Abstract
Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity.
Highlights
Alternative splicing of pre-mRNA is an essential mechanism in the regulation of differential gene expression that can produce functionally distinct proteins from a single gene based on the developmental or physiological state of the cells in a multicellular organism [1,2]
To investigate whether these epigenetic marks are associated with male-specific splicing of II mRNA-binding protein (Imp) pre-mRNA, we performed RNAi knockdown of several histone methyl transferases (HMTases) such as ASH2, EZH2, SETD2, and DOT1L known to modify H3K4, H3K27, H3K36, and H3K79, respectively, in embryos
Higher distributions of H3K79me2 were observed in males than in females across Imp in negative control individuals (Figure 3C, left panel), DOT1L knockdown exerted a marked influence on the accumulation of H3K79me2, with an at least 10-fold reduction throughout the regions examined in males (Figure 3C, right panel)
Summary
Alternative splicing of pre-mRNA is an essential mechanism in the regulation of differential gene expression that can produce functionally distinct proteins from a single gene based on the developmental or physiological state of the cells in a multicellular organism [1,2]. Recent studies estimate that 90% of human genes are alternatively spliced [3], and several thousand different mRNA isoforms can be produced from a single gene. Many examples describe how alternative splicing regulates gene expression, the mechanisms involved are less well understood [4,5,6,7,8]. Recent provocative studies point to a key function of chromatin structure and histone modification in alternative splicing regulation [9,10,11]. A genome-wide study across different species revealed that
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