Fig 2: m6A binding is dispensable for YTHDC2 function. (A) Multiple sequence alignment obtained using Clustal Omega (Sievers et al. 2011) 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 m6A-binding residues. (B) Fluorescence anisotropy binding curves for the interaction of the mouse YTHDC2 YTH domain (YTHWT) or the YTH domain carrying m6A-binding pocket mutations W1375A and L1380A (YTHW1375A/L1380A) with m6A-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 (Kd). (C) Bar graph showing Kd (mean ± SD) obtained from B. (D) The YTH domain (gray highlight) of the CRISPR/Cas9-induced Ythdc2WLA 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) Ythdc2WLA/WLA and control littermates. The number of seminiferous tubules counted from each animal is indicated (n). Bars indicate mean and SD.

Fig 3: YTHDC2 domain architecture and structure of its YTH domain.(A) Schematic of mouse YTHDC2 domain structure (not to scale). Sequence motifs characteristic of superfamily 2 DExH-box helicases (I, Ia, II, III, IV, V, VI) within the helicase core domain are indicated, along with sequence logos from Clustal Omega alignments of 157 superfamily 2 DExH-box helicases. The height of each stack of residues reflects the degree of conservation, and the height of each amino acid symbol within each stack is proportional to the frequency of a residue at that position. Amino acids are colored according to their physico-chemical properties (hydrophilic (blue), neutral (green), and hydrophobic (black)). (B) Clustal Omega alignments of sequences around helicase motifs I and II from YTHDC2 proteins of the indicated species. The position of the ketu mutation is indicated. The residues are shaded based on percent agreement with the consensus. Sequence logos were generated from Clustal Omega alignments of YTHDC2 homologs from 201 species and are colored as in panel A. (C) Cladogram of Clustal Omega protein sequence alignments of mouse and human YTHDC2 paralogs. The tree was rooted using vaccinia virus NPH-II sequence and D. melanogaster Bgcn is included for comparison. DHX29 is an RNA helicase that promotes translation initiation of mRNAs with structured 5’ untranslated regions (Pisareva et al., 2008). DHX57 is an uncharacterized protein of unknown function. DHX36 has G-quadruplex unwinding activity for DNA and RNA and is involved in antiviral responses to dsRNA (Vaughn et al., 2005; Yoo et al., 2014). DHX9 (also known as RNA helicase A and DNA helicase II) has both DNA and RNA helicase activities and multiple cellular functions, including in genome stability and in viral and cellular RNA metabolism (Friedemann et al., 2005; Lin et al., 2012). DHX30 is a poorly characterized protein required for cell viability during mouse embryonic development (Zheng et al., 2015). And TDRD9 forms a complex with MIWI2 involved in piRNA-directed transposon silencing in the male germline (Shoji et al., 2009; Wenda et al., 2017). (D) Clustal Omega alignment of YTH domain sequences. Inverted triangles in auburn indicate residues that make up the hydrophobic pocket that binds m6A. Residues boxed in auburn are required for Mmi1 interaction with RNA (Wang et al., 2016). (E) NMR structure of the YTH domain from human YTHDC2, generated by the RIKEN Structural Genomics/Proteomics Initiative (Endo et al., 2007) (PDB ID: 2YU6). (F) Structure of the m6A binding pocket is conserved in YTHDC2. The solution structure of YTH domain from human YTHDC2 (green) is shown superimposed on the crystal structure of the RNA-bound YTH domain from human YTHDC1 (pink; PDB ID: 4R3I) (Xu et al., 2014). The m6A nucleotide and the hydrophobic amino acid residues lining the binding pocket are shown at a higher magnification on the right. Protein accession numbers for sequence logos in panel A are in Supplementary file 2; all other protein accession numbers are in Supplementary file 3.

Fig 4: YTHDC2 localization in wild-type and mutant testes.(A and B) YTHDC2 and SYCP3 immunofluorescence on testis sections from 2-month-old Ythdc2em1/em1, Ythdc2ketu/ketu, and their wild-type littermates. Arrowheads indicate SYCP3-positive spermatocytes. Scale bars represent 100 μm. (C) YTHDC2 and SYCP3 immunofluorescence on testis sections from an adult B6 male. Approximate seminiferous epithelial cycle stages (based on SYCP3 and DAPI staining patterns) are provided. S, Sertoli cell; Spg, spermatogonia; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diplotene spermatocyte; RS, round spermatid; ES, elongating spermatid. Scale bar represents 15 μm.

Fig 5: ketu mice harbor a point mutation in Ythdc2.(A) SNP genotypes of five G3 ketu mutants (a, b, c, d, e; from Figure 1C) obtained using the Illumina Medium Density Linkage Panel. The single 30.59-Mbp region of B6 SNP homozygosity that is shared between mutants is highlighted in pink. (B) Top: Schematic of Ythdc2 (as predicted by Ensembl release 89) showing the locations of the ENU-induced lesion and the gRNA used for CRISPR/Cas9-targeting. Bottom: The density of ENCODE long RNA-sequencing reads (release 3) from adult testis within a window spanning from 3500 bp upstream to 200 bp downstream of Ythdc2. The vertical viewing range is 0–50; read densities exceeding this range are overlined in pink. (C) The ketu and CRISPR/Cas9-induced (em1) alleles of Ythdc2. (D) Ythdc2 and Meioc expression level estimate (mean reads per kilobase per million mapped reads (RPKM) values provided by ENCODE; included in Figure 2—source data 1) in adult and embryonic tissues.10.7554/eLife.30919.005Figure 2—source data 1.Ythdc2 and Meioc RPKM values plotted in Figure 2D.