Journal: Frontiers in Veterinary Science

Article Title: METTL14 alleviates heat stress in Hu sheep involves enhancing fatty acid oxidation while reducing lipid deposition

doi: 10.3389/fvets.2025.1732947

Figure Lengend Snippet: Changes in expression of methylation-related genes and immunofluorescence assays with and without HS in primary hepatocytes. (A) Expression of METTL3 , METTL14, and WTAP in the RNA-seq of primary hepatocytes after HS; (B) Expression of FTO and ALKBH5 in the RNA-seq of primary hepatocytes after HS; (C) Expression of YTHDC2 , YTHDF2 and YTHDF3 in the RNA-seq of primary hepatocytes after HS; (D) Immunofluorescence assays of METTL14 and YTHDC2 with no HS and post HS. * p < 0.05, ** p < 0.01.

Article Snippet: Cultured hepatocytes were fixed with 4% paraformaldehyde for 15 min, washed with PBS, permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, washed with PBS, and blocked with PBS containing 5% FBS and 0.3% TritonX-100 for 1 h. Thereafter, Primary antibodies against METTL14 (Proteintech, Rosemont, IL, USA) and YTHDC2 (Proteintech) were applied overnight at 4 °C.

Techniques: Expressing, Methylation, Immunofluorescence, RNA Sequencing

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet: (A) Regimen of tamoxifen treatment in ≥ 8-week-old Ythdc2 fl/− Ddx4 -Cre ERT2 male mice. See also . (B) Testis weight (mean ± s.d.) of control ( Ythdc2 fl/− , n = 16) and Ythdc2 iKO males (n = 10 at 2 dpt; 16 at 4 dpt; 3 at 6 dpt, 8 dpt, or 10 dpt). (C) Western blot analysis of cell cycle regulators in control ( Ythdc2 fl/− ), Ythdc2 iKO , and Ythdc2 −/− testes. (D) Histology of control and Ythdc2 iKO (2 dpt) testes. Five stages of seminiferous tubules are shown (VIII-XII). Abbreviations: PreL, preleptotene; Lep, leptotene; Zyg, zygotene; Pa, pachytene; Dip, diplotene; Met, metaphase; RS, round spermatid; ES, elongating spermatid; CS, condensing spermatids. Arrowheads indicate apparently apoptotic pachytene spermatocytes, which are characterized by strong but diffuse eosin stain in the nucleus. Insets show the boxed pachytene spermatocytes with higher magnification in stages VIII-X. Loss of diplotene spermatocytes at the stage XI Ythdc2 iKO tubule is demarcated by a dashed blue line. Scale bar, 50 μm. See also . (E) Surface nuclear spread analysis of control and Ythdc2 iKO (2 dpt) spermatocytes. Absence of TC pachytene cells in control is marked with a large symbol. A severe loss of diplotene cells in Ythdc2 iKO (2 dpt) is indicated by a small symbol. TC, telomere clustering. (F) The plot shows the percentage of each type of spermatocytes in control and Ythdc2 iKO testes (mean ± s.d.) is shown. Eighty-two to 887 cells per mouse and three males per genotype per time point were analyzed. Scale bar, 10 μm. (G) A schematic diagram of spermatocytes in control and Ythdc2 iKO (2 dpt) testes. Lines indicate the presence of spermatocytes. A thinner dash line at 2 dpt (iKO) indicates a severe reduction in the number of diplotene spermatocytes.

Article Snippet: The cDNA fragment encoding the N-terminal 239 aa of mouse YTHDC2 was amplified by PCR from the Ythdc2 plasmid (NM_001163013) (Cat. No. MR226474, OriGene Technologies) using these two primers: 5'-CGCGGATCCATGTCCAGGCCGAGCAG-3' and 5'-CCCAAGCTTTGTGGTCTTTCCAGACCCAGT-3', and was cloned into the BamHI and HindIII sites of the pQE30 vector.

Techniques: Control, Western Blot, Staining

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet: (A) TUNEL analysis of paraffin-embedded tissue sections from control and Ythdc2 iKO (2 dpt) testes. The acrosome morphology shown by SP10 immunofluorescence staining was used for seminiferous tubule staging. Nuclear DNA was stained with DAPI. Arrowheads indicate TUNEL-positive pachytene cells. The dashed line in stage IX Ythdc2 iKO demarcates pachytene cells (the inner layer) from leptotene cells (the outer layer). Abbreviations: PreL, preleptotene; Lep, leptotene; Pa, pachytene; RS, round spermatid; ES, elongating spermatid. Scale bar, 50 μm. (B) Percentage of TUNEL-positive tubules from control and Ythdc2 iKO testes at 2 dpt. The mean ± s.d. values were plotted. Two males per genotype (control and Ythdc2 iKO ) were analyzed. Three hundred twenty-three to three hundred ninety-eight tubules were counted for each mouse. (C) Quantification of TUNEL-positive cells in TUNEL-positive tubules (mean ± s.d.) from control and Ythdc2 iKO at 2 dpt. Two males per genotype (control and Ythdc2 iKO ) were analyzed. One hundred seventy-six to two hundred eighty-nine tubules were counted for each mouse.

