## 2 Results and Discussion
### Discussion  
Imp in _B. mori_ has been identified as a male-specific RNA-binding protein involved in the regulation of male-specific splicing of _Bmdsx_[27]. The pre-mRNA of _Imp_ undergoes sex-specific \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**siRNA** & **Injected** & **Early or mid-stage** & **Late-stage** & \\  & **eggs** & **embryonic lethal** & **embryonic lethal** & \\ \hline N. C. siRNA & 115 & 58 (50.4\%) & 32 (27.8\%) & 25 (21.7\%) \\ DOT1L siRNA & 263 & 188 (71.5\%) & 74 (28.1\%) & 1 (0.4\%) (male) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Effects of DOTIL siRNA injection on egg development. RNA processing. In male cells, male-specific _Imp_ mRNA contains exon 8 and the distal promoter poly(A) site choice, whereas non-sex-specific polyadenylation occurs at the proximal promoter poly(A) site downstream of exon 7. Here, we found that _DOT1L_ knockdown affects male-specific splicing of _Imp_ pre-mRNA, leading to loss of male-specific _Imp_ expression (Figure 2B,C). In support of this result, higher distributions of H3K79 me2 were observed in males than in females or in _DOT1L_ knockdown males across _Imp_ (Figure 3B,C). Strong link between enrichment of H3K79 me2 and DOT1L-dependent inclusion of alternative exons was also observed in _Bm-Sxl_ (Figure 4). Comparative analysis of RNAP II processivity indicated that higher distribution of H3K79 me2 across _Imp_ was correlated to increased RNAP II processivity at this locus (Figure 5B). Inhibition experiments using inhibitors of RNAP II elongation suggested that the higher elongation rate was tightly associated with male-specific RNA processing of _Imp_ pre-mRNA (Figure 5D). Together, our data suggest that H3K79 me2 accumulation along _Imp_ is associated with male-specific alternative RNA processing in _Imp_ mRNA production, resulting from increased RNAP II processivity. The alternative RNA processing pattern of _Imp_ pre-mRNA closely resembles that found in _Drosophila polo_ pre-mRNA. _Polo_, which is a cell cycle gene, also contains a proximal and a distal poly(A) site in the 3'untranslated region (UTR) to produce alternative mRNA that differ in their 3'UTR length [45]. In a mutant _Drosophila_ strain that displays a reduced RNAP II elongation rate, RNAP II occupancy along _polo_ is altered and the proximal poly(A) site is used 3.5-fold more efficiently than in wild-type flies [46]. An increase in proximal poly(A) site usage was also observed in five other alternatively polyadenylated transcripts in _Drosophila_. These results in _Drosophila_ show that the kinetics of RNAP II can determine alternative poly(A) site selection. As shown in Figure 5D, significant reduction in the _Imp\({}^{M}\)/Imp\({}^{C}\)_ ratio was observed when the male cells were treated with inhibitors of RNAP II elongation. This result indicates that drug-induced repression of transcript elongation leads to the reduction in utilization of distal poly(A) site, resulting in the relative increase in proximal poly(A) site usage. Slow RNAP II presumably exposes the proximal poly(A) site on the nascent transcript to the polyadenylation machinery for a longer time before RNAP II transcribes the distal poly(A) site [46]. Therefore, the proximal poly(A) site is processed before the distal poly(A) site is transcribed, suggests a \"first come, first served\" mechanism. This resembles the extra domain I (EDI) alternative splicing mechanism described previously, whereby a slow RNAP II preferentially included the normally excluded alternative EDI exon because it allowed ample assembly time for the spliceosome machinery [36,47]. Eleven-nineteen lysine-rich leukemia gene (ELL) family proteins are essential components of the super elongation complex (SEC) and increase the catalytic rate of transcription elongation by RNA polymerase II [48, 49, 50]. ELL2 knockdown by siRNA affects the alternative pre-mRNA processing of the immunoglobulin heavy chain (IgH) gene, which is accompanied by reduced H3K4 and H3K79 methylation [50].