Figure 6. Bcas2 is involved in pre-mRNA splicing of functional genes during spermatogenesis.

(a) RNA-seq results of alternative sites in genes related to spermatogenesis using IGV software. Red arrowheads indicate splicing sites. (b) RT-PCR analysis of alterative splicing patterns of the changed splicing genes in control and Bcas2^F/−;Vasa-Cre testes of P9 mice with Gapdh as the internal control. RT-PCR was performed with specific primers (Supplementary Table 6) in three independent experiments. (c) Real-time RT-RCR verified the changed splicing genes with Hprt as the internal control. Dazl-FL and Ehmt2-FL represent the full-length isoform and Dazl-Δ8 and Ehmt2-Δ10 denote the short form that lacked exon 8 and exon 10, respectively. An alternative first exon of Hmga1 was shown by Hmga1-E1. Dazl+7 primers specifically recognized intron 7 of Dazl. Dazl, Ehmt2 and Hmga1 were detected using primers at the common region (except for the splicing site) of both isoforms (*P<0.05; **P<0.01; ***P<0.001, n>5). Error bars represent s.e.m. (d) Western blotting analysis of the expression of two isoforms of DAZL in control and Bcas2^F/−;Vasa-Cre testes of P9 and P12 mice with GAPDH as the loading control. (e and f) Relative abundances of DAZL-FL (e) and DAZL-total (f) in control and Bcas2^F/−;Vasa-Cre testes from P9 and P12 mice were determined by Western blotting analyses of four independent experiments. Error bars represent s.e.m.