Article Snippet: The cDNA fragment encoding the N-terminal 239 aa of mouse YTHDC2 was amplified by PCR from the Ythdc2 plasmid (NM_001163013) (Cat. No. MR226474, OriGene Technologies) using these two primers: 5'-CGCGGATCCATGTCCAGGCCGAGCAG-3' and 5'-CCCAAGCTTTGTGGTCTTTCCAGACCCAGT-3', and was cloned into the BamHI and HindIII sites of the pQE30 vector.

Techniques: TUNEL Assay, Control, Immunofluorescence, Staining

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet: (A) Volcano plot of expression changes in Ythdc2 iKO versus control ( Ythdc2 fl/− ; HT) pachytene spermatocytes. Only coding genes (13,754) with an average expression of ≥ 50 normalized counts across all the samples are plotted. (B) MA plot of expression changes. The differentially expressed (up and downregulated) genes are shown as red dots. See also and . (C) Expression of differentially expressed genes in various wild-type spermatogenic populations. SSC, spermatogonial stem cells; SC, spermatocytes; RS, round spermatids. ANOVA was performed (p value < 2 × 10 −16 for both upregulated and downregulated genes) and followed by TukeyHSD (p value < 0.001). (D) Expression comparison of genes in Ythdc2 iKO versus control ( Ythdc2 fl/− ; HT) pachytene cells that are enriched or depleted of m 6 A in juvenile testes . (E) Overlap of YTHDC2 RIP-seq targets with the differentially expressed genes in Ythdc2 iKO pachytene cells. (F) Overlap of YTHDC2 RIP-seq targets with the differentially expressed genes in Ythdc2 iKO pachytene cells.

Article Snippet: The cDNA fragment encoding the N-terminal 239 aa of mouse YTHDC2 was amplified by PCR from the Ythdc2 plasmid (NM_001163013) (Cat. No. MR226474, OriGene Technologies) using these two primers: 5'-CGCGGATCCATGTCCAGGCCGAGCAG-3' and 5'-CCCAAGCTTTGTGGTCTTTCCAGACCCAGT-3', and was cloned into the BamHI and HindIII sites of the pQE30 vector.

Techniques: Expressing, Control, Comparison

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet: (A) Immunofluorescence of α-tubulin, SUN1, SYCP2 in intact control ( Ythdc2 fl/− ) and Ythdc2 iKO (2 dpt) pachytene spermatocytes. Scale bar, 10μm. See also . (B) Nuclear spread analysis of pachytene, diplotene, and diakinesis/metaphase I (dia/met) spermatocytes after treatment with DMSO or nocodazole. TC, telomere clustering. Scale bar, 10μm. (C) Percentage of spermatocytes (mean ± s.d.) in control ( Ythdc2 fl/− ) and Ythdc2 iKO (2 dpt) after treatment with DMSO or nocodazole. Only pachytene, diplotene, and diakinesis/metaphase I spermatocytes are included. Five hundred thirty-two to nine hundred eighty-five cells were counted per genotype per treatment group. Two males per genotype per experiment were used. The experiments were performed three times (n = 3). n.s., non-significant. See also .

Article Snippet: The cDNA fragment encoding the N-terminal 239 aa of mouse YTHDC2 was amplified by PCR from the Ythdc2 plasmid (NM_001163013) (Cat. No. MR226474, OriGene Technologies) using these two primers: 5'-CGCGGATCCATGTCCAGGCCGAGCAG-3' and 5'-CCCAAGCTTTGTGGTCTTTCCAGACCCAGT-3', and was cloned into the BamHI and HindIII sites of the pQE30 vector.

Techniques: Immunofluorescence, Control

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet: (A) Requirement of YTHDC2 at the leptotene and pachytene stages. The red cross designates the stage of meiotic arrest in the Ythdc2 or Meioc mutants. Ythdc2 or Meioc global knockout mutants display the same early meiotic arrest, which is also present in the Ythdc2 iKO ( Ythdc2 fl/− Ddx4 -Cre ERT2 ) mutant. In contrast, Ythdc2 iKO pachytene cells undergo apoptosis at the late pachytene stage. The seminiferous tubule stage is shown in Roman numerals. See also . (B) Illustration of telomere distribution in wild-type and Ythdc2 iKO pachytene spermatocytes. Two pairs of homologous chromosomes are depicted. The SUN/KASH proteins that connect telomeres with microtubules are not shown. In contrast with the random distribution of telomeres in wild-type, telomeres in the Ythdc2 iKO pachytene cell are clustered to one pole, where microtubules converge and the cytoplasm is expanded.

Article Snippet: The cDNA fragment encoding the N-terminal 239 aa of mouse YTHDC2 was amplified by PCR from the Ythdc2 plasmid (NM_001163013) (Cat. No. MR226474, OriGene Technologies) using these two primers: 5'-CGCGGATCCATGTCCAGGCCGAGCAG-3' and 5'-CCCAAGCTTTGTGGTCTTTCCAGACCCAGT-3', and was cloned into the BamHI and HindIII sites of the pQE30 vector.

Techniques: Knock-Out, Mutagenesis

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet:

Article Snippet: The cDNA fragment encoding the N-terminal 239 aa of mouse YTHDC2 was amplified by PCR from the Ythdc2 plasmid (NM_001163013) (Cat. No. MR226474, OriGene Technologies) using these two primers: 5'-CGCGGATCCATGTCCAGGCCGAGCAG-3' and 5'-CCCAAGCTTTGTGGTCTTTCCAGACCCAGT-3', and was cloned into the BamHI and HindIII sites of the pQE30 vector.

