## 2 Results and Discussion
### Results
#### 2.1.2. \(Dotil\) Knockdown Affects Male-Specific Splicing of _Imp_ Pre-mRNA  
The above results indicate that \(DOTIL\) knockdown led to the loss of male-specific _Imp_ expression in males. Two possible explanations may account for this phenomenon: \(DOTIL\) knockdown repressed _Imp_ transcription or downregulation of \(DOTIL\) inhibited the splicing between exons 7 and 8 in _Imp_ pre-mRNA. To examine these possibilities, we performed comparative analyses of \(\textit{Imp}^{M}\) and the _Imp_ transcript common to both sexes (\(\textit{Imp}^{C}\)). RT-PCR analyses using primers described in Figure 2A demonstrated that \(DOTIL\) knockdown had little or no effect on the \(\textit{Imp}^{C}\) mRNA expression (Figure 2B). In contrast, \(\textit{Imp}^{M}\) transcript was not detected in a male with the \(DOTIL\) expressed to \(<\)10% of that in a  
Figure 1: The effect of histone methyltransferase (HMTase) knockdown on sex-specific splicing of _Imp_. (**A**) Quantification of HMTase mRNA expression 4 days after siRNA injections using quantitative reverse transcription (qRT)-PCR. _Elongation factor 2_ (_EF-2_) served as an internal standard. Error bar: SD; \(n=8\)–24 individuals. **\(p<0.01\), Student’s \(t\)-test; (**B**) A schematic diagram of alternative splicing in _Imp_ pre-mRNA. Exons are numbered and displayed as boxes. The gray box indicates the male-specific exon. The V-shaped lines above and below the diagram denote the splice variants observed in males and females. _Imp_ contains two poly(A) sites. The proximal promoter site located within intron 7 is utilized in a non-sex-specific manner. The distal promoter site is selected in a male-specific manner and exists near the end of exon 8. The arrows indicate the approximate location of primers used for RT-PCR in **C**; **(C)** The male-specific _Imp_ mRNA (\(\textit{Imp}^{M}\)) was detected by RT-PCR and analyzed in a 1% agarose gel. The upper panel depicts expression of \(\textit{Imp}^{M}\), and the lower panel shows the amplification of the _Actin3_ transcript, which served as a positive control for RNA extraction and RT-PCR. Sex identification of each egg was performed by PCR amplification of the W-specific random amplified polymorphic DNA (RAPD) marker _Rikishi_. The expression levels of targeted HMTases relative to the negative control embryos in each individual examined are indicated below each lane. control male (Figure 2B, lane 2). Next, using qRT-PCR, \(\mathit{Imp}^{M}\) mRNA expression was compared with that of the \(\mathit{Imp}^{C}\) mRNA. As shown in Figure 2C, extremely low expression of \(\mathit{Imp}^{M}\) mRNAs were observed in all males with \(\mathit{DOT1L}\) levels \(<\)10% of those in control males relative to \(\mathit{Imp}^{C}\) mRNA. These results indicate that \(\mathit{DOT1L}\) knockdown exclusively decreased the expression of the \(\mathit{Imp}^{M}\) transcript. To rule out the possibility that down-regulation of \(\mathit{DOT1L}\) by RNAi affects \(\mathit{Imp}\) RNA transcription, qRT-PCR was performed to measure the \(\mathit{Imp}^{C}\) mRNA level that represents total \(\mathit{Imp}\) mRNA expression. As shown in Figure 2D, the \(\mathit{Imp}^{C}\) mRNA level in \(\mathit{DOT1L}\) knockdown males with \(\mathit{DOT1L}\) expression \(<\)10% of that in control males was decreased to a level less than one-third of that in control males (Figure 2D, left panel). In contrast, a more prominent reduction in \(\mathit{Imp}^{M}\) mRNA was observed in the same \(\mathit{DOT1L}\) knockdown males (Figure 2D, right panel). These results indicate that \(\mathit{DOT1L}\) knockdown affects \(\mathit{Imp}\) RNA transcription but the male-specific splicing of \(\mathit{Imp}\) is repressed more severely by DOT1 depletion, leading to the loss of the \(\mathit{Imp}^{M}\) expression. These results highlight the link between DOT1L and male-specific alternative splicing of \(\mathit{Imp}\) pre-mRNA.