippet: YTHDC1 aberrantly expressed in HNSCC and was associated with cancer cell stemness. (a) The relative mRNA expression of YTHDC1 in tumor and normal tissues in HNSCC in TCGA database. The comparison of the YTHDC1 mRNA expression in (b) individual tumor grades, (c) HPV infection status, and (d) different nodal metastasis status. (e) The Pearson correlation between YTHDC1 and SOX2 mRNA expression in HNSCC according to TCGA data. (f) The Pearson correlation between YTHDC1 and BMI1 mRNA expression in HNSCC according to the TCGA data.

Article Snippet: The following antibodies were used in the experiment: GAPDH (Proteintech, 10494-1-AP), YTHDC1 (Cell Signaling Technology, 54737S), BMI1 (Abcam, ab269678), SOX2 (Abcam, ab97959), OCT4 (Abcam, ab18976), and NANOG (Abcam, ab109250).

Techniques: Expressing, Comparison, Infection

Journal: Stem Cells International

Article Title: YTHDC1 Promotes Stemness Maintenance and Malignant Progression in Head and Neck Squamous Cell Carcinoma

doi: 10.1155/2022/7494354

Figure Lengend Snippet: YTHDC1 was overexpressed in HNSCC patients. (a) Representative graph of YTHDC1 staining in tumor and normal tissues of HNSCC patients (left) and the IHC score quantification (right) ( n = 6). Scale bar, 100 μ m. (b) The YTHDC1 expression in patients HNSCC tissues and normal tissues detected by immunoblotting analysis ( n = 3). (c) The SOX2 and BMI1 protein expression in patient HNSCC tissues and normal tissues detected by immunoblotting analysis ( n = 3).

Article Snippet: The following antibodies were used in the experiment: GAPDH (Proteintech, 10494-1-AP), YTHDC1 (Cell Signaling Technology, 54737S), BMI1 (Abcam, ab269678), SOX2 (Abcam, ab97959), OCT4 (Abcam, ab18976), and NANOG (Abcam, ab109250).

Techniques: Staining, Expressing, Western Blot

Journal: Stem Cells International

Article Title: YTHDC1 Promotes Stemness Maintenance and Malignant Progression in Head and Neck Squamous Cell Carcinoma

doi: 10.1155/2022/7494354

Figure Lengend Snippet: Inhibition of the YTHDC1 expression significantly limits tumor cell stemness maintenance, migration, and proliferation. (a) Western blotting of YTHDC1 in six HNSCC cell lines. (b) Western blotting of YTHDC1 in SCC9 and HN4 with or without YTHDC1 knockdown. (c) qPCR data of YTHDC1 in SCC9 and HN4 with or without YTHDC1 knockdown. (d) SOX2 and BMI1 protein expression in SCC9 and HN4 with or without YTHDC1 knockdown. (e) The OCT4 and NANOG protein expression in SCC9 and HN4 with or without YTHDC1 knockdown. (f). SCC9 and HN4 cell proliferation ability was suppressed after the YTHDC1 expression was decreased. (g) Sphere formation ability of SCC9 and HN4 cell lines was inhibited after the YTHDC1 expression was suppressed. (h) Representative images and quantification of migration assay of SCC9 and HN4 cell lines with or without YTHDC1 knockdown. (i) Representative images and quantification of colony formation assay of SCC9 and HN4 cell lines with or without YTHDC1 knockdown. (j). Tumor xenograft experiment in nude mice detects the effect of YTHDC1 on tumor growth in vivo ( n = 6 in each group). (k) Tumor growth curve. The quantitative value of tumor volumes is plotted, and tumor volumes are measured once two days after injected. (l) Scatter diagram shows tumor weight.

Article Snippet: The following antibodies were used in the experiment: GAPDH (Proteintech, 10494-1-AP), YTHDC1 (Cell Signaling Technology, 54737S), BMI1 (Abcam, ab269678), SOX2 (Abcam, ab97959), OCT4 (Abcam, ab18976), and NANOG (Abcam, ab109250).