Techniques: Recombinant, Software

Journal: Cell reports

Article Title: YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis

doi: 10.1016/j.celrep.2021.110110

Figure Lengend Snippet:

Article Snippet: Rabbit anti-YTHDC2 , Novus Biologicals , Cat# NBP1–85089; RRID: AB_11034900.

Techniques: Recombinant, Software

Journal: JCI Insight

Article Title: Pathogenic variants in the human m 6 A reader YTHDC2 are associated with primary ovarian insufficiency

doi: 10.1172/jci.insight.154671

Figure Lengend Snippet: ( A ) Pedigrees of kindred with YTHDC2 variants (upper panel: p.P856R, lower panel: p.E377*). Solid symbols indicate affected family members. Genotype is indicated underneath tested family members. ( B ) Domains of the human YTHDC2 protein. Domains include an R3H domain; 2 RecA-like domains, RecA1: ATP-binding DEAD-like helicase domain (DExDc) and RecA2: helicase superfamily C-terminal domain (HELICc); 2 ankyrin repeats (506–535 and 539–568) comprising the ankyrin domain (ANK); a helicase-associated domain (HA2), an oligonucleotide/oligosaccharide-binding fold domain (OB-fold), and a YTH (YT521-B homology) m 6 A-dependent RNA binding domain (YTH). The DExDc, ankyrin repeats, and HELICc domains make up the helicase core of this protein. Domain positions (in amino acids) are annotated below the schematic. The position of the 2 pathogenic variants (p.E377*; p.P856R); the ketu mouse mutation (mouse: p.H327R; human: p.H312R) (Jain et al. 2017, ref. ); and another Ythdc2 –/– knockout mouse (deletion of 50 amino acids within exon 7) (Wojtas et al. 2017, ref. ) are indicated. ( C ) Amino acid conservancy in the region of YTHDC2 surrounding codon 856. Yellow asterisks represent complete conservation among the species shown. ( D ) The m 6 A methyltransferase complex. The m 6 A modification on mRNA is mediated by “writers” and demethylases (“erasers”). “Readers” recognize the m 6 A modification and allow execution of its functions. One key reader is YTHDC2 , with its known cofactors MEIOC and XRN .

Article Snippet: RNA was extracted as described previously and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and reverse-transcribed using the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan Fast Advanced MasterMix (Applied Biosystems) and TaqMan assays ( YTHDC2: Hs00403320_m1; MEIOC : Hs00403320_m1; Applied Biosystems) on an ABI StepOne Plus System (Applied Biosystems).

Techniques: Binding Assay, RNA Binding Assay, Mutagenesis, Knock-Out, Modification

Journal: JCI Insight

Article Title: Pathogenic variants in the human m 6 A reader YTHDC2 are associated with primary ovarian insufficiency

doi: 10.1172/jci.insight.154671

Figure Lengend Snippet: ( A ) Violin plots depicting normalized counts for YTHDC2 , MEIOC , and XRN1 in the developing ovary and testis at 4 developmental stages (4 samples at stages CS22/23, 9 wpc, 11 wpc, 15/16 wpc). ( B ) Quantitative reverse transcriptase PCR (qRT-PCR) expression (log 2 ) of YTHDC2 in developing ovary and testis compared with reference ( ACTB ) and relative to expression in an index CS22 ovary sample. Four fetal ovary and fetal testis samples were included at CS22/CS23, 9 wpc, 11 wpc, 15/16 wpc, and 19/20 wpc. ( C ) qRT-PCR expression (log 2 ) of MEIOC in developing ovary and testis compared with reference ( ACTB ) and relative to the expression in a CS22 ovary sample. Four fetal ovary and fetal testis samples were included at CS22/CS23, 9 wpc, 11 wpc, 15/16 wpc, and 19/20 wpc. ( D ) qRT-PCR mean expression (log 2 ) of YTHDC2 in 4 adult ovary samples and 4 adult testis samples from individuals without gonadal insufficiency. B – D represent mean expression of triplicates, and error bars indicate mean ± SEM. qRT-PCR experiments were performed 3 times; representative data from 1 experiment shown. Differences in mean expression between sample groups were assessed using multiple t test analysis (* P < 0.05; ** P < 0.01); correction for multiple comparisons was performed using Holm-Šídák. CS, Carnegie stage. ( E ) Uniform manifold approximation and projection representation of distinct germ cell clusters ( n = 8867 germ cells) identified by single-cell RNA sequencing of human fetal gonads between 6 and 21 wpc. Key ovary clusters are annotated. PGCs, primordial germ cells; GCs, germ cells. ( F ) Visium spatial transcriptomic expression of YTHDC2 in a 17 wpc human fetal ovary. EG, extragonadal. ( G ) Immunohistochemistry of YTHDC2 in a 16 wpc human fetal ovary showing increased staining in the central region containing meiotic cells and decreased staining in primordial germ cells at the periphery in the cortex of the fetal ovary (PGCs).

