Figure 9: MEIOC/YTHDC2 complex binds meiotic mRNA.
(a) Western blot analysis of co-IP of MEIOC and YTHDC2 in postnatal testes. (b) MEIOC coiled-coil domain interacts with YTHDC2. Upper panel: schematic representation of FLAG-tagged MEIOC complete protein and deletion mutants overexpressed in HEK-293 cells. Numbers in square brackets indicate the deleted amino acids, which are also represented with dotted lines. Blue boxes indicate the coiled-coil domain. Lower panel: co-IP was performed with anti-FLAG antibody, and samples were subjected to western blotting with anti-FLAG and anti-YTHDC2 antibodies. Deletion of the N terminus (ΔN1 and ΔN2) of MEIOC did not abolish the interaction with YTHDC2, whereas deletion of the C terminus of MEIOC (ΔC) or the second helix of the coiled-coil domain (ΔH2) abolished this interaction (c). Confocal acquisitions of MEIOC (red), YTHDC2 (green) and DAPI (blue) staining in spermatocytes. Scale bar, 10 μm. (d) Meioc+/+ and Meioc−/− 8- and 16-d.p.p. testis sections were stained for YTHDC2 (green), SYCP3 (red) and DAPI (blue). Scale bar, 40 μm. (e) Western blot analysis of YTHDC2 in mouse testis protein extracts at the indicated postnatal ages (days post-partum). β-actin/ACTB was used as a control. Ad, adult. (f) RT–qPCR analysis of mRNA bound after IP assays using anti-YTHDC2 antibody (YTH) or IgG in testicular protein extracts. Statistical analysis compares mRNA fold enrichment/input levels with that of Gapdh. Mean±s.e.m.; n=6. **P<0.01, ***P<0.001 (Student’s t-test).
