Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: To create pgW1-YTHDF2-2A-GFP, the YTHDF2 ORF was PCR amplified from pcDNA-flag-YTHDF2 (Addgene, #52300).

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: To create pgW1-YTHDF2-2A-GFP, the YTHDF2 ORF was PCR amplified from pcDNA-flag-YTHDF2 (Addgene, #52300).

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Immunostaining of YTHDF2 in control and sALS patient spinal cord samples. Scale bar= 50 µm. ( B ) Quantification of YTHDF2 immunoreactivity in spinal cord neurons from control (n= 117 neurons) and sALS (n= 193 neurons) samples. Plot shows mean +/- SD, color coded by sample. ****p< 0.0001 via Mann-Whitney test. ( C ) Strategy used to create isogenic iPSCs expressing native TDP43(WT)-Dendra2 or TDP43(M337V)-Dendra2. ( D ) Representative images of untransduced (grey) and transduced (green) iNeurons expressing shRNA against YTHDF2 (shYTHDF2) and a GFP reporter. Time of death (red circles) for each cell is used to determine cumulative risk of death, plotted in ( E ) and ( F ). Scale bar= 20µm. shRNA-mediated knockdown of YTHDF2 significantly extended the survival of TDP43(M337V)-Dendra2 iNeurons ( E ; †p= 8.42×10 −12 , HR= 6.25; ***p= 4.82×10 −9 , HR=0.32; #p= 0.08, HR= 1.84) as well as mutant C9ORF72 iNeurons ( F , †p= 1.42×10 −11 , HR= 2.85; ***p= 1.42×10 −16 , HR= 0.32). ns= not significant. Values in ( E , F ) calculated by Cox proportional hazards analysis, with a minimum 3 biological replicates.

Article Snippet: To create pgW1-YTHDF2-2A-GFP, the YTHDF2 ORF was PCR amplified from pcDNA-flag-YTHDF2 (Addgene, #52300).

Techniques: Immunostaining, Control, MANN-WHITNEY, Expressing, shRNA, Knockdown, Mutagenesis

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: Cell Research

Article Title: Suppression of m 6 A reader Ythdf2 promotes hematopoietic stem cell expansion

doi: 10.1038/s41422-018-0072-0

Figure Lengend Snippet: Ythdf2 KO leads to an increase in phenotypic HSCs in mice. a Deletion of YTHDF2 in the HSPCs of Mx1-cre;Ythdf2f/f conditional KO (cKO) mice. b Western blotting (left) and histogram (right) showing intracellular flow validation of KO Ythdf2 in mouse HSPCs. c Representative flow plots of HSPCs in BM from wt and Ythdf2 KO mice (n = 5 for each group). d, e Frequency in total nucleated cells (TNCs) (d) and absolute cell number (e) of HSPCs in BM from wt and Ythdf2 KO mice (n = 5 for each group). f Absolute number of BM TNCs from wt and Ythdf2 KO mice (n = 5 for each group). g, h Absolute number of committed progenitors (g) and lineage cells (h) in BM of wt and Ythdf2 KO mice (n = 5 for each group). Data shown as mean ± SEM. Unpaired t-test. n.s. nonsignificant. CMP, common myeloid progenitor; GMP, granulocyte-macrophageprogenitor; MEP, megakaryocyte-erythrocyte progenitor; CLP, common lymphoid progenitor; Erythro, erythrocyte

Article Snippet: Anti-YTHDF2 (MBL, 1: 500) and anti-DCP1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life Technologies, catalog number 003118) and incubated with the specimens at 4 °C overnight.

