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Figure Lengend Snippet: YTHDC2 binds preferentially to m6A-marked RNA transcripts. (A) In vitro probe pulldown assay in HeLa cells overexpressing FLAG-YTHDC2 showing YTHDC2 binds preferentially to probe with m6A on GGACU. GGYCU Probe sequence: 5′-CGUGGYCUGGCU-B-3′ (Y = m6A or A, B = biotin) ACYGA Probe sequence: 5′-GAUACYGAGAAG-B-3′ (Y = m6A or A, B = biotin). Labels: relative protein expression. (B) Consensus motif of Ythdc2 binding identified by HOMER of CLIP-seq of Ythdc2 in mouse testes. (C) Overlap of Ythdc2 RIP-seq genes and genes containing m6A in young mouse testes. Targets of YTHDC2 are defined as genes enriched in the RIP: Log2(RIP/input) ≥ 1. Non-targets are defined as genes depleted in the RIP: Log2(RIP/input) ≤ −1. Genes with m6A are defined as genes enriched in the m6A IP: Log2(m6A IP/input) ≥ 2.

Article Snippet: The antibodies used in this study (listed in the format of name (catalogue; supplier; dilution) were as follows: Goat anti-YTHDC2 (Sc-249370; Santa-Cruz; IP/20μg; Western Blot/1:1 000); Rabbit anti-Ythdc2 (A303-026A; BETHYL; Western Blot/1:2 000); Rabbit anti-m 6 A (202003; Synaptic Systems; IP/2.5 μg); Rabbit anti-eIF3a (3411; Cell Signaling Technology; Western Blot/1:2 000); Mouse anti-FLAG M2-peroxidase HRP (A8592; Sigma; Western Blot/1:10 000); Goat anti-GAPDH HRP (A00192-100; GenScript; Western Blot/1:5 000); Mouse anti-DDX4 (ab27591; Abcam; IF/1:200); Mouse anti-γ-H2A.X (ab26350; Abcam; IF/1:100); Mouse anti-SYCP3 (ab97672; Abcam; IF/1:100); Rabbit anti-SYCP3 (ab15093; Abcam; IF/1:100); Rabbit anti SYCP1 (ab15090; Abcam; IF/1:100); Mouse anti-SMC3 (ab128919; Abcam; Western Blot/1:2 000, IF/1:200); Rabbit anti-cleaved caspase3 (9661; CST; IHC/1:200); Rabbit anti-Cep76 (gift from Dr Xueliang Zhu, Institute of Biochemistry and Cell Biology, SIBS, CAS, Western Blot/1:2 000); Mouse anti-PLZF (sc-28319; Santa-Cruz; IF/1:200); Rabbit anti-Lin28 (13456-1-AP; Proteintech; IHC/1:200); Rabbit anti-SOX9 (ab5535; Chemicon; IF/1:200); Rabbit anti-IMP3 (IGF2BP3) (A303-426A; Bethyl; Western Blot/1:1 000); Rabbit anti-YBX1 (ab76149; Abcam; Western Blot/1: 1 000); Rabbit anti-MOV10 (A301-571A; Bethyl; Western Blot/1:1 000); Rabbit anti-XRN1 (A300-443A; Bethyl; Western Blot/1:1 000); Mouse anti-Tubulin(AT819; Beyotime; Western Blot/1:10 000); Donkey anti-Rabbit IgG-HRP (sc-2313; Santa-Cruz; Western Blot/1:2 000); Donkey anti-Goat IgG-HRP (sc-2033; Santa-Cruz; Western Blot/1:2 000).

