Fig. 1

Figure 1. YTHDC2 binds to the 3′ UTR of protein-coding genes through a U-rich motif (A) Cartoon showing domain organization of mouse YTHDC2 protein. The 3xFLAG tag sequence was inserted in the mouse Ythdc2 gene locus between the OB-fold and the YTH domain. Immunofluorescence detection with anti-FLAG (green) and anti-SYCP3 (red) antibodies in adult (P60) testes sections of the Ythdc2FLAG knockin mice. Scale bar in μm is indicated. (B) Testes from Ythdc2FLAG mice (P15) were used to perform iCLIP experiment to identify the YTHDC2-bound RNAs. Binding sites were identified as loci having a pile-up of reads with 3′ ends terminating at the potential crosslinking site or by reads crossing it but having an increased rate of indels. Pie charts showing proportion of YTHDC2 binding sites in different RNA biotypes, and the location of the YTHDC2 binding sites over different RNA features. The coverage of iCLIP reads over a representative target is shown. Notice the enrichment on the 3′ UTR. (C) Heatmap showing z-scores of the iCLIP read coverage per binned YTHDC2-bound transcripts. See also Table S2. (D) Metaplot showing an average iCLIP read coverage over YTHDC2 target transcripts. (E) Scatterplot and the correlation analysis of the number of YTHDC2 binding sites against 3′ UTR length. (F) Sequence logo showing a U-rich motif centered on the YTHDC2 binding site (BS) in a 5-mer window. Scatterplot comparing the 3′ UTRs and CDS frequency of all the pentamers centered on the YTHDC2 crosslink site (mostly a uridine). Top seven 5-mers found at the crosslink site are highlighted. See also Figure S1G. (G) Histogram showing a distribution of the total number of binding sites per transcript. (H) Gene ontology analysis of YTHDC2 targets. Only terms with the adjusted p value < 0.5 were plotted. (I) Violin plots showing the distribution of expression counts (TPM + 0.0001) of all genes (green) in spermatocytes from P15 wild-type mouse testes, next to the distribution of expression counts (TPM + 0.0001) of YTHDC2 targets (orange). Three biological replicates are shown. Although transcripts bound by YTHDC2 have a broad expression profile, they show a slightly higher expression. (J) Distribution of putative m6A methylation sites (DRACH) and a U-rich sequence (UUUU) in a 200-nt window flanking the YTHDC2 binding site (position 0). See also Figures S1E and S1F. (K) A modeled m6A nucleotide in the hydrophobic pocket of the YTH domain of human YTHDC2 from (Wojtas et al., 2017). Key residues required for m6A recognition are indicated. Mutation of W1360 is shown to abolish m6A binding. The corresponding W1375 in the Ythdc2YTH was mutated to assess physiological relevance of the YTH domain in vivo. See also Figure S1H. (L) Eosin and hematoxylin-stained adult testis and ovary sections from heterozygous and homozygous Ythdc2YTH knockin mutant mice. No difference was observed. Scale bar in μm is indicated. (M) Ribo-depleted RNA-seq (in biological triplicates) analysis of FACS-purified 2n (spermatogonia) and 4n (spermatocytes) cells from mouse testes of heterozygous and homozygous Ythdc2YTH knockin mutant mice. Volcano plots show the differential gene expression between the genotypes. Genes showing significant differential expression (padj < 0.05 and log2FC > 1.5 or log2FC < −1.5) are marked in blue. Significantly dysregulated pseudogenes are masked.