Techniques: Western Blot

Journal: Cell Research

Article Title: Suppression of m 6 A reader Ythdf2 promotes hematopoietic stem cell expansion

doi: 10.1038/s41422-018-0072-0

Figure Lengend Snippet: Ythdf2 KO results in expansion of functional HSCs in mice. a Experimental scheme for limiting dilution transplantation assay (LDA) to determine the frequency of functional HSCs. b Primary LDA to determine the CRU frequency by ELDA (Extreme Limiting Dilution Analysis) at 16 weeks post transplantation (n = 10 per group). c Competitive reconstitution assay by transplanting 200 K whole bone marrow (WBM) cells with 200 K rescue cells into irradiated recipients (n = 10 for each group). d, e Frequency in TNC (d) and absolute cell number (e) of donor-derived HSPCs in BM from transplantation recipient mice as c (n = 10 for each group). f, g Absolute cell number of donor-derived (CD45.2+) committed progenitors (f) and lineage cells (g) in BM from primary 200 K BM transplantation recipient mice (n = 9–10 for each group). h Secondary LDA to determine the long-term CRU frequency by ELDA at 16 weeks after secondary transplantation (n = 10). Data shown as mean ± SEM. Unpaired t-test. n.s. nonsignificant

Article Snippet: Anti-YTHDF2 (MBL, 1: 500) and anti-DCP1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life Technologies, catalog number 003118) and incubated with the specimens at 4 °C overnight.

Techniques: Functional Assay, Limiting Dilution Transplantation Assay, Transplantation Assay, Reconstitution Assay, Irradiation, Derivative Assay

Journal: Cell Research

Article Title: Suppression of m 6 A reader Ythdf2 promotes hematopoietic stem cell expansion

doi: 10.1038/s41422-018-0072-0

Figure Lengend Snippet: YTHDF2 functions as an m6A reader and regulates HSC gene expression by mediating mRNA decay. a Schematic of irCLIP-seq workflow. b YTHDF2-binding motif identified by MEME with all irCLIP peaks found in all three replicates. c Pie chart depicting the fraction of YTHDF2-binding peaks in each of five transcript segments. d GO enrichment analysis of YTHDF2 targets from intersect genes of three YTHDF2 irCLIP-seq replicates. e Representative tracks of Tal1 harboring m6A peaks and YTHDF2 irCLIP peaks. Coverage of m6A immunoprecipitation and input fragments indicated in red and grey, respectively. YTHDF2 irCLIP reads highlighted in yellow. Arrows indicate the overlapped peaks between m6A-seq and irCLIP-seq. f qPCR analysis of total mRNA of sorted LSK cells from wt and Ythdf2 KO mice. All Ct values were first normalized to Actb control (not m6A-tagged) and the ratio (Ythdf2 KO over wt) was then calculated. (n = 3). g Representative images (left) and quantification (right) of staining intensity of wt (n = 65) and Ythdf2 KO (n = 54) HSPCs for TAL1 (green). h Fluorescence in situ hybridization of Tal1 mRNA (red) and fluorescence immunostaining of DCP1a (P-body marker) (magenta), YTHDF2 (green) in wt and Ythdf2 KO HSPCs. Arrows indicate colocalized staining. Scale bars, 5 μm. i Quantification of Tal1 mRNA and DCP1a co-localization in sorted LSK cells from wt and Ythdf2 KO mice. Percentage indicates the average frequency of the Tal1 mRNA that colocalized with DCP1a over total Tal1 mRNA level in each LSK cells (n = 9–17). Data shown as mean ± SEM. Unpaired t-test

Article Snippet: Anti-YTHDF2 (MBL, 1: 500) and anti-DCP1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life Technologies, catalog number 003118) and incubated with the specimens at 4 °C overnight.