Techniques: Inhibition, Expressing, Migration, Western Blot, Knockdown, Colony Assay, In Vivo, Injection

Journal: Stem Cells International

Article Title: YTHDC1 Promotes Stemness Maintenance and Malignant Progression in Head and Neck Squamous Cell Carcinoma

doi: 10.1155/2022/7494354

Figure Lengend Snippet: Validation of the role of YTHDC1 in regulating stemness maintenance at the single cell level. (a) The UMAP plot of scRNA-seq cell data labeled by cell type. (b) The UMAP plot of the YTHDC1 expression in all the clusters. (c) The UMAP plot of cancer cells from HNSCC tissues shows the YTHDC1 expression pattern. (d) The Pearson correlation between YTHDC1 and SOX2 mRNA expression according to the single cell RNA data. (e) The Pearson correlation between YTHDC1 and BMI1 mRNA expression according to the single cell RNA data. (f) Volcano plot shows differentially expressed genes between YTHDC1 high and YTHDC1 low cells. (g) GO analysis of the upregulated genes in the YTHDC1 high expression cell population. (h) KEGG analysis of the upregulated genes in the YTHDC1 high expression cell population. (i) Violin plot depicts the expression of stem cell-related markers of YTHDC1 high and YTHDC1 low cells.

Article Snippet: The following antibodies were used in the experiment: GAPDH (Proteintech, 10494-1-AP), YTHDC1 (Cell Signaling Technology, 54737S), BMI1 (Abcam, ab269678), SOX2 (Abcam, ab97959), OCT4 (Abcam, ab18976), and NANOG (Abcam, ab109250).

Techniques: Biomarker Discovery, Labeling, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m 6 A modification

doi: 10.1074/jbc.RA119.011556

Figure Lengend Snippet: pncRNA-D is highly m6A-modified, and the modification destabilizes pncRNA-D. A, the interaction between pncRNA-D and TLS was detected by RIP assay using TLS antibody with HeLa cells before and after osmotic stress (n = 5). B, Western blotting analysis using HeLa NE with or without 0.4 m sorbitol treatment. The arginine-methylated TLS level was compared with the total TLS level. ACTB was used as a loading control. A representative image of three independent experiments is shown. C, RIP assay with HeLa cell after osmotic stress or irradiation by m6A antibody (n = 5). D, RIP assay as in A with indicated antibodies of m6A recognition proteins. pncRNA-D bound to each recognition protein was measured by RT-qPCR (n = 3). E, relative expression level (normalized to GAPDH) of pncRNA-D after actinomycin D treatment with HeLa cells treated with siRNAs of negative control, METTL3, or YTHDC1. Expression levels at 1, 2, and 3 h after actinomycin D treatment are shown. Expression levels at 0 h in control cells were set to a value of 1.0 (n = 5). *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Article Snippet: The antibodies used were anti-METTL3 (15073-1-AP, Proteintech), anti-YTHDC1 (A305-096A, Bethyl Laboratories), anti-mono and di-methylarginine (7E6, ab412, Abcam), and anti-TLS/FUS (611385, BD Biosciences) for primary antibodies at the concentration of 1:2,000, anti-rabbit IgG horseradish peroxidase–linked antibody (7074S, Cell Signaling), and anti-mouse Igs/horseradish peroxidase (P0161, DAKO) for secondary antibodies at the concentration of 1:5,000.

Techniques: Modification, Western Blot, Methylation, Irradiation, Quantitative RT-PCR, Expressing, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m 6 A modification

doi: 10.1074/jbc.RA119.011556

Figure Lengend Snippet: m6A modification inhibits interaction between pncRNA-D and TLS by competitive binding of YTHDC1. A, RIP assay by TLS antibody with HeLa cells treated with siRNAs of negative control, METTL3, or YTHDC1 (n = 5). *, p < 0.05; **, p < 0.01. B, schematic drawing of full-length pncRNA-D and position of GGACU. The results of Western blotting analysis after RNA pulldown assay were cited from our previous paper in 2016 (9). 20-nt biotinylated fragment around GGACU was generated with (fragment m6A) or without (fragment A) m6A modification. C and D, RNA pulldown assay using fragment A or m6A incubated with GST–TLS (C) or HeLa nuclear extract (D). RNA-bound proteins were detected by the indicated antibodies. Representative images of three individual experiments are shown. The band intensity of TLS bound to fragment A or fragment m6A in HeLa NE or YTHDC1 KD HeLa NE was quantified by ImageJ (RRID:SCR_003070) (D, right) (n = 3); *, p < 0.05. E, RNA pulldown assay using fragment m6A incubated with GST–TLS and various concentration of strep-YTHDC1. Fragment m6A was incubated with GST–TLS and strep-YTHDC1 at the indicated amount, and the RNA bound GST–TLS was detected by Western blotting analysis.