Article Snippet: RNA was extracted as described previously and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and reverse-transcribed using the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan Fast Advanced MasterMix (Applied Biosystems) and TaqMan assays ( YTHDC2: Hs00403320_m1; MEIOC : Hs00403320_m1; Applied Biosystems) on an ABI StepOne Plus System (Applied Biosystems).

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, RNA Sequencing, Immunohistochemistry, Staining

Journal: JCI Insight

Article Title: Pathogenic variants in the human m 6 A reader YTHDC2 are associated with primary ovarian insufficiency

doi: 10.1172/jci.insight.154671

Figure Lengend Snippet: ( A ) qRT-PCR mean expression (log 2 ) of YTHDC2 , SYCP2L , and TRIM9 in the peripheral leukocytes of patients with p.P856R (missense) and p.E377* (nonsense) YTHDC2 variants compared with reference ( ACTB ) and relative to the expression in patients with POI who have no known YTHDC2 variants. Samples were processed in triplicate and error bars indicate mean ± SEM (** P < 0.01, ANOVA 1 way comparing gene expression between the 3 groups). Experiments were performed 3 times and representative data from 1 experiment are shown. ( B ) Cellular localization studies of either WT or mutant p.P856R YTHDC2 together with MEIOC in HeLa cells. Original magnification, ×100.

Article Snippet: RNA was extracted as described previously and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and reverse-transcribed using the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan Fast Advanced MasterMix (Applied Biosystems) and TaqMan assays ( YTHDC2: Hs00403320_m1; MEIOC : Hs00403320_m1; Applied Biosystems) on an ABI StepOne Plus System (Applied Biosystems).

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Mutagenesis

Journal: JCI Insight

Article Title: Pathogenic variants in the human m 6 A reader YTHDC2 are associated with primary ovarian insufficiency

doi: 10.1172/jci.insight.154671

Figure Lengend Snippet: ( A ) The HA2 domain is shown in green and codon P856 is indicated by an arrow. The RNA-binding domain is shown with the bound RNA in orange. ( B ) P856R can establish strong hydrogen bond interactions with surrounding residues (D855,T859, Q905, Q908, R937, R959, N967), which is predicted to increase stability and to reduce flexibility of the P856R protein. ( C ) The radius of gyration (RoG) indicates that P856R mutant protein is more compact than WT protein (left panel), and there is an increased number of protein-RNA hydrogen bonds in the P856R protein compared with WT (right panel), suggesting the P856R protein to be more stable and less flexible than WT. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. **** P < 0.0001, unpaired t test. ( D ) Root-mean square deviation (RMSD) of alpha carbons at the protein and nucleic acid (NA) level for both WT and P856R mutant YTHDC2. Global movement is lower in the P856R mutant protein compared with WT. ( E ) Replacement of proline at position 856 with arginine may also alter the electrostatic charge of YTHDC2.

Article Snippet: RNA was extracted as described previously and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and reverse-transcribed using the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan Fast Advanced MasterMix (Applied Biosystems) and TaqMan assays ( YTHDC2: Hs00403320_m1; MEIOC : Hs00403320_m1; Applied Biosystems) on an ABI StepOne Plus System (Applied Biosystems).

Techniques: RNA Binding Assay, Mutagenesis

Journal: JCI Insight

Article Title: Pathogenic variants in the human m 6 A reader YTHDC2 are associated with primary ovarian insufficiency

doi: 10.1172/jci.insight.154671

Figure Lengend Snippet: Although the exact role of partners is still incompletely understood, the YTHDC2-MEIOC-XRN1 complex would stabilize meiotic transcripts and/or degrade mitotic transcripts by binding to m 6 A-marked mRNA, promoting a normal mitosis to meiosis transition. At pachytene, YTHDC2 and other DExH helicases may functionally partner with PIWIL proteins within cytoplasmic RNA germ cell granules and may regulate piRNA activity, which is required for normal meiotic timing and progression.

Article Snippet: RNA was extracted as described previously and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and reverse-transcribed using the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan Fast Advanced MasterMix (Applied Biosystems) and TaqMan assays ( YTHDC2: Hs00403320_m1; MEIOC : Hs00403320_m1; Applied Biosystems) on an ABI StepOne Plus System (Applied Biosystems).

Techniques: Binding Assay, Activity Assay

Journal: Biochemical pharmacology

Article Title: m 6 A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2.

doi: 10.1016/j.bcp.2021.114766

Figure Lengend Snippet: Fig. 4. Posttranscriptional regulation of CES2 by RNA methylation via m6A reader protein YTHDC2. The stability of CES2 mRNA in siRNA-transfected HepG2 cells was examined (A-C). HepG2 cells were treated with 10 ng/µL α-amanitin 48 h after transfection with siMETTL3 and siMETTL14 (A), siFTO (B), siALKBH5 (C), or siControl (A-C). Total RNA was prepared after 0, 12, 24, and 36 h. The CES2 mRNA level was determined by using real-time RT-PCR. The CES2 mRNA levels at time 0 (the time of addition of α-amanitin) in each treatment were assigned values of 100%. YTHDC2 mRNA, YTHDF2 mRNA (D), CES2 mRNA (E), and CES2 protein (F) levels in siYTHDC2- or siYTHDF2-transfected HepG2 cells were determined by real-time RT-PCR and Western blotting. The mRNA and protein levels were normalized to β-actin levels. The values represent the levels relative to siControl. (G) Cell lysates from HepG2 cells were immunoprecipitated with an anti-human YTHDC2 antibody or normal rabbit IgG. An electropherogram of the PCR amplicon using primers for CES2 mRNA is shown. The length of the PCR product was 316 bp. Each point and column represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.01 compared with siControl.

