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: Genes & Development

Article Title: YTHDC2 control of gametogenesis requires helicase activity but not m 6 A binding

doi: 10.1101/gad.349190.121

Figure Lengend Snippet: m 6 A binding is dispensable for YTHDC2 function. ( A ) Multiple sequence alignment obtained using Clustal Omega of YTH domains (gray highlight) from mouse YTHDC2 (Uniprot B2RR83) and all five human YTH domain-containing proteins: YTHDC2 (Uniprot Q9H6S0), YTHDC1 (Uniprot Q96MU7), YTHDF1 (Uniprot Q9BYJ9), YTHDF2 (Uniprot Q9Y5A9), and YTHDF3 (Uniprot Q7Z739). Pink boxes indicate positions of m 6 A-binding residues. ( B ) Fluorescence anisotropy binding curves for the interaction of the mouse YTHDC2 YTH domain (YTH WT ) or the YTH domain carrying m 6 A-binding pocket mutations W1375A and L1380A (YTH W1375A/L1380A ) with m 6 A-modified or nonmodified RNA. Data points (arbitrary units [a.u.]) represent mean ± SD from three technical replicates and are fitted with a one site total binding equation (GraphPad Prism 9) to obtain the dissociation constant ( K d ). ( C ) Bar graph showing K d (mean ± SD) obtained from B . ( D ) The YTH domain (gray highlight) of the CRISPR/Cas9-induced Ythdc2 WLA allele with nucleotide and amino acid changes indicated. ( E ) Ratios of testis weight to body weight for 10- to 24-wk-old mice. ( F ) Bouin's fixed and periodic acid Schiff (PAS)-stained seminiferous tubule sections from adult (14 wk) testes are shown. (Se) Sertoli cells, (Sg) spermatogonia, (Sc) spermatocytes, (rSt) round spermatids, (eSt) elongated spermatids. ( G ) Adult (14-wk) testis sections stained with TUNEL and hematoxylin. ( H ) Quantification of TUNEL staining in three littermate pairs of adult (10- and 14-wk) Ythdc2 WLA/WLA and control littermates. The number of seminiferous tubules counted from each animal is indicated ( n ). Bars indicate mean and SD.

Article Snippet: We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920).

Techniques: Binding Assay, Sequencing, Fluorescence, Modification, CRISPR, Staining, TUNEL Assay

Journal: Genes & Development

Article Title: YTHDC2 control of gametogenesis requires helicase activity but not m 6 A binding

doi: 10.1101/gad.349190.121

Figure Lengend Snippet: Optimized YTHDC2 CLIP in juvenile and adult testes. ( A ) Autoradiograph images of YTHDC2 CLIP samples labeled with T4 polynucleotide kinase and γ- 32 P ATP using normal and optimized conditions. Frozen tissue powder from 8-dpp, 10-dpp, and adult (8.5-wk) testes was subjected to UV cross-linking (XL) and either high RNase (+++) or low RNase (+) digestion conditions prior to IP of YTHDC2. RNA fragments were subsequently used for cDNA library preparation and analysis. ( B ) Biotype of RNA with YTHDC2 CLIP peaks. ( C ) Distribution of YTHDC2 CLIP peak locations across genic regions. ( D ) Metatranscript coverage profiles of YTHDC2 CLIP tags on protein-coding transcripts. The Y -axis indicates the percentage of YTHDC2 CLIP tags mapped at the indicated transcript positions (in nucleotides [nt]) when anchored to translation start ( left ), translation stop ( middle ), or transcript end ( right ). Unique CLIP tags obtained after collapsing PCR duplicates were used for analysis. ( E ) YTHDC2-binding motifs enriched in cross-linking-induced truncation sites (CITSs) by MEME-ChIP motif analysis . Fifty-nucleotide-long sequences flanking CITSs ( P < 0.001) on both 5′ and 3′ ends were used for motif analysis. Significant motifs ( E -value ≤ 0.05) were clustered by similarity and ordered by E -value using MEME-ChIP. Shown are the three most significant motifs from the most significant cluster ( left panel; E -value ≤ 7.2 × 10 −1799 ) and the next most significant cluster ( right panel; E -value ≤ 1.2 × 10 −466 ). ( F ) Overlap of top CLIP transcripts between mice of different ages. ( G ) Functional clusters of GO terms ( P < 0.05) enriched in the top YTHDC2 CLIP targets. P -values are indicated by bar colors and are listed in or adjacent to bars.