Article Snippet: The antibodies used were anti-METTL3 (15073-1-AP, Proteintech), anti-YTHDC1 (A305-096A, Bethyl Laboratories), anti-mono and di-methylarginine (7E6, ab412, Abcam), and anti-TLS/FUS (611385, BD Biosciences) for primary antibodies at the concentration of 1:2,000, anti-rabbit IgG horseradish peroxidase–linked antibody (7074S, Cell Signaling), and anti-mouse Igs/horseradish peroxidase (P0161, DAKO) for secondary antibodies at the concentration of 1:5,000.

Techniques: Modification, Binding Assay, Negative Control, Western Blot, Generated, Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m 6 A modification

doi: 10.1074/jbc.RA119.011556

Figure Lengend Snippet: Deletion of GGACU motif in pncRNA-D decreases m6A modification level and alters interaction with RNA-binding proteins, but not RNA stability. A, sequences deleted by CRISPR/Cas9 are described. HAP1 clones named Δ20 and Δ46 lack 20 and 46 nucleotides around GGACT, respectively. B, RIP assay with WT or GGACT deleted HAP1 cells by anti-m6A antibody (n = 5). C, relative expression level (normalized to GAPDH) of pncRNA-D and CCND1 of WT or GGACT-deleted HAP1. Expression levels in control cells were set to a value of 1.0 (n = 5). D, luciferase reporter assay with control or GGACT deleted promoter region of CCND1 (n = 4). E, relative expression level (normalized to GAPDH) of pncRNA-D after actinomycin D treatment in WT or GGACT deleted HAP1. Expression levels of 1 and 2 h after actinomycin D treatment are shown. Expression levels at 0 h in control cells were set to a value of 1.0. F, RIP assay as in B with antibodies against TLS or YTHDC1. pncRNA-D bound to each protein was measured by RT-qPCR (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.005 in all the figures.

Article Snippet: The antibodies used were anti-METTL3 (15073-1-AP, Proteintech), anti-YTHDC1 (A305-096A, Bethyl Laboratories), anti-mono and di-methylarginine (7E6, ab412, Abcam), and anti-TLS/FUS (611385, BD Biosciences) for primary antibodies at the concentration of 1:2,000, anti-rabbit IgG horseradish peroxidase–linked antibody (7074S, Cell Signaling), and anti-mouse Igs/horseradish peroxidase (P0161, DAKO) for secondary antibodies at the concentration of 1:5,000.

Techniques: Modification, RNA Binding Assay, CRISPR, Clone Assay, Expressing, Luciferase, Reporter Assay, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m 6 A modification

doi: 10.1074/jbc.RA119.011556

Figure Lengend Snippet: pncRNA-D rescues cell cycle arrest caused by decreased m6A modification level. A, HeLa cells were sorted into G0/G1, S, and G2/M phase cells by FACS (top panel), and the expression level of CCND1 and pncRNA-D was measured in each fraction (bottom panel). The expression level of pncRNA-D in G0/G1 phase control cells was set to a value of 1.0 (n = 5). B, histograms of FACS after indicated treatments. Representative figures of three distinct experiments are shown by histograms. Overlaid graph of HeLa cells treated with si-pncRNAD in addition to si-METTL3, si-YTHDC1, or 0.4 m sorbitol treatment are shown in light blue histograms. C and D, population of each fraction of experiments in B were analyzed statistically for siRNA treated cells (C) or 0.4 m sorbitol treated cells (D). E, relative expression level of CCND1 after siRNA treatments. Expression level of in control cells was set to a value of 1.0 (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Article Snippet: The antibodies used were anti-METTL3 (15073-1-AP, Proteintech), anti-YTHDC1 (A305-096A, Bethyl Laboratories), anti-mono and di-methylarginine (7E6, ab412, Abcam), and anti-TLS/FUS (611385, BD Biosciences) for primary antibodies at the concentration of 1:2,000, anti-rabbit IgG horseradish peroxidase–linked antibody (7074S, Cell Signaling), and anti-mouse Igs/horseradish peroxidase (P0161, DAKO) for secondary antibodies at the concentration of 1:5,000.