Article Snippet: The rabbit anti-human YTHDC2 polyclonal antibody was from Bethyl Laboratories (Montgomery, TX).

Techniques: Methylation, Transfection, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Amplification

Journal: PLoS ONE

Article Title: Identification and Validation of a Multigene Predictor of Recurrence in Primary Laryngeal Cancer

doi: 10.1371/journal.pone.0070429

Figure Lengend Snippet: mRNA target selection from the 30 probes included in the prognostically relevant signature and TaqMan® gene expression assay description.

Article Snippet: 10 , 205836_s_at , YTH domain containing 2 , NM_022828 , YTHDC2 , 64848 , , 0,0981884 , 1,04 , yes , , NM_022828.3 , Hs00967276_g1 , 75 , 29–30 , Accepted.

Techniques: Selection, Gene Expression, Membrane, Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: Identification and Validation of a Multigene Predictor of Recurrence in Primary Laryngeal Cancer

doi: 10.1371/journal.pone.0070429

Figure Lengend Snippet: 30-probe set model annotations.

Article Snippet: 10 , 205836_s_at , YTH domain containing 2 , NM_022828 , YTHDC2 , 64848 , , 0,0981884 , 1,04 , yes , , NM_022828.3 , Hs00967276_g1 , 75 , 29–30 , Accepted.

Techniques: Binding Assay, Membrane, Sequencing

Journal: PLoS ONE

Article Title: Identification and Validation of a Multigene Predictor of Recurrence in Primary Laryngeal Cancer

doi: 10.1371/journal.pone.0070429

Figure Lengend Snippet: Individual and profiled gene expression effects on DFS.

Article Snippet: 10 , 205836_s_at , YTH domain containing 2 , NM_022828 , YTHDC2 , 64848 , , 0,0981884 , 1,04 , yes , , NM_022828.3 , Hs00967276_g1 , 75 , 29–30 , Accepted.

Techniques: Gene Expression

Journal: Clinical and Translational Medicine

Article Title: Targeting Zfp36 to combat cardiac hypertrophy: Insights into ferroptosis pathways

doi: 10.1002/ctm2.70247

Figure Lengend Snippet: Zinc finger protein 36 (Zfp36) through the Ythdc2/SLC7A11/glutathione (GSH)‐dependent ferroptosis pathway improve cardiac hypertrophy. (A‒H) The mRNA and protein expression levels of SLC7A11 ( n = 4‒5). (I) RNA immunoprecipitation (RIP)‐PCR analysed the m 6 A modification of SLC7A11 mRNA ( n = 3). (J) RNA‐pull down verified Ythdc2 bond to SLC7A11 mRNA ( n = 3). (K) RIP analysed the binding relation between Ythdc2 and SLC7A11 mRNA ( n = 3). (L) Co‐localisation of Ythdc2 and SLC7A11 mRNA in cardiomyocyte. (M) Real‐time PCR analysed the mRNA expression levels of SLC7A11 treated with or not Act D in over‐expression or knockdown Ythdc2 ( n = 3‒4). (N‒Q) Protein expression levels of SLC7A11 in mice hearts ( n = 4‒6). (R and S) GSH was negatively regulated by Ythdc2 in cardiac hypertrophy ( n = 3). (T‒U) Zfp36 regulates GSH level by Ythdc2 ( n = 3‒4). Statistical analysis was performed with Student's t ‐test or one‐way analysis of variance (ANOVA). Results presented as mean ± standard deviation (SD). * p < .05; ** p < .01.

Article Snippet: Cy3‐labelled mRNA probes for Ythdc2 and SLC7A11, designed by GenePharma, were hybridised overnight according to the manufacturer's instructions.

Techniques: Expressing, RNA Immunoprecipitation, Modification, Binding Assay, Real-time Polymerase Chain Reaction, Over Expression, Knockdown, Standard Deviation

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet: Negative correlation between YTHDC2 and RUNX2 in BMSCs osteogenesis. A-B. The mRNA and protein expression of YTHDC2 in BMSCs quantified by qRT-PCR and Western blot. C-D. The mRNA and protein expression of RUNX2 in BMSCs quantified by qRT-PCR and Western blot. (*P < 0.05).

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet: Knockdown of YTHDC2 promoted BMSCs osteogenic differentiation and RUNX2 expression. BMSCs were transfected with YTHDC2-siRNA (YTHDC2-si) or negative control-siRNA (NC), after which they were cultured in OM for 7 days. A-B . The qRT-PCR and Western blot results of YTHDC2 expression in BMSCs. C-D . ALP staining and alizarin red staining of BMSCs. E-F . The qRT-PCR and Western blot results of YTHDC2 expression in BMSCs. (*P < 0.05).