Article Snippet: We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920).

Techniques: Autoradiography, Labeling, cDNA Library Assay, Binding Assay, Functional Assay

Journal: Genes & Development

Article Title: YTHDC2 control of gametogenesis requires helicase activity but not m 6 A binding

doi: 10.1101/gad.349190.121

Figure Lengend Snippet: YTHDC2 binds distinct transcripts at distinct positions relative to the m 6 A mark. ( A ) Venn diagrams showing overlap between the top YTHDC2 CLIP targets and transcripts containing m 6 A in testes. Transcripts containing the top 250 m 6 A peaks identified by reanalysis of raw data from are shown in dark pink, and the list of the top 500 m 6 A-containing transcripts reported by are shown in light pink. CLIP data from 8 dpp, 10 dpp, and adults were compared with m 6 A data from 8-, 12-, and 20-dpp testes, respectively. ( B ) Metatranscript coverage profiles of YTHDC2 CLIP unique tags and reads from m 6 A IPs on protein-coding transcripts. The Y -axis indicates the percentage of total CLIP tags or IP reads mapped within 1 kb of the translation start sites ( left ), translation stop sites ( middle ), or the ends of transcripts ( right ). ( C ) Bar graph showing the distribution of distances between m 6 A peaks and YTHDC2 CLIP peaks when present on the same transcript. The Y -axis shows the percentage of total m 6 A peaks that are within a defined distance from a YTHDC2 CLIP peak ( X -axis). (nt) Nucleotides. ( D ) Proportion of YTHDC2 CITSs containing either the m 6 A consensus motif GGACU or UKU (where K is U or G) within 5 or 10 nt. ( E ) Immunoblot analysis of YTHDC2 protein levels in testis extracts from adult (20- and 24-wk) Ythdc2 WLA/WLA and wild-type animals. Immunoblot for β-actin served as a loading control. ( F ) Quantification of YTHDC2 protein levels in E . Mean values from three mice per genotype are plotted. SD are indicated, and P -value is listed (Student's t -test). ( G ) Gene expression (reads per thousand mapped [RPKM]) in testes from adult (20- and 24-wk) Ythdc2 WLA/WLA and Ythdc2 +/WLA littermates. Three mice per genotype were analyzed. Gray dots represent transcripts with comparable values between Ythdc2 +/WLA and Ythdc2 WLA/WLA . Purple stars denote transcripts with significantly altered expression in Ythdc2 WLA/WLA compared with Ythdc2 +/WLA (fold change ≥ 1.2 and adjusted P -value < 0.05). Green dots show the top YTHDC2 CLIP targets in adults. ( H , I ) Comparison of YTHDC2 CLIP between Ythdc2 +/+ and Ythdc2 WLA/WLA testes from adult animals (20 and 24 wk). Scatter plots show the correlation of total YTHDC2 CLIP unique tag numbers (obtained by independent CLIP assays in three animals per genotype) on individual transcripts ( H ) or CLIP peaks ( I ). A pseudocount of one was added for calculating log 2 scores used in the X - and Y -axes. Each black dot represents a transcript ( H ) or a YTHDC2 CLIP peak ( I ). Each green dot in I represents a top ranked CLIP peak (peak height ≥ 16) in both Ythdc2 +/+ and Ythdc2 WLA/WLA .

Article Snippet: We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920).

Techniques: Western Blot, Expressing

Journal: Genes & Development

Article Title: YTHDC2 control of gametogenesis requires helicase activity but not m 6 A binding