Techniques: Modification, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m 6 A modification

doi: 10.1074/jbc.RA119.011556

Figure Lengend Snippet: pncRNA-D negatively regulates cell proliferation. A–C, cell proliferation assay of HeLa cells with indicated siRNA treatments (A) or induced RNAs (C) or HAP1 cells with or without GGACU deletion (B). The numbers of living cells after the indicated number of days after treatments were evaluated. *, p < 0.05; **, p < 0.01; ***, p < 0.005. D, model of TLS–pncRNA-D inhibitory effect on CCND1 expression caused by cellular stresses. Normally, pncRNA-D is highly methylated, and this modification leads to pncRNA-D degradation, and interaction with YTHDC1 inhibits binding to TLS. Therefore, histone acetyltransferase activity of CBP is maintained, and CCND1 is activated. However, once the cell gets damaged by cellular stresses, pncRNA-D expression is enhanced, and decreased methylation stabilizes it and thereafter recruits TLS to the promoter. Interaction with pncRNA-D enables TLS to inhibit histone acetyltransferase (HAT) activity of CBP and subsequently reduces the expression of CCND1, which results in cell cycle arrest.

Article Snippet: The antibodies used were anti-METTL3 (15073-1-AP, Proteintech), anti-YTHDC1 (A305-096A, Bethyl Laboratories), anti-mono and di-methylarginine (7E6, ab412, Abcam), and anti-TLS/FUS (611385, BD Biosciences) for primary antibodies at the concentration of 1:2,000, anti-rabbit IgG horseradish peroxidase–linked antibody (7074S, Cell Signaling), and anti-mouse Igs/horseradish peroxidase (P0161, DAKO) for secondary antibodies at the concentration of 1:5,000.

Techniques: Proliferation Assay, Expressing, Methylation, Modification, Binding Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m 6 A modification

doi: 10.1074/jbc.RA119.011556

Figure Lengend Snippet: Primers used in the study

Article Snippet: The antibodies used were anti-METTL3 (15073-1-AP, Proteintech), anti-YTHDC1 (A305-096A, Bethyl Laboratories), anti-mono and di-methylarginine (7E6, ab412, Abcam), and anti-TLS/FUS (611385, BD Biosciences) for primary antibodies at the concentration of 1:2,000, anti-rabbit IgG horseradish peroxidase–linked antibody (7074S, Cell Signaling), and anti-mouse Igs/horseradish peroxidase (P0161, DAKO) for secondary antibodies at the concentration of 1:5,000.

Techniques: ChIP-chip, CRISPR, Luciferase, Over Expression

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 2. YTHDC1 Knockdown Prolongs the Duration of the Early Stage of Mitosis

Article Snippet: Doxycycline (Dox)-inducible cell lines expressing FLAG and HA-tagged wild-type or mutant YTHDC1 (A549/YTHDC1-WT or -W377A/W428A) were established as previously described.11) Plasmids pcDNA3-FLAG-HA-hYTHDC1 was a gift from Samie Jaffrey (Addgene plasmid # 85167; http://n2t.net/ addgene:85167; RRID:Addgene_85167).12) FLAG-HA epitopetagged YTHDC1 was subcloned into pENTR4-no-ccDB (686-1) [a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17424; http://n2t.net/addgene:17424; RRID: Addgene_17424)].13) The small interfering RNA (siRNA)-resistant YTHDC1 plasmid was created by introducing a silent mutation into the siYTHDC1#1 target site by PCR using the following primers: (sense) 5′-CTGGTCTCCAAGCCACTGAGCTCATCTGTTAGC-3′ and (antisense) 5′-CTGCCTCGAATGGACAGAAGGCTTTTGTCG-3′.