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Knockdown, Expressing, Transfection, Negative Control, Cell Culture, Quantitative RT-PCR, Western Blot, Staining

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet: YTHDC2 regulated RUNX2 expression through m6A modification. BMSCs were cultured in osteogenic medium (OM) or growth medium (GM) for 7 days. A. m6A-IP showed OM-induced BMSCs had more m6A modification on RUNX2 mRNA. B. YTHDC2-IP showed YTHDC2 tightly bound to RUNX2 m6A. C. Immunofluorescence staining showed negative correlation between YTHDC2 and RUNX2. D. Fluorescence intensity of OM-induced and GM-induced BMSCs. E. The mechanism of YTHDC2 potential effects on BMSCs osteogenesis through m6A modification on RUNX2 mRNA. (*P < 0.05).

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Expressing, Modification, Cell Culture, Immunofluorescence, Staining, Fluorescence

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet:

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Sequencing

Journal: Heliyon

Article Title: YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

doi: 10.1016/j.heliyon.2023.e18876

Figure Lengend Snippet:

Article Snippet: The YTHDC2 interference fragment and negative control fragment (Synbio Technologies, China) were co-transfected with Lipofectamine 2000 reagent to downregulate the target gene expression level in BMSCs according to the manufacturer's instructions.

Techniques: Sequencing

Journal: Science Advances

Article Title: RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

doi: 10.1126/sciadv.abq2945

Figure Lengend Snippet: ( A ) Identification of YTHDC2-associated partners by immunoprecipitation (IP) experiments with either YTHDC2 or normal immunoglobulin antibodies from P12 mouse testes. Immunoprecipitated YTHDC2 protein complexes were separated by SDS-PAGE, and the gel was stained with silver before mass spectrometry (MS). Testis lysates were prepared from mouse at P12 ( n = 8). ( B ) Selective YTHDC2-associated protein candidates identified by MS analysis from P12 mouse testes. ( C ) Immunoprecipitation of YTHDC2 from mouse testis lysates at P21 and Western blot with anti-YTHDC2, anti-MEIOC, and anti-RBM46 antibodies (top). RBM46 protein was immunoprecipitated with anti-RBM46 antibodies with or without RNases, followed by Western blot analysis with indicated antibodies (bottom). ( D ) Full-length GFP- Rbm46 and FLAG- Ythdc2 or HA- Meioc expression constructs were cotransfected in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG or anti-HA antibodies and Western blot analysis with indicated antibodies. IB, immunoblotting. ( E ) GFP-tagged RBM46 mutants with deleted fragments were coexpressed with FLAG-YTHDC2 in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibodies before Western blot analysis with GFP antibodies. ( F ) GST pull-down assay showing that in vitro translated YTHDC2 does not bind to GST-RBM46. GST-RBM46 was expressed in E. coli and purified with glutathione beads. YTHDC2 with N-terminal His tag was in vitro translated. ( G and H ) Immunofluorescence analysis of Rbm46 -HA knock-in mice with anti-HA antibodies and the acrosome marker PNA that defines different stages of spermatogenesis. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. Lower panels show magnification image of single cell in the upper panels. Spg, spermatogonia; PL, pre-leptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene; RS, round spermatids; ES, elongating spermatids. Scale bars, 5 μm.

Article Snippet: GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega).

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Western Blot, Expressing, Construct, Pull Down Assay, In Vitro, Purification, Immunofluorescence, Knock-In, Marker

Journal: Science Advances

Article Title: RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

doi: 10.1126/sciadv.abq2945

Figure Lengend Snippet: ( A ) RBM46 and YTHDC2 exhibit a similar distribution pattern along the length of mRNA. ( B ) RBM46 eCLIP mRNA targets substantially overlapped with YTHDC2 mRNA targets by CLIP-seq in P10 testis. In total, 53 and 52% of P10 YTHDC2–binding sites were co-occupied by RBM46 within a 100- and 70-nt window, respectively. ( C ) Top motif at best-scored YTHDC-binding sites from P10 and adult YTHDC2 CLIP-seq using HOMER. Sequences within ±100 nt relative to YTHDC2-binding sites were used for de novo motif analysis. ( D ) RBM46-binding motif AAUCAUGU is the top motif within a 100-nt window centered on YTHDC2 U-rich motifs in both P10 and adult testis. Motif analysis by HOMER was performed with best-scored P10 and adult YTHDC2 CLIP targets. ( E ) Position and distance between RBM46 and YTHDC2 binding on their shared targets. ( F ) Schematic illustrations for the function of RBM46-MEIOC-YTHDC2 complex during spermatogenesis. RNA binding protein RBM46 forms an ancient posttranscriptional network with MEIOC and YTHDC2 to recognize and destabilize mitotic transcripts for a successful meiotic entry.

Article Snippet: GST-RBM46 fusion protein or GST alone in the lysates was immobilized on precleared MagneGST particles (Promega) and then incubated with in vitro translated YTHDC2 protein in binding (Promega).

