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

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Multiple alignments of Ythdf1, Ythdf2 & Ythdf3 proteins, calculated using the Clustal Omega tool. The area of YTH-domain is highlighted in red. Ythdf1-Ythdf3 protein sequence similarity is 70.11%, Ythdf1-Ythdf2 is 67.15%, and Ythdf2-Ythdf3 is 67.78%. b)□ Phylogenetic tree of the protein sequences of Ythdf1, Ythdf2 and Ythdf3, based on the UCSC database. The three readers appear together in vertebrates, possibly due to whole genome duplication. c)□ CRISPR-Cas9 targeting strategy for knocking-out Ythdf readers in vivo. d)□ KO validation using PCR, showing successful primer integration in clones #12 (Ythdf1); #36, #46 (Ythdf2); #1, #3 & #4 (Ythdf3).

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Sequencing, CRISPR, In Vivo, Biomarker Discovery, Clone Assay

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Gross morphology of Cre+ and Cre−(control) female ovaries. Cre+ females show a smooth shape that lacks the typical follicular morphology. b) H&E staining of an ovary, showing a severe abnormality in Mettl3 f/f Vasa-Cre+ females. c) Number of pups per plug produced by mating Mettl3 f/f Vasa-Cre+ females, compared to Mettl3 f/f Vasa-Cre−control females. The fathers in both cases are WT. A significant difference between Cre+ and Cre−female fertility is observed (p<0.0001, Mann-Whitney test). d) H&E staining of ovaries, showing normal morphology in Mettl3 f/f Zp3-Cre+ ovaries. e) Number of pups per plug produced by mating a Mettl3 f/f Zp3-Cre+ female, compared to a Mettl3 f/+ Zp3-Cre+ control female. The fathers in both cases are WT. A significant difference between f/f and f/+ female fertility is observed (p<0.0001, Mann-Whitney test). f) Number of oocytes per mouse produced by mating Mettl3 f/f Zp3-Cre+ females, compared to Mettl3 f/f Zp3-Cre−control females. The fathers in both cases are WT. A significant difference between the number of oocytes of f/f Cre+ and f/f Cre−is observed (p<0.0002, Mann-Whitney test). g) Top: Experimental design - Mett3 f/f Zp3-Cre+ and Cre−as control underwent hormone priming, flush, fixation and staining for tubulin. Bottom: Number of oocytes observed in the different stages of meiosis. In the control, most of the oocytes were in MI stage, in KO (Cre+) all of the observed oocytes were in the GV state. h) Staining examples of oocytes in the different stages of meiosis as observed in KO (Cre+) and control (Cre−). i) Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes after hormone priming (PMS & hCG).

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Control, Staining, Produced, MANN-WHITNEY, Immunostaining

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Gross morphology of testis and epididymis of Mettl3f/f Vasa-Cre+ and Mettl3f/f Vasa-Cre−males. Cre+ males show a massive decrease in testis and epididymis size compared to Cre−control. b) Number of pups per plug produced by Mettl3f/f Vasa-Cre+ males, compared to Mettl3f/f Vasa-Cre−control males. The mothers in both cases were WT. In this case there is a significant hypofertility of the KO (p<0.0001, Mann-Whitney test). c) Gross morphology of testis and epididymis of Mettl3f/f Stra8-Cre+ and Mettl3f/f Stra8-Cre−males. Cre+ males show a reduced-size testis and epididymis compared to Cre−control. d) Same as in (b), for Stra8-Cre, showing a significant hypofertility of the KO (p<0.0001, Mann-Whitney test). e) Vasa, Stra8 and Prm1 are expressed during spermatogenesis, in different stages, as indicated. f) Same as in (b), for Prm1-Cre, showing no significant difference between Cre+ and Cre−male fertility. g) Number of pups per plug produced by Ythdf2 −/− males, compared to Ythdf2 +/− control males. The mothers in both cases were WT. A significant difference between the fertility of KO and heterozygous males is observed (p<0.0001, Mann-Whitney test). h) Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in seminiferous tubules, showing that each of the proteins is expressed at different stages of spermatogenesis.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Control, Produced, MANN-WHITNEY, Immunostaining