doi: 10.1101/gad.349190.121

Figure Lengend Snippet: Molecular effects of complete YTHDC2 loss on direct RNA targets during meiotic entry. ( A ) Scatter plots showing log 2 fold changes for the mRNA input and ribosome protected fragments (RPFs) as analyzed by anota2seq in whole testes from Ythdc2 knockouts compared with wild-type or heterozygous littermates at 8 and 10 dpp. Three pairs of knockout and wild-type or heterozygous littermates were examined for each age. The default anota2seq parameter of translational efficiency (TE; i.e. RPF/input) fold change ≥ 1.2 (threshold denoted by solid black lines) was applied. Transcripts meeting this criterion are shown in light pink. Transcripts that met this criterion and also had an adjusted P -value < 0.1 were considered to have significantly different TEs and are shown in dark pink. ( B ) Venn diagrams illustrating the almost complete lack of overlap of top YTHDC2 CLIP targets with transcripts that show TE fold change ≥ 1.2 (irrespective of P -value; light-pink transcripts in A ) at 8 and 10 dpp. ( C ) Comparison of the change in TE in Ythdc2 knockout at the indicated ages for nontargets and the top 250 (based on CLIP peak height) YTHDC2 targets, categorized according to the location of CLIP peaks. ( D ) Change in steady-state mRNA levels of the top 250 (based on CLIP peak height) YTHDC2 targets in Ythdc2 knockout compared with wild type at 8 and 10 dpp. The results of Mann–Whitney tests are shown in C and D . (ns) Not significant ( P > 0.05), (***) P < 0.001.

Article Snippet: We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920).

Techniques: Knock-Out, MANN-WHITNEY

Journal: Genes & Development

Article Title: YTHDC2 control of gametogenesis requires helicase activity but not m 6 A binding

doi: 10.1101/gad.349190.121

Figure Lengend Snippet: YTHDC2 ATPase mutation disrupts meiotic progression. ( A ) Schematic of mouse YTHDC2 domain structure ( top ) and sequences around motif II (gray highlight) within the helicase core domain ( bottom ) are shown. The amino acid change (pink box) and corresponding nucleotide changes in motif II of the TALEN-induced Ythdc2 EQ allele are indicated. ( B ) Testis weight to body weight ratios for 5- to 36-wk-old mice. ( C ) Bouin's fixed and PAS-stained seminiferous tubule sections from 9-wk-old animals harboring Ythdc2 EQ and Ythdc2 knockout ( Ythdc2 − ) alleles and from wild-type littermates. (Sc) Spermatocytes, (St) spermatids, (A) cells with compacted apoptotic-like nuclei, (red arrow) tubules with only a single layer of cells (spermatogonia plus Sertoli cells). ( D ) Quantification of the proportion of seminiferous tubule types in testes from Ythdc2 +/EQ , Ythdc2 −/EQ , and wild-type littermates of the indicated ages. Normal tubule types are categorized based on the meiotic prophase I stage of cell layers present within them as judged by SYCP3 and γH2AX staining. Cell layers are as follows: empty with no SYCP3 staining (E), leptonema or zygonema (LZ), pachynema (P), pachynema or diplonema (PD), round spermatids (RS), and elongating spermatids (ES). Abnormal tubule types in Ythdc2 +/EQ and Ythdc2 −/EQ that contained cells with fragmented and intense SYCP3 staining indicative of dying cells were also quantified. ( E ) Example images of tubule categories quantified in D taken from 24-dpp mice. Arrowheads indicate presumptive dying cells (based on SYCP3 staining). ( F ) Quantification of TUNEL staining (images in Supplemental Fig. S3A ) in Ythdc2 +/EQ , Ythdc2 −/EQ , and control littermates. Wild-type animals with 0% tubules containing ≥10 TUNEL-positive cells are indicated with an asterisk. Each lane represents one animal, and the number of seminiferous tubules counted is indicated ( n ). ( G ) Quantification of pH3 staining in seminiferous tubules from Ythdc2 +/EQ , Ythdc2 −/EQ , and control littermates of the indicated ages. pH3-positive cells were categorized based on their location within the tubule (luminal or peripheral). The number of seminiferous tubules counted is indicated ( n ), and bars show mean and SD for the distribution of pH3-positive cells that are luminally located. ( H ) Example images from 24-dpp mice of tubules quantified in G . Arrowheads point to pH3-positive cells that were categorized as being luminally located ( top ) or located near the periphery ( bottom ). ( Top right ) A rare tubule type containing one luminally-located pH3-positive cell is shown for Ythdc2 +/EQ . In D , F , and G , adult Ythdc2 +/EQ animals were 5 and 14 wk old, and Ythdc2 −/EQ animals were 8–9 wk old.

Article Snippet: We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920).

Techniques: Mutagenesis, Staining, Knock-Out, TUNEL Assay