Techniques: Knockdown

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 3. The Spindle Assembly Checkpoint Is Involved in the YTHDC1 Knockdown-Induced Delay in Mitotic Progression

Article Snippet: Doxycycline (Dox)-inducible cell lines expressing FLAG and HA-tagged wild-type or mutant YTHDC1 (A549/YTHDC1-WT or -W377A/W428A) were established as previously described.11) Plasmids pcDNA3-FLAG-HA-hYTHDC1 was a gift from Samie Jaffrey (Addgene plasmid # 85167; http://n2t.net/ addgene:85167; RRID:Addgene_85167).12) FLAG-HA epitopetagged YTHDC1 was subcloned into pENTR4-no-ccDB (686-1) [a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17424; http://n2t.net/addgene:17424; RRID: Addgene_17424)].13) The small interfering RNA (siRNA)-resistant YTHDC1 plasmid was created by introducing a silent mutation into the siYTHDC1#1 target site by PCR using the following primers: (sense) 5′-CTGGTCTCCAAGCCACTGAGCTCATCTGTTAGC-3′ and (antisense) 5′-CTGCCTCGAATGGACAGAAGGCTTTTGTCG-3′.

Techniques: Knockdown

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 4. YTHDC1 Supports Mitotic Progression Partly through Regulation of Centrosomal Abundance of Aurora A

Article Snippet: Doxycycline (Dox)-inducible cell lines expressing FLAG and HA-tagged wild-type or mutant YTHDC1 (A549/YTHDC1-WT or -W377A/W428A) were established as previously described.11) Plasmids pcDNA3-FLAG-HA-hYTHDC1 was a gift from Samie Jaffrey (Addgene plasmid # 85167; http://n2t.net/ addgene:85167; RRID:Addgene_85167).12) FLAG-HA epitopetagged YTHDC1 was subcloned into pENTR4-no-ccDB (686-1) [a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17424; http://n2t.net/addgene:17424; RRID: Addgene_17424)].13) The small interfering RNA (siRNA)-resistant YTHDC1 plasmid was created by introducing a silent mutation into the siYTHDC1#1 target site by PCR using the following primers: (sense) 5′-CTGGTCTCCAAGCCACTGAGCTCATCTGTTAGC-3′ and (antisense) 5′-CTGCCTCGAATGGACAGAAGGCTTTTGTCG-3′.

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: N 6 -Methyladenosine Reader Protein YTHDC1 Supports Proper Mitotic Progression Partly through Regulation of TPX2-Aurora A Signaling.

doi: 10.1248/bpb.b24-00542

Figure Lengend Snippet: Fig. 5. YTHDC1 Represses the Centrosomal Abundance of Aurora A through the Regulation of TPX2 Expression

Article Snippet: Doxycycline (Dox)-inducible cell lines expressing FLAG and HA-tagged wild-type or mutant YTHDC1 (A549/YTHDC1-WT or -W377A/W428A) were established as previously described.11) Plasmids pcDNA3-FLAG-HA-hYTHDC1 was a gift from Samie Jaffrey (Addgene plasmid # 85167; http://n2t.net/ addgene:85167; RRID:Addgene_85167).12) FLAG-HA epitopetagged YTHDC1 was subcloned into pENTR4-no-ccDB (686-1) [a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17424; http://n2t.net/addgene:17424; RRID: Addgene_17424)].13) The small interfering RNA (siRNA)-resistant YTHDC1 plasmid was created by introducing a silent mutation into the siYTHDC1#1 target site by PCR using the following primers: (sense) 5′-CTGGTCTCCAAGCCACTGAGCTCATCTGTTAGC-3′ and (antisense) 5′-CTGCCTCGAATGGACAGAAGGCTTTTGTCG-3′.

Techniques: Expressing

Journal: BMC cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma.

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Fig. 2 Representative histology sections show high (A, D, G) and low (B, E, H) expression levels of METTL3, METTL14 and YTHDC1 visualized by immunohistochemistry; hematoxylin (blue) was used for nuclear staining (bright field image, 400xmagnification). Kaplan–Meier estimates show a significantly shorter 5-year survival (p < 0.05) in patients with high expression of METTL3, (F) METTL14, and (I) YTHDC1. Prognostic significance remained after correction for multiple testing (q < 0.1). Scale bar = 20 um