Techniques: Binding Assay, RNA Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Expression profiles and characterization of circYthdc2. A Schematic diagram of circRNA translation ways and its selection conditions. B Strategies used for circRNA-seq and ribosome profiling (Ribo-seq). The gray strips represented the total circRNAs by circRNA-Seq. The orange strips represented the differential circRNAs upon SCRV treatment. The red strips represented the circRNAs with potential translation ability by Ribo-seq. C We confirmed the head-to-tail splicing of circYthdc2 in the circYthdc2 RT-PCR product by Sanger sequencing. D RT-PCR validated the existence of circYthdc2 in MKC and MIC cell lines. CircYthdc2 was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. E The expression of circYthdc2 and linear Ythdc2 mRNA in both MKC and MIC cell lines was detected by RT-PCR assay followed by nucleic acid electrophoresis or qPCR assay in the presence or absence of RNase R. All data represented the three independent triplicated experiments

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Expressing, Selection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Negative Control, Nucleic Acid Electrophoresis

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Activity Assay, Clone Assay, Construct, Plasmid Preparation, FLAG-tag, Expressing, Transfection, Infection, Immunofluorescence, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: CircYthdc2 and Ythdc2-170aa inhibit host antiviral innate immunity. A and B The schematic diagram of siRNAs ( A ) and circYthdc2 overexpression plasmid structure ( B ). C qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MIC cells treated with siRNAs. qPCR analysis of circYthdc2 and linear Ythdc2 mRNA in MKC cells overexpressing circYthdc2. D and E qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-circYthdc2 ( D ) and transfected in MKC cells with circYthdc2 or pLC5-circ and Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG after SCRV infection ( E ). F circYthdc2 and Ythdc2-170aa promote SCRV replication. MIC cells transfected with NC or si-circYthdc2 and MKC cells were transfected with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. G MKC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MKC cells were transfected with Linear-FLAG-Ythdc2-170aa or pcDNA3.1-FLAG. H Effect of circYthdc2 on cell viability after SCRV infection. MIC cells were transfected with NC or si-circYthdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay was measured. I Ythdc2-170aa counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING, MAVS, TRIF, and TBK1 expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa. J Relative protein levels of STING in MIC cells after transfected with NC or si-circYthdc2 and in MKC cells with pLC5-circ or circYthdc2 and pcDNA3.1-FLAG or Linear-FLAG-Ythdc2-170aa or Linear-FL-Ythdc2-AG. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Over Expression, Plasmid Preparation, Expressing, Transfection, Infection, RNA Expression, Staining, Viability Assay, Luciferase, Cotransfection

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Ythdc2 inhibits host antiviral innate immunity. A Relative protein and mRNA levels of Ythdc2 in MIC cells after transfected with NC or si-circYthdc2. B Relative protein levels of STING in MIC cells after transfected with NC or si-Ythdc2 and in MKC cells with pcDNA3.1 or Ythdc2. C and D qPCR assays were performed to determine the expression levels of IFN1, TNF-α, Mx1, ISG15, and Viperin in MIC cells transfected with NC or si-Ythdc2 ( C ) and transfected in MKC cells with pcDNA3.1 and Ythdc2 after SCRV infection ( D ). E Ythdc2 counteracts the negative effect of STING. Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, pcDNA3.1, Ythdc2. F Ythdc2 promotes SCRV replication. MIC cells transfected with NC or si-Ythdc2 and MKC cells were transfected with pcDNA3.1 or Ythdc2 for 24 h, respectively, then infected with SCRV at 24 h. The qPCR analysis was conducted for intracellular and supernatant SCRV RNA expression. (G) MIC cells seeded in 48-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC cells were transfected with NC or si-Ythdc2. H Effect of Ythdc2 on cell viability after SCRV infection. MIC cells was transfected with NC or si-Ythdc2 for 24 h and then treated with SCRV for 24 h. Cell viability assay were measured. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Transfection, Expressing, Infection, Luciferase, Cotransfection, Plasmid Preparation, RNA Expression, Staining, Viability Assay

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. A MKC cells were transfected with Flag-Ythdc2 and Flag-circYthdc2 plasmids, the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed; MIC cells were silence Ythdc2 or circYthdc2, and the cells were treated with 10 μM CHX for a different time before immunoblot analysis was performed B MKC cells were transfected with Linear-Flag-Ythdc2-170aa or Flag-circYthdc2 or Flag-Ythdc2 plasmids, after 42 h, the cells were treated with DMSO or 10 μM MG132 for 6 h before immunoblot analysis was performed. C Flag-Ythdc2 and Linear-Flag-Ythdc2-170aa were cotransfected with GFP-STING into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2 and STING or Ythdc2-170aa and STING cellular localization. Original magnification is 630. D Immunoprecipitation and immunoblot analysis of Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa with Myc-STING, in EPC cells. IP, immunoprecipitation. E Upper panel: Schematic diagram of Ythdc2-△HELICc plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, Flag-Ythdc2-△HELICc or Linear-Flag-Ythdc2-170aa with Myc-STING, in MKC cells. IP, immunoprecipitation. F Schematic diagram of STING, STING-△TM, STING-△N, STING-△C plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-Ythdc2, with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. H Immunoprecipitation and immunoblot analysis of Linear-Flag-Ythdc2-170aa with Myc-STING, Myc-STING-△TM, Myc-STING-△N, Myc-STING-△C in MKC cells. IP, immunoprecipitation. I Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. J Coimmunoprecipitation analysis of STING ubiquitination in EPC cells transfected with Myc-STING or HA-ubiquitin-WT in the presence of control vector, Flag-Ythdc2, or Flag-Ythdc2-△HELICc, or Linear-Flag-Ythdc2-170aa expression plasmid. IP, immunoprecipitation. K Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-Ythdc2 or Linear-Flag-Ythdc2-170aa expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the three independent triplicated experiments