Techniques: In Vitro, Sequencing, Expressing, Binding Assay

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Figure Lengend Snippet: YTHDC2 affects the translation efficiency and mRNA abundance of its targets. (A) Relative mRNA expression of Smc3, normalized to Rn18s, in 8.5 d.p.p. Ythdc2+/+ and Ythdc2−/− testes. Error bars, mean ± sd, n = 3. **P < 0.01 (Student's t-test). (B) Translation efficiency of Smc3 in 8.5 d.p.p. Ythdc2+/+ and Ythdc2−/− testes, calculated as: translation efficiency = Smc3 protein level / Smc3 mRNA expression level. Smc3 protein level was determined by western blot and normalized to tubulin. Error bars, mean ± sd, n = 3. **P < 0.01 (Student's t-test). (C) Constructs in the tethering reporter assay. Effector: the YTH domain of YTHDC2 was replaced with λ peptide (N-YTHDC2-λ), which binds with high affinity to the BoxB RNA motif. Reporter: 5 BoxB domains were fused to the 3′ UTR of firefly luciferase mRNA (F-Luc-5BoxB). Renilla luciferase (R-Luc) mRNA was used as an internal control to normalize luciferase signals from different samples. (D) mRNA level of F-Luc normalized to R-Luc 4 h post F-Luc induction. Error bars, mean ± sd, n = 4. **P < 0.01 (Student's t-test). (E) Translation efficiency of F-Luc normalized to R-Luc 4 h post F-Luc induction. Error bars, mean ± sd, n = 4. ****P < 0.0001; (Student's t-test). (F) Cumulative frequency of log2-fold changes of RNA expression upon Ythdc2 depletion, in RIP targets and non-targets. P value was calculated using two-sided Mann-Whitney U test. (G) Top GO terms in GO analysis of components of protein complex containing YTHDC2 obtained from tandem affinity purification followed by mass spectrometry.

Article Snippet: The antibodies used in this study (listed in the format of name (catalogue; supplier; dilution) were as follows: Goat anti-YTHDC2 (Sc-249370; Santa-Cruz; IP/20μg; Western Blot/1:1 000); Rabbit anti-Ythdc2 (A303-026A; BETHYL; Western Blot/1:2 000); Rabbit anti-m 6 A (202003; Synaptic Systems; IP/2.5 μg); Rabbit anti-eIF3a (3411; Cell Signaling Technology; Western Blot/1:2 000); Mouse anti-FLAG M2-peroxidase HRP (A8592; Sigma; Western Blot/1:10 000); Goat anti-GAPDH HRP (A00192-100; GenScript; Western Blot/1:5 000); Mouse anti-DDX4 (ab27591; Abcam; IF/1:200); Mouse anti-γ-H2A.X (ab26350; Abcam; IF/1:100); Mouse anti-SYCP3 (ab97672; Abcam; IF/1:100); Rabbit anti-SYCP3 (ab15093; Abcam; IF/1:100); Rabbit anti SYCP1 (ab15090; Abcam; IF/1:100); Mouse anti-SMC3 (ab128919; Abcam; Western Blot/1:2 000, IF/1:200); Rabbit anti-cleaved caspase3 (9661; CST; IHC/1:200); Rabbit anti-Cep76 (gift from Dr Xueliang Zhu, Institute of Biochemistry and Cell Biology, SIBS, CAS, Western Blot/1:2 000); Mouse anti-PLZF (sc-28319; Santa-Cruz; IF/1:200); Rabbit anti-Lin28 (13456-1-AP; Proteintech; IHC/1:200); Rabbit anti-SOX9 (ab5535; Chemicon; IF/1:200); Rabbit anti-IMP3 (IGF2BP3) (A303-426A; Bethyl; Western Blot/1:1 000); Rabbit anti-YBX1 (ab76149; Abcam; Western Blot/1: 1 000); Rabbit anti-MOV10 (A301-571A; Bethyl; Western Blot/1:1 000); Rabbit anti-XRN1 (A300-443A; Bethyl; Western Blot/1:1 000); Mouse anti-Tubulin(AT819; Beyotime; Western Blot/1:10 000); Donkey anti-Rabbit IgG-HRP (sc-2313; Santa-Cruz; Western Blot/1:2 000); Donkey anti-Goat IgG-HRP (sc-2033; Santa-Cruz; Western Blot/1:2 000).

Techniques: Expressing, Western Blot, Construct, Reporter Assay, Luciferase, RNA Expression, MANN-WHITNEY, Affinity Purification, Mass Spectrometry

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Figure Lengend Snippet: Ythdc2-deficient mice display defects in testes. (A) RT-PCR analysis of Ythdc2 mRNA levels in various organs of adult mice. (B) Schematic diagram of sgRNAs targeting at Ythdc2 locus. Two founders were generated by micro-injection of sgRNA/Cas9 mRNA into one-cell embryos. PAM sequences are underlined and highlighted in green. sgRNA targeting sites are in red. Mutant sequence are in blue. (C) Testes and epididymis from adult Ythdc2−/− mice are smaller in size than wild type. (D) The testis/body weight ratio was calculated for adult Ythdc2+/+ and Ythdc2−/− mice. Error bars, mean ± sd, n = 4, biological replicates ***P < 0.001 (Student's t-test) (E) Periodic Acid Schiff (PAS) staining of adult testes and epididymis. Scale bar, 50 μm.