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Epitranscriptomic addition of m 6 A regulates HIV-1 RNA stability and alternative splicing

doi: 10.1101/2021.02.23.432449

Figure Lengend Snippet: (A-C) 293T cells transfected with either empty vector (Ctrl) or a YTHDF2 expression plasmid (+DF2) were infected with the NL4-3-NLuc reporter virus and collected at 24 hpi for (A) Western blot detection of over-expressed YTHDF2 or (B) qRT-PCR analysis of viral RNA expression, n=8. (C) Quantification of the virally encoded NLuc protein as a measure of viral replication at 24 hpi (n=8) and 48 hpi (n=4). (D) 293T cells were co-transfected with NL4-3-NLuc along with expression vectors expressing FLAG-tagged YTHDF readers or agmA3G and treated with the HIV-1 protease inhibitor indinavir. Lysates of the virus producer cells (upper panel) and purified virions from the supernatant media (two lower panels) were analyzed by Western blot for presence of the FLAG-tagged YTHDF or agmA3G protein and the HIV-1 Gag protein. Statistical analysis by two-tailed Student’s T test, *p<0.05, **p<0.01, error bars=SD.

Article Snippet: YTHDC1 mRNA knockdown in 293T cells was performed using siRNAs (Origene # SR314128) transfected using Lipofectamine RNAiMax (Invitrogen).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Western Blot, Quantitative RT-PCR, RNA Expression, Protease Inhibitor, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: Epitranscriptomic addition of m 6 A regulates HIV-1 RNA stability and alternative splicing

doi: 10.1101/2021.02.23.432449

Figure Lengend Snippet: PAR-CLIP was performed on 293T cells transfected with FLAG-GFP or FLAG-YTHDC1, and infected with HIV-1, by immunoprecipitating protein-RNA complexes using a FLAG antibody. Sequencing reads were mapped to the HIV-1 genome, with two independent repeats of YTHDC1 PAR-CLIP (DC1 lanes) shown alongside previously published YTHDF1 & YTHDF2 PAR-CLIP (DF1 & DF2 lanes) and m 6 A mapping results (PA-m 6 A-seq lane) [ , ]. An overview of the whole HIV-1 genome is shown in (A), with the 3’end (3’ of 7,500bp) shown in (B) where the major m 6 A sites are located. Locations of the m 6 A motif 5’ - RRACH-3’ are shown in the bottom lane, with a diagram of the splice donors and acceptors relative to the HIV-1 genome map shown beneath. Significant YTHDC1 peaks called by PARalyzer shown as blue bars beneath each DC1 lane in (B). PARalyzer-called peaks that overlap with m 6 A sites and 5’-RRACH-3’ motifs are highlighted in yellow.

Article Snippet: YTHDC1 mRNA knockdown in 293T cells was performed using siRNAs (Origene # SR314128) transfected using Lipofectamine RNAiMax (Invitrogen).

Techniques: Transfection, Infection, Sequencing

Journal: bioRxiv

Article Title: Epitranscriptomic addition of m 6 A regulates HIV-1 RNA stability and alternative splicing

doi: 10.1101/2021.02.23.432449

Figure Lengend Snippet: 293T cells transfected with non-targeting (siCtrl, gray) or YTHDC1-targeting (siDC1, light blue) siRNAs were co-transfected with a YTHDC1 expression vector (+DC1, dark blue) or empty vector. HIV-1 single cycle infections were performed for the following analyses: (A) Viral Gag protein expression assayed by Western blot, co-stained with a YTHDC1 antibody. (B) Viral RNA levels assayed by qRT-PCR, n=3, with the m 6 A-free host NONO mRNA as a control. A representive Western blot shown in the top left inset depicts the validation of YTHDC1 levels for the samples used in this panel. (C-F) Subcellular fractionation assay of infected siCtrl and siDC1 cells. (C) Western blot validation of fractionation, stained for YTHDC1, nuclear Lamin A/C, and the cytosolic protein GAPDH. (D-F) HIV-1 transcript alternative splice forms were quantified by qRT-PCR with primers targeting unspliced (unspli, U5-gag), and the D1/A1, and D4/A7 splice junctions, calculated as fold change of siDC1 over siCtrl in the nuclear (D), and cytosolic fraction (E). (F) The same RNA quantification as panels (D-E) calculated as percent nuclear and percent cytoplasmic. Statistical analysis used Student’s T test, error bars=SD, *<0.05, **<0.01.

Article Snippet: YTHDC1 mRNA knockdown in 293T cells was performed using siRNAs (Origene # SR314128) transfected using Lipofectamine RNAiMax (Invitrogen).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Quantitative RT-PCR, Fractionation, Infection