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: 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: 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: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

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, Staining

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: 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: Biochemistry

Article Title: In vitro selection with a site-specifically modified RNA library reveals the binding preferences of N 6 -methyladenosine (m 6 A) reader proteins

doi: 10.1021/acs.biochem.9b00485

Figure Lengend Snippet: The 10 most abundant m 6 A-centered 5-mers and 11-mers enriched upon in vitro binding selections with YTH readers. 5-mers and 11-mers shared between selections are highlighted in yellow and purple, respectively.

Article Snippet: Protein expression and purification Plasmids encoding cDNA for YTH proteins were obtained from Addgene: YTHDC1 ( {"type":"entrez-protein","attrs":{"text":"NP_001026902.1","term_id":"72534750","term_text":"NP_001026902.1"}} NP_001026902.1 ) (#85167) 46 , YTHDF1 ( {"type":"entrez-protein","attrs":{"text":"NP_060268.2","term_id":"31377750","term_text":"NP_060268.2"}} NP_060268.2 ) (# 70087) 5 , and YTHDF2 ( {"type":"entrez-protein","attrs":{"text":"NP_057342.2","term_id":"116812575","term_text":"NP_057342.2"}} NP_057342.2 ) (# 52300) 5 .

Techniques: In Vitro, 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: 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: Protein-Protein interactions, RNA Sequencing, 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, 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, ChIP-qPCR, 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: Genes & Development

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

doi: 10.1101/gad.348508.121

Figure Lengend Snippet: The nuclear m 6 A reader YTHDC1 binds HIV-1 RNA at previously mapped m 6 A sites. PAR-CLIP was performed on 293T cells transfected with FLAG-GFP or FLAG-YTHDC1, and infected with HIV-1. Sequencing reads were mapped to the HIV-1 genome, with two independent repeats of YTHDC1 PAR-CLIP (DC1 lanes) shown alongside previously published YTHDF1 and YTHDF2 PAR-CLIP (DF1 and DF2 lanes) and m 6 A mapping results (PA-m 6 A-seq lane) ( ; ). A schematic of the HIV-1 genome is shown in A , with the 3′ end (3′ of 7500 bp) shown in B where most m 6 A sites are located. Locations of the m 6 A motif 5′ - RRACH-3′ are shown in the bottom lane, with the location of viral splice donors and acceptors indicated. Significant YTHDC1 peaks called by PARalyzer are shown as blue bars below 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: Cells in six-well plates were transfected with nontargeting (siCtrl) or YTHDC1-targeted siRNAs (siDC1; three YTHDC1 siRNAs premixed at a 1:1:1 ratio; Origene SR314128) using Lipofectamine RNAiMax (Invitrogen), with the media changed the next day.

Techniques: Transfection, Infection, Sequencing

Journal: Genes & Development

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

doi: 10.1101/gad.348508.121

Figure Lengend Snippet: YTHDC1 regulates HIV-1 gene expression with no detectable effect on RNA nuclear export. 293T cells transfected with nontargeting (siCtrl, gray) or YTHDC1-targeting (siDC1, light blue) siRNAs or were 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, costained 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 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 cytosolic GAPDH. ( D – F ) HIV-1 transcript alternatively spliced isoforms 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 in D and E calculated as percent nuclear and percent cytoplasmic. ( G ) Stability of RNA transcripts in siCtrl and siDC1 cells assayed by treating infected cells at 2 dpi with ActD and the viral RNA levels from the indicated time points analyzed by qRT-PCR and shown as percentage of the RNA level at time point 0. ( H ) Production of nascent HIV-1 transcripts in infected siCtrl and siDC1 cells was measured by pulsing cells with the nucleoside analog 4SU for 1.5 h. The 4SU+ transcripts were then isolated and quantified by qRT-PCR. Statistical analysis used Student's t- test. Error bars indicate SD. (*) P < 0.05, (**) P < 0.01.

Article Snippet: Cells in six-well plates were transfected with nontargeting (siCtrl) or YTHDC1-targeted siRNAs (siDC1; three YTHDC1 siRNAs premixed at a 1:1:1 ratio; Origene SR314128) using Lipofectamine RNAiMax (Invitrogen), with the media changed the next day.

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

Journal: Genes & Development

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

doi: 10.1101/gad.348508.121

Figure Lengend Snippet: YTHDC1 regulates the alternative splicing of HIV-1 RNAs. Viral transcript spliced isoforms in infected siCtrl, siDC1, and +DC1 cells 48 hpi were analyzed by PrimerID RNA-seq. n = 3. ( A ) Schematic of HIV-1 splice donors and acceptors relative to the three major classes of spliced viral RNAs. Top arrows depict the primers used to amplify sequences for RNA-seq, including the common 5′ forward primer in blue, the reverse 4-kb primer in green, and the reverse 1.8-kb primer in red spanning the D4/A7 splice junction. A random reverse primer was also used but is not shown. ( B ) Spliced transcripts are given as a percentage of all transcripts (1.8-kb + 4-kb class read counts/total read counts). ( C ) Percent of fully spliced transcripts over all spliced transcripts (1.8-kb/[1.8-kb + 4-kb] read counts). ( D – F ) Splice acceptor usage assayed using the common forward primer in conjunction with the random reverse primer ( D ), 1.8-kb reverse primer ( E ), and 4-kb reverse primer ( F ). Use of acceptor A3 results in long transcripts that are biased against when amplifying using the 1.8- or 4-kb reverse primers, A3 usage is thus only shown with the random reverse primer. ( G ) Percent occurrence of D1/A7 splices. ( H ) Percent occurrence of A2-using spliced RNAs that subsequently splice from D3. Statistical analysis used Student's t- test. Error bars indicate SD. (*) P < 0.05, (**) P < 0.01.

Article Snippet: Cells in six-well plates were transfected with nontargeting (siCtrl) or YTHDC1-targeted siRNAs (siDC1; three YTHDC1 siRNAs premixed at a 1:1:1 ratio; Origene SR314128) using Lipofectamine RNAiMax (Invitrogen), with the media changed the next day.

Techniques: Infection, RNA Sequencing Assay