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Plasmid Preparation, Control, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: N6-methyladenosine modification mediates circYthdc2 translation polypeptides. A Upper panel: Schematic diagram of Flag-circYthdc2-m 6 A-mut plasmid construction. Lower panel: MKC cells were transfected with Flag-circYthdc2 or Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. B Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with m 6 A modification-related genes, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. C Left panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with METTL3 or METTL14, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Middle panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with FTO or YTHDF1, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. Right panel: Myc-STING and Flag-circYthdc2 were cotransfected into MKC cells with YTHDF3 or Ythdc2, respectively, and then the protein levels of Myc-STING and Flag-circYthdc2 were detected. D Relative RNA levels of circYthdc2 in MKC cells after transfected with pcDNA3.1, METTL3, METTL14, YTHDF1, YTHDF3, FTO, and Ythdc2, respectively. E The m 6 A level alteration of circYthdc2 upon METTL3 or FTO overexpression was examined by MeRIP-qPCR. MKC cells were transfected with vector or METTL3 or FTO plasmid for 48 h. F The level of circYthdc2 upon YTHDF1 or YTHDF3 or Ythdc2 overexpression were examined by RIP-qPCR. MKC cells were transfected with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. G The protein level of YTHDF1 or YTHDF3 or Ythdc2 was examined by RNA pulldown. MKC cells were transfected MS2-GFP, MS2-circYthdc2 or MS2-circYthdc2-m 6 A-mut with vector or YTHDF1 or YTHDF3 or Ythdc2 plasmid for 48 h. H Relative luciferase activities were detected in MKC after cotransfection with STING expression plasmid, pRL-TK Renilla luciferase plasmid, luciferase reporters, circYthdc2, METTL3, METTL14, FTO, YTHDF1, YTHDF3. I Coimmunoprecipitation analysis of STING ubiquitination in MKC cotransfected with Myc-STING, Flag-circYthdc2 or m 6 A modification-related genes expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiquitin-K48 plasmids. All data represented the mean ± SE from three independent triplicated experiments. *, p < 0.05; **, p < 0.01

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Modification, Plasmid Preparation, Transfection, Western Blot, Over Expression, Luciferase, Cotransfection, Expressing, Ubiquitin Proteomics

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Ythdc2-170aa is highly conserved in structure and function in vertebrates. A CircYthdc2 exists in Miichthys miiuy , Nibea albiflora , Sciaenops ocellatus , Larimichthys ocellatus , Xenopus tropicalis (GenBank accession no. XM_031893156.1), Bufo gargarizans (GenBank accession no. XM_044275926.1), Podarcis muralis (GenBank accession no. XM_028748342.1), Numida meleagris (GenBank accession no. XM_021380759.1), Mus musculus (GenBank accession no. NM_001163013.1), Homo sapiens (GenBank accession no. NM_022828.5), and is composed of exon 13 to exon 18, with a length of 634nt. We confirmed the head-to-tail splicing of hsa -circYthdc2 in the hsa -circYthdc2 RT-PCR product by Sanger sequencing. B Sequence alignment of circYthdc2 from teleost fish to mammals. C Amino acid sequence alignment of circYthdc2 translated polypeptides from teleost fish to mammals. D HEK293 cells were transfected with vector or hsa -Flag-circYthdc2, hsa -Flag-circYthdc2-ATG-mut, hsa -Flag-circYthdc2-m 6 A-mut plasmids, after 48 h, the immunoblot analysis was performed. E HEK293 cells were transfected STING and hsa -Flag-circYthdc2 and hsa -Flag-circYthdc2-m 6 A-mut with METTL3 or METTL14 or YTHDF1 or FTO plasmids, after 48 h, the immunoblot analysis was performed. F HEK293 cells were transfected STING and hsa -Flag-circYthdc2 with si- has -METTL3 or si- has -METTL14 or si- has -YTHDF1 or si- has -FTO, after 48 h, the immunoblot analysis was performed. All data represented the three independent triplicated experiments

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Plasmid Preparation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape

doi: 10.1007/s00018-024-05148-9

Figure Lengend Snippet: Schematic diagram of the mechanism underlying Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING. Under normal circumstances, circYthdc2 does not translate to produce polypeptides. When SCRV virus infects the host, the pathway of circYthdc2 translating polypeptides is activated. There are two pathways for circYthdc2 to be translated into polypeptides, the one is IRES-mediated translation pathway and another m 6 A modification mediated translation pathway. In addition, Ythdc2 will preferentially promote the RNA degradation of circYthdc2 when circYthdc2 is produced in large quantities. Ythdc2-170aa and Ythdc2 both could promote the STING protein degradation and represses STING-mediated antiviral responses, thereby regulating viral replication. Ythdc2-170aa and Ythdc2 both promoted K11 and K48-linked ubiquitination of STING, thereby inhibited the antiviral responses and help the virus escape

Article Snippet: The antibody against STING was diluted at 1: 500 (Abcam); The antibody against Ythdc2 was diluted at 1: 500 (Abcam); the antibody against Ythdc2-170aa was diluted at 1: 200 (GenScript); anti-Flag, anti-HA, anti-Myc, and anti-Tubulin monoclonal antibody were diluted at 1: 2,000 (Sigma); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000.

Techniques: Ubiquitin Proteomics, Virus, Modification, Produced