Techniques: Expressing, Binding Assay, Immunoprecipitation, Staining, Fluorescence, In Situ Hybridization, Immunostaining, Marker

Journal: Cell Research

Article Title: Suppression of m 6 A reader Ythdf2 promotes hematopoietic stem cell expansion

doi: 10.1038/s41422-018-0072-0

Figure Lengend Snippet: Role of YTHDF2 in human cord blood HSCs by m6A-seq and RNA-seq analysis. a Metagene profiles depicting sequence coverage in windows surrounding the TSS (left) and stop codon (right). Coverage of m6A IP and control (input) fragments indicated in red and grey, respectively. b Pie chart presenting the fraction of m6A peaks in each of five non-overlapping transcript segments. c Venn diagram showing shared and unique m6A-tagged genes in mouse and hUCB HSPCs. d GO enrichment analysis of m6A-tagged transcripts shared in both mouse and hUCB HSPCs. e Representative tracks of HOXB4 harboring m6A peaks. Color codes are the same as in a. f Lentivirus mediated YTHDF2 KD in hUCB CD34+ HSPCs for RNA-seq. g Cumulative distribution of log2 (fold change) for m6A-marked genes (purple line) and non-m6A-marked genes (black line) with control and YTHDF2 KD hUCB CD34+ cells. h Representative coverage plots from the RNA-seq analysis, showing increased reads of m6A-tagged gene HOXB4 but not a non-m6A-tagged gene ACTB in YTHDF2 KD compared with control hUCB CD34+ cells. i Relative mRNA expression levels of non-m6A labeled ACTB (as control) and m6A-marked transcription factor related to stem cell self-renewal in control and YTHDF2 KD hUCB CD34+ cells. RPKM from RNA-seq analysis were normalized to controls. Adjusted P-value indicated. n.s. nonsignificant

Article Snippet: Anti-YTHDF2 (MBL, 1: 500) and anti-DCP1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life Technologies, catalog number 003118) and incubated with the specimens at 4 °C overnight.

Techniques: RNA Sequencing Assay, Sequencing, Expressing, Labeling

Journal: Cell Research

Article Title: Suppression of m 6 A reader Ythdf2 promotes hematopoietic stem cell expansion

doi: 10.1038/s41422-018-0072-0

Figure Lengend Snippet: YTHDF2 KD facilitates expansion of human cord blood HSCs ex vivo. a, b Fold change of frequency (a) and absolute number (b) of indicated cells in YTHDF2 KD over control cells after 7 days culture. c CFU output from transduced CD34+ CD38− hUCB cells and images of CFU the granulocyte-erythrocyte-monocyte-megakaryocytes (GEMMs) (scale bar, 200 μm). d Images of burst forming unit-erythroid (BFU-E) (left) and colony-forming unit-granulocyte/macrophage (CFU-GM) (right) from 7 days cultured control or YTHDF2 KD hUCB cells (scale bar, 200 μm). Three independent cord blood samples were used and this was repeated twice for a to d. e Apoptosis analysis of CD34+ CD38- cells in 7-day cultures of transduced CD34+ hUCB cells by Annexin V staining (n = 3 independent CB samples). Data shown as mean ± SEM. Unpaired t-test. n.s. nonsignificant

Article Snippet: Anti-YTHDF2 (MBL, 1: 500) and anti-DCP1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life Technologies, catalog number 003118) and incubated with the specimens at 4 °C overnight.

Techniques: Ex Vivo, Cell Culture, Staining

Journal: Cell Research

Article Title: Suppression of m 6 A reader Ythdf2 promotes hematopoietic stem cell expansion

doi: 10.1038/s41422-018-0072-0

Figure Lengend Snippet: YTHDF2 KD facilitates expansion of human cord blood functional long-term HSCs. a Experimental scheme for measuring frequency of HSCs after in vivo expansion. b Representative flow plots of hCD45+ GFP+ reconstitution from primary recipient mice receiving the highest two cell doses. hCD45, human CD45 (c) hCD45+ GFP+ engraftment in BM from the primary recipient mice that received the highest two doses (n = 8). d HSC frequency determined by primary LDA. Dashed lines indicate 95% confidence intervals. e Representative flow plots of hCD45+ GFP+ reconstitution from secondary recipient mice receiving the highest two cell doses. f hCD45+ GFP+ engraftment in BM from secondary recipient mice that received the highest two doses (n = 6). g HSC frequency determined by secondary LDA. Dashed lines indicate 95% confidence intervals. Data shown as mean ± SEM. Unpaired t-test. n.s. nonsignificant

Article Snippet: Anti-YTHDF2 (MBL, 1: 500) and anti-DCP1a (Santa Cruz, SC100706, 1:200) antibodies were diluted with antibody diluent reagent buffer (Life Technologies, catalog number 003118) and incubated with the specimens at 4 °C overnight.

Techniques: Functional Assay, In Vivo

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: The expression of YTHDF2 in ovarian cancer tissues. a Relative expression of YTHDF2 in ovarian cancer tissues and normal ovarian tissues. b The relationship between the expression level of YTHDF2 and clinical stage. c The relationship between the expression level of YTHDF2 and pathological grade. d The relationship between the expression level of YTHDF2 and metastasis. All experiments were carried out in triplicate and the results were presented as means ± SE. * P < 0.05, ** P < 0.001, t-test. N normal ovarian tissue, C ovarian cancer tissues

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Expressing

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: YTHDF2 significantly downregulates global mRNA m6A levels to promotes EOC cell proliferation and migration. a The mRNA and protein level of YTHDF2 was downregulated after transfection of YTHDF2 siRNA. b Knocking down YTHDF2 increased global mRNA m6A level. (C)CCK8 assay result showed knock-down of YTHDF2 decreased proliferation of ovarian cancer cells. d Knock-down of YTHDF2 promoted apoptosis of ovarian cancer cells. e Knock-down of YTHDF2 inhibited migration of ovarian cancer cells. f The mRNA and protein level of YTHDF2 was upregulated after overexpression of YTHDF2. g overexpression of YTHDF2 decreased global mRNA m6A level. (H)CCK8 assay result showed upregulated of YTHDF2 increased proliferation of ovarian cancer cells. (I) overexpression of YTHDF2 inhibited apoptosis of ovarian cancer cells. j overexpression of YTHDF2 promoted migration of ovarian cancer cells. All experiments were carried out in triplicate and the results were presented as means ± SE. * P < 0.05, ** P < 0.001, t-test

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Migration, Transfection, CCK-8 Assay, Over Expression

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: YTHDF2 is the direct target gene of miR-145. a Scatter diagram showing. YTHDF2 expression and miR-145 expression by qRT-PCR and their correlations (R 2 = 0.914, P = 0.000) in 31 EOC tissue samples. b qRT-PCR results showed a negative correlation between YTHDF2 and miR-145 in both SKOV3 and 3AO cells. c qRT-PCR showed that transfection of miR-145 mimic rescued miR-145 level in SKOV3 and 3AO cells. d The mRNA and protein levels of YTHDF2 decreased after overexpression of miR-145. e The expression level of miR-145 increased after overexpression of YTHDF2. f Luciferase reporter assays showed that miR-145 targeted YTHDF2 directly. All experiments were carried out in triplicate and the results were presented as means ± SE. *P < 0.05, **P < 0.001, t-test

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Luciferase

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: Overexpression of YTHDF2 rescues miR-145-induced reduction of proliferation and migration in EOC. a overexpression of YTHDF2 reversed the proliferation repressed by overexpression of miR-145. b miR-145 promoted apoptosis of ovarian cancer cells, which was counteracted by overexpression of YTHDF2. c overexpression of YTHDF2 reversed the migration repressed by overexpression of miR-145. d The effect of overexpression of miR-145 on the global mRNA m6A levels was offset by overexpression of YTHDF2. e The expression of YTDHF2 at mRNA levels after overexpression of miR-145 and YTHDF2. f The expression of YTDHF2 at protein levels after overexpression of miR-145 and YTHDF2. All experiments were carried out in triplicate and the results were presented as means ± SE. * P < 0.05, ** P < 0.001, t-test

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Over Expression, Migration, Expressing