Article Snippet: The antibodies used in this study (listed in the format of name (catalogue; supplier; dilution) were as follows: Goat anti-YTHDC2 (Sc-249370; Santa-Cruz; IP/20μg; Western Blot/1:1 000); Rabbit anti-Ythdc2 (A303-026A; BETHYL; Western Blot/1:2 000); Rabbit anti-m 6 A (202003; Synaptic Systems; IP/2.5 μg); Rabbit anti-eIF3a (3411; Cell Signaling Technology; Western Blot/1:2 000); Mouse anti-FLAG M2-peroxidase HRP (A8592; Sigma; Western Blot/1:10 000); Goat anti-GAPDH HRP (A00192-100; GenScript; Western Blot/1:5 000); Mouse anti-DDX4 (ab27591; Abcam; IF/1:200); Mouse anti-γ-H2A.X (ab26350; Abcam; IF/1:100); Mouse anti-SYCP3 (ab97672; Abcam; IF/1:100); Rabbit anti-SYCP3 (ab15093; Abcam; IF/1:100); Rabbit anti SYCP1 (ab15090; Abcam; IF/1:100); Mouse anti-SMC3 (ab128919; Abcam; Western Blot/1:2 000, IF/1:200); Rabbit anti-cleaved caspase3 (9661; CST; IHC/1:200); Rabbit anti-Cep76 (gift from Dr Xueliang Zhu, Institute of Biochemistry and Cell Biology, SIBS, CAS, Western Blot/1:2 000); Mouse anti-PLZF (sc-28319; Santa-Cruz; IF/1:200); Rabbit anti-Lin28 (13456-1-AP; Proteintech; IHC/1:200); Rabbit anti-SOX9 (ab5535; Chemicon; IF/1:200); Rabbit anti-IMP3 (IGF2BP3) (A303-426A; Bethyl; Western Blot/1:1 000); Rabbit anti-YBX1 (ab76149; Abcam; Western Blot/1: 1 000); Rabbit anti-MOV10 (A301-571A; Bethyl; Western Blot/1:1 000); Rabbit anti-XRN1 (A300-443A; Bethyl; Western Blot/1:1 000); Mouse anti-Tubulin(AT819; Beyotime; Western Blot/1:10 000); Donkey anti-Rabbit IgG-HRP (sc-2313; Santa-Cruz; Western Blot/1:2 000); Donkey anti-Goat IgG-HRP (sc-2033; Santa-Cruz; Western Blot/1:2 000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Generated, Injection, Mutagenesis, Sequencing, Staining

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Figure Lengend Snippet: Ythdc2-deficient mice display defects in meiotic prophase I. (A) RT-qPCR analysis of Ythdc2 mRNA levels in wild type testes over time, normalized to Rn18s as an internal control. Error bars, mean ± sd, n = 3, technical replicates (Student's t-test). (B) Ythdc2+/+ and Ythdc2−/− spermatocyte chromosome spreads at 15.0 d.p.p. stained for SYCP1 (red) and SYCP3 (green). Scale bar, 10 μm. (C) TUNEL assay of testes sections from 8.5 d.p.p. to 10.5 d.p.p.. DNA was stained with Hoechst (blue). Scale bar, 20 μm. (D) Quantification of apoptotic cells in Ythdc2+/+ (black columns) and Ythdc2−/− (grey columns) testes at 8.5 to 10.5 d.p.p.. Error bars, mean ± sd, n = 3, biological replicates *P < 0.05, *P < 0.01 (Student's t-test). (E) Cleaved Caspase-3 staining of testes sections at 8.5–10.5 d.p.p.. Scale bar, 20 μm.