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Left: statistics of KO offspring received from crossing heterozygous mice from each of the indicated strains (Ythdf1 +/− , Ythdf2 +/− , Ythdf3 +/− ). Right: distribution of Ythdf2 WT, HET and KO offspring in E13.5, two days postnatal (DPN), and 30 DPN (compared to expected ratios). b) Crossing strategy for generating triple-heterozygous mice, which were further crossed, and their offspring, statistics are presented in panel (c). c) Percentage of genotypes received by crossing triple-heterozygous mice, out of 200 pups tested 30 DPN. Red - observed percentage, grey - expected under null assumption. No pups with Ythdf2-KO genotype survived 30 DPN. In the Ythdf2 +/− genotype, pups with KO in either Ythdf1 or Ythdf3 were born at a sub-Mendelian ratio. Chi-square test p-values are indicated. d) H&E staining showing the impaired morphology of triple-KO E7.5 embryos, compared to WT control.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Number of pups per plug produced by mating Ythdf1-KO males, compared to Ythdf1-HET males. The mothers in both cases are WT. Here there is no significant difference between KO and HET male fertility (Mann-Whitney test). b)□ Number of pups per plug produced by mating Ythdf1-KO females, compared to Ythdf1-HET females. The fathers in both cases are WT. Here there is no significant difference between KO and HET female fertility (Mann-Whitney test). c)□ Number of pups per plug produced by mating Ythdf3-KO males, compared to Ythdf3-HET males. The mothers in both cases are WT. Here there is no significant difference between KO and HET male fertility (Mann-Whitney test). d)□ Number of pups per plug produced by mating Ythdf3-KO females, compared to Ythdf3-HET females. The fathers in both cases are WT. Here there is no significant difference between KO and HET female fertility (Mann-Whitney test). e)□ H&E staining of seminiferous tubules showing a normal morphology in Ythdf1-KO and Ythdf3-KO males. f)□ The morphology of Ythdf1-KO and Ythdf3-KO flushed oocytes appears to be normal, similar to the Ythdf1-heterozygous (HET) and Ythdf3-HET flushed oocytes.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Produced, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes after hormone priming (PMS & hCG). b)□ Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes after PMS & HCG - negative control (NC), without primary antibody. c)□ Immunostaining of Ythdf1, Ythdf2 and Ythdf3 in ICR WT oocytes without hormone priming.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Immunostaining, Negative Control

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a) Immunostaining of Ythdf1, Ythdf2, and Ythdf3 in KH2 mESCs, showing a protein expression in the cytosolic compartment of the cell. b) Cell growth curve of all KO lines and WT control. Cells were grown on mouse feeders, in serum/LIF conditions. c) Brightfield and immunostaining of Nanog (green), Esrrb (red) and DAPI (blue), in KO cells (single, triple and Mettl3) and WT control, showing that all cell lines express Nanog and Esrrb. d) Teratomas generated by the KO cell line and by WT control. Single-KO cell lines show all germ layers, while triple-KO teratomas as poorly differentiated. e) Immunostaining of triple-KO and WT control with Oct4 (red), Foxa (green), Tuj1 (purple) and DAPI (blue). Triple-KO contains patches of Oct4 staining, unlike WT teratomas. f) Alkaline phosphatase (AP) staining of disassociated teratomas from Triple-KO and WT control, showing a greater AP staining in the triple-KO cells. g) RT-PCR of pluripotent genes (left) and differentiation genes (right), measured in WT and KO EBs, and in WT mESCs as a control. In the triple-KO EBs, pluripotent markers are higher than the control and differentiation markers are lower than the WT control.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Immunostaining, Expressing, Control, Generated, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ CRISPR-Cas9 targeting strategy for knocking out Ythdf readers in mESC cell lines. b)□ Sequencing validation of the single-KO lines. c)□ IGV browser view showing the missing fragments in the KO of Ythdf1, Ythdf2 & Ythdf3.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: CRISPR, Sequencing, Biomarker Discovery

Journal: bioRxiv

Article Title: Context-dependent functional compensation between Ythdf m6A readers

doi: 10.1101/2020.06.03.131441

Figure Lengend Snippet: a)□ Targets of Ythdf1, Ythdf2 and Ythdf3 highly overlap targets that were published before in human cancer cell lines. b)□ Sequence logo of the GGACT containing motif which appears in 14% of YTHDF targets (enrichment fold-change 1.89, p<1e-24). c)□ Distribution of Ythdf peaks in various genomic entities, showing that the three readers have a tendency to bind 3' UTR, particularly Ythdf2. d)□ Significant overlap of Ythdf targets, with m 6 a-methylated genes. e)□ Significant overlap between Ythdf1, Ythdf2 and Ythdf3 targets f)□ Enrichment of Ythdf targets that were identified in mESCs, to early embryo genes (Gao et al. 2017), showing significant overlap with blastocyte genes.

Article Snippet: The following primary antibodies were used: Ythdf2 (AVIVA SYSTEMS BIOLOGY, ARP67917_P050), Ythdf3 (Santa Cruz, SC-87503), Cnot1 (Proteintech, 14276-1-A) and Hsp90 (Epitomics, 1492-1).

Techniques: Sequencing, Methylation