Effect of RNAi-mediated knockdown of the Bombyx mori transformer-2 gene on the sex-specific splicing of Bmdsx pre-mRNA

Abstract

In Drosophila melanogaster, transformer-2 (tra-2) is essential for female differentiation and is known to induce female-specific splicing of doublesex (dsx). The function of Bmtra-2, the Bombyx mori homolog of tra-2, on the other hand remains to be elucidated. As an initial step to learn about the biological function of Bmtra-2, we determined whether Bmtra-2 is capable of inducing the female-specific splicing of Drosophila dsx. RNAi-mediated knockdown of Bmtra-2 using Bombyx cultured cells transiently transfected with a dsx minigene revealed that Bmtra-2 can induce female-specific splicing of Drosophila dsx. To examine the role Bmtra-2 plays in regulating sex-specific splicing of Bmdsx pre-mRNA, we used an RNAi approach to reduce BmTra-2 expression in the early embryo. Embryos injected with dsRNAs or siRNAs targeted to Bmtra-2 showed no variation in the sex-specific splicing pattern of Bmdsx pre-mRNA. RNAi knockdown of Bmtra-2 in the early embryo caused abnormal testis formation. Taken together, these results indicate that Bmtra-2 is required for normal testis development, but is not involved in regulating the sex-specific splicing of Bmdsx pre-mRNA, even though it is capable of inducing the female-specific splicing of Drosophila dsx.

Lepidopteran Sex Determination: A Cascade of Surprises

“…Also, RNAi of Bmtra2 in Bombyx eggs does not affect female development but causes abnormal development of testis in males. Hence, Bmtra2 has a role in early testis morphogenesis, but it does not play a role in Bombyx sex determination [Suzuki et al, 2012].…”

“…Due to the selection of 3 different 3 ′ splice sites within the second intron and 2 different splice sites within the seventh intron, Bmtra2 pre-mRNA is alternatively spliced to produce 6 different isoforms. All 6 deduced BmTRA2 proteins contain 1 RNA recognition motif domain flanked by RS (Arg/Ser) domains, a typical characteristic feature of the Drosophila TRA2 protein [Suzuki et al, 2012]. RNAi-mediated knockdown of all Bmtra2 isoforms in B. mori cultured cell lines does not affect sex-specific splicing of a Bmdsx mini gene [Suzuki et al, 2012].…”

“…All 6 deduced BmTRA2 proteins contain 1 RNA recognition motif domain flanked by RS (Arg/Ser) domains, a typical characteristic feature of the Drosophila TRA2 protein [Suzuki et al, 2012]. RNAi-mediated knockdown of all Bmtra2 isoforms in B. mori cultured cell lines does not affect sex-specific splicing of a Bmdsx mini gene [Suzuki et al, 2012]. Also, RNAi of Bmtra2 in Bombyx eggs does not affect female development but causes abnormal development of testis in males.…”

Conserved Domains in the Transformer Protein Act Complementary to Regulate Sex-Specific Splicing of Its Own Pre-mRNA

“…Total RNA was extracted from 3 adult females and from BmN cells using ISOGEN (Nippon Gene) according to a protocol described previously [Suzuki et al, 2012]. Poly (A)+RNA was further purified from the total RNA extracted from BmN cells using a Magnosphere Ultra Pure mRNA Purification kit (TaKaRa) according to the manufacturer's instruction.…”

“…Bopp [2010] hypothesized that in Drosophila Sex-lethal has been added upstream of transformer and has taken over auto-regulation, making the TRA-CAM domain of transformer obsolete. To examine this hypothesis, we investigated the functional relationship between the TRA-CAM domain and the autoregulation of tra using an in vitro splicing assay system in cultured cells derived from the silkworm, Bombyx mori , which was originally developed in our previous study [Suzuki et al, 2012[Suzuki et al, , 2015. Our results demonstrated that TRA enriches on the male-specific exon (exon 2b) that contains putative TRA/TRA-2 binding sites (also called dsxRE) to achieve female-specific splicing of tra in the housefly as predicted by a previous study [Hediger et al, 2010].…”