Article Snippet: The antibodies used in this study (listed in the format of name (catalogue; supplier; dilution) were as follows: Goat anti-YTHDC2 (Sc-249370; Santa-Cruz; IP/20μg; Western Blot/1:1 000); Rabbit anti-Ythdc2 (A303-026A; BETHYL; Western Blot/1:2 000); Rabbit anti-m 6 A (202003; Synaptic Systems; IP/2.5 μg); Rabbit anti-eIF3a (3411; Cell Signaling Technology; Western Blot/1:2 000); Mouse anti-FLAG M2-peroxidase HRP (A8592; Sigma; Western Blot/1:10 000); Goat anti-GAPDH HRP (A00192-100; GenScript; Western Blot/1:5 000); Mouse anti-DDX4 (ab27591; Abcam; IF/1:200); Mouse anti-γ-H2A.X (ab26350; Abcam; IF/1:100); Mouse anti-SYCP3 (ab97672; Abcam; IF/1:100); Rabbit anti-SYCP3 (ab15093; Abcam; IF/1:100); Rabbit anti SYCP1 (ab15090; Abcam; IF/1:100); Mouse anti-SMC3 (ab128919; Abcam; Western Blot/1:2 000, IF/1:200); Rabbit anti-cleaved caspase3 (9661; CST; IHC/1:200); Rabbit anti-Cep76 (gift from Dr Xueliang Zhu, Institute of Biochemistry and Cell Biology, SIBS, CAS, Western Blot/1:2 000); Mouse anti-PLZF (sc-28319; Santa-Cruz; IF/1:200); Rabbit anti-Lin28 (13456-1-AP; Proteintech; IHC/1:200); Rabbit anti-SOX9 (ab5535; Chemicon; IF/1:200); Rabbit anti-IMP3 (IGF2BP3) (A303-426A; Bethyl; Western Blot/1:1 000); Rabbit anti-YBX1 (ab76149; Abcam; Western Blot/1: 1 000); Rabbit anti-MOV10 (A301-571A; Bethyl; Western Blot/1:1 000); Rabbit anti-XRN1 (A300-443A; Bethyl; Western Blot/1:1 000); Mouse anti-Tubulin(AT819; Beyotime; Western Blot/1:10 000); Donkey anti-Rabbit IgG-HRP (sc-2313; Santa-Cruz; Western Blot/1:2 000); Donkey anti-Goat IgG-HRP (sc-2033; Santa-Cruz; Western Blot/1:2 000).

Techniques: Quantitative RT-PCR, Staining, TUNEL Assay

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Figure Lengend Snippet: (A, B) Immunoprecipitation with α-YTHDC2 from P12 and P18 wild-type mouse testis extracts containing either an RNase Inhibitor (-RNase) or RNase A (+RNase). Western blots probed with α-YTHDC2, α-MEIOC, α-PABPC1, α-FMR1, α-CAPRIN1, α-PTBP1, α-EWS, α-MOV10, α- YBX1, α-G3BP2 and α-UPF1. (C-H) Immunofluorescence images of testis cross-sections from P18 (C-E) control ( +/+; Ythdc2 flox /Δ ) and (F-H) Ythdc2 cKO ( UBC-CreERT2; Ythdc2 flox /Δ ) mice stained for MEIOC (green) and SYCP3 (red). Scale bars: 50 µm. (D, E) High-magnification images of (D) pachytene spermatocytes in boxed region 1 in panel C′ and (E) leptotene spermatocytes in boxed region 2 in panel C′. (G, H) High-magnification image of (G) pachytene spermatocytes in boxed region 1 in panel F′ and (H) leptotene spermatocytes in boxed region 2 in panel F′. Arrows: MEIOC protein in perinuclear puncta in pachytene spermatocytes. (I-I′′′) High-magnification immunofluorescence images of spermatocytes from a P18 Ythdc2 cKO mouse testis cross-section stained for MEIOC (green), MIWI (blue) and DCP1A (red). Arrows: puncta in the cytoplasm of pachytene spermatocytes. See also Figure 4 – source data 1 and Figure 4 – source data 2.

Article Snippet: The following antibodies were used for immunoblot: rabbit anti-YTHDC2 (1:1,000, A303-026A, (RRID:AB_10754785), Bethyl laboratories), rabbit anti-MEIOC (1:2000 immunoblot, David Page Lab), goat anti-RENT1/UPF1 (1:2,000, A300-038A, (RRID:AB_2288326), Bethyl laboratories), rabbit anti-EWS (1:2,000, A300-417A, (RRID:AB_420957), Bethyl laboratories), rabbit anti-MOV10 (1:2,000, A301-571A, (RRID:AB_1040002), Bethyl laboratories), rabbit anti-G3BP2 (1:2,000, A302-040A, (RRID:AB_1576545), Bethyl laboratories), rabbit anti-YBX1 (1:2,000, A303-231A, (RRID:AB_10951283), Bethyl laboratories), mouse anti-PTBP1 (1:2,000, Thermo), rabbit anti-PABPC1 (1:1,000, ab21060, (RRID:AB_777008, Abcam), rabbit anti-FMR1 (1:2,000, A305-200A, (RRID:AB_2631593), Bethyl laboratories), rabbit anti-CAPRIN1 (1:2,000, HPA018126, (RRID:AB_1849929), Sigma).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining