Journal: Advanced Science

Article Title: Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

doi: 10.1002/advs.202301538

Figure Lengend Snippet: YTHDF3 deficiency inhibits RNA TE in mouse oocytes. a) The in vitro PB1 emission rates of mouse oocytes from the control groups and the m6A‐related gene depletion groups. Each dot represents a single biological replicate. p ‐Values were calculated with Student's t‐test for paired samples. b) Immunofluorescence verifying the depletion of YTHDF3 by Trim‐Away. Scale bar, 50 µm. The right panel shows the quantification of YTHDF3 protein levels. The average intensity of the control group oocytes was set as 1.0. Each dot represents a single oocyte analyzed. p ‐Value was calculated with two‐tailed Mann–Whitney test. c) Immunofluorescence verifying the expression of YTHDF3 in young and aged mouse GV oocytes. Scale bar, 50 µm. The right panel shows the quantification of YTHDF3 protein levels. The average intensity of young mouse oocytes was set as 1.0. Each dot represents a single oocyte analyzed. p ‐Value was calculated with two‐tailed Mann–Whitney test. d) Scatter plot showing the changes in gene translation and transcription in YTHDF3‐KD oocytes and control oocytes. The Pearson correlation coefficient = −0.196. e) Cumulative distribution of total RNA expression (log 2 TPM); the red line denotes the control group, and the blue line represents the YTHDF3‐KD group. f) Cumulative distribution of TE. The red line denotes the control group, and the blue line represents the YTHDF3‐KD group. g) Scatter plot showing the RNA TE alterations of YTHDF3‐KD oocytes compared with the control group. Red and blue dots denote up‐ and down‐regulated genes, respectively. Upregulated, FC>1.5; downregulated, FC<0.67. h) Gene set enrichment analysis of TE showing the TE downregulated genes enriched in the hallmark of the G2/M checkpoint and TE upregulated genes enriched in the hallmark of oxidative phosphorylation. i) Bar plots showing the numbers of high‐TE genes (TE>2) and low‐TE genes (TE<0.5) in the YTHDF3‐KD group and the control group oocytes, respectively. PB1, polar body‐1. TE, translational efficiency. FC, fold change. YTHDF3‐KD, YTHDF3 knockdown. Ns, no significant difference. * p < 0.05, **** p < 0.0001.

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955‐1‐AP),

Techniques: In Vitro, Control, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Expressing, RNA Expression, Phospho-proteomics, Knockdown

Journal: Advanced Science

Article Title: Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

doi: 10.1002/advs.202301538

Figure Lengend Snippet: YTHDF3 modulates RNA translation efficiency in an m6A‐dependent manner. a) Gene set enrichment analysis demonstrating that the TE of m6A‐enriched RNA was significantly decreased upon YTHDF3 depletion. b) Bar plots showing the numbers of up‐ (FC>1.5) and down‐regulated (FC<0.67) genes for m6A‐enriched genes or genes not enriched by m6A, respectively. Pink denotes m6A‐enriched genes. Blue denotes genes not enriched by m6A. c) Venn diagram portraying the overlap of YTHDF3 target genes among three independent RIP‐seq biological replicates. d) Motif identified by HOMER within YTHDF3 RIP‐seq peaks in HEK293T cells. e) Gene set enrichment analysis showing the TE alterations of YTHDF3‐binding RNA upon YTHDF3 depletion. f) Gene set enrichment analysis showing the TE alterations of m6A‐enriched YTHDF3‐binding RNA upon YTHDF3 depletion. g) Venn diagram showing the overlap of m6A‐modified YTHDF3 target genes between the differential TE genes in YTHDF3‐KD oocytes and the differential TE genes in aged mouse oocytes. h) The RNA TE log 2 fold change in the 449 overlapping genes (described in g) in aged mouse oocytes and YTHDF3‐depleted oocytes. i) The RNA translational level changes in 302 downregulated TE genes (described in h) in aged mouse oocytes and YTHDF3‐depleted oocytes. j) Immunofluorescence verifying the expression of HELLS in the control group and the YTHDF3‐depleted group oocytes. Scale bar, 50 µm. A screenshot of the nucleus is shown separately at the bottom. The right panel shows the quantification of the HELLS protein level. The average intensity of the control group oocytes was set as 1.0. Each dot represents a single oocyte analyzed. p ‐Value was calculated with two‐tailed Mann‐Whitney test. k) Schematic representation of wild‐type (YTHDF3‐WT) and mutant (YTHDF3‐Mut) YTHDF3 constructs. l) Western blot demonstrating the expression of HELLS in HEK293T cells transfected with empty vector or wild‐type or mutant Flag‐tagged YTHDF3 plasmid. GAPDH was used as the negative control. The left panel presents a representative Western blot image. The right panel shows the quantification of the HELLS protein level. The average intensity of the NC group was set as 1.0. Each dot represents a single biological replicate. p ‐Value was calculated with two‐tailed Mann–Whitney test. m) HEK293T cells were cotransfected with NC, YTHDF3‐WT, or YTHDF3‐Mut plasmids, and luciferase reporter plasmids carrying the HELLS 3’UTR, and luciferase activity was measured. p‐ Value was calculated with two‐tailed Mann–Whitney test. TE, translational efficiency. FC, fold change. HOMER, Hypergeometric Optimization of Motif EnRichment. RIP, RNA immunoprecipitation. YTHDF3‐KD, YTHDF3 knockdown. NC, negative control. Ns, no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955‐1‐AP),

Techniques: Binding Assay, Modification, Immunofluorescence, Expressing, Control, Two Tailed Test, MANN-WHITNEY, Mutagenesis, Construct, Western Blot, Transfection, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, RNA Immunoprecipitation, Knockdown

Journal: Advanced Science

Article Title: Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

doi: 10.1002/advs.202301538

Figure Lengend Snippet: The changes of RNA translation efficiency in aged human oocytes. a) Scatter plot showing the changes in gene translation and transcription during oocyte aging. b) Bar plots showing the numbers of upregulated TE genes and downregulated TE genes in young and aged mouse/human GV oocytes, respectively. Upregulated, FC>1.5; downregulated, FC<0.67. c) Bar plots showing the numbers of high‐TE genes (TE>2) and low‐TE genes (TE<0.5) in young and aged mouse/human GV oocytes, respectively. d) Violin plots showing the TE changes in the genes of the group not enriched by m6A and of the m6A‐enriched group in aged human oocytes compared with young human oocytes. p ‐Value was calculated with Student's t‐test for independent samples. e) Gene set enrichment analysis demonstrating the correlation of TE alterations and YTHDF3 target RNA in aged human oocytes. f) Violin plots showing the TE changes in genes of the papCPE‐containing group and in genes of the group not containing papCPE in aged human oocytes compared with young human oocytes. p ‐Value was calculated with Student's t‐test for independent samples. g) Violin plots showing the TE changes in genes of the CPE‐containing group and in genes of the group not containing CPE in aged human oocytes compared with young human oocytes. p ‐Value was calculated with Student's t‐test for independent samples. h) Violin plots showing the TE changes in four groups of genes in aged human oocytes compared with young human oocytes. p ‐Values were calculated with one‐way ANOVA and Bonferroni post hoc test. i) Violin plots showing the TE changes in four groups of genes in aged human oocytes compared with young human oocytes. p ‐Values were calculated with one‐way ANOVA and Bonferroni post hoc test. j) Transcriptional and translational expression levels of the YTHDF3 in human/mouse GV oocytes. Data are shown as the mean ± SEMs. p ‐Values were calculated with Student's t‐test for independent samples. k) Transcriptional and translational expression levels of the HELLS in human/mouse GV oocytes. Data are shown as the mean ± SEMs. p‐Values were calculated with Student's t‐test for independent samples. l) Representative RBPs enriched in 3’UTR of the genes in human oocytes that potentially regulated RNA TE. Data are shown as the means±SEMs. p ‐Values were calculated with Student's t‐test for independent samples. FC, fold change. TE, translational efficiency. CPEs, cytoplasmic polyadenylation elements. papCPE, CPEs within 100 bp of PASs. RBPs, RNA binding proteins. Ns, no significant difference. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Antibodies used in this study are listed as follows: YTHDF3 (Novus Biologicals, 94636), HELLS (Proteintech, 11955‐1‐AP),

Techniques: Expressing, RNA Binding Assay