Spliceosomal Protein Gene BmSPX Regulates Reproductive Organ Development in Bombyx mori.

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori. In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes (BmMasc and BmIMP) and reduced the female-type splice isoform of Bmdsx, which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx. Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B (Adb-B), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.

P-element Somatic Inhibitor Protein Binding a Target Sequence in dsx Pre-mRNA Conserved in Bombyx mori and Spodoptera litura.

Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.

Long Non-Coding RNA Bmdsx-AS1 Effects on Male External Genital Development in Silkworm.

Long non-coding RNAs (lncRNAs) have been suggested to play important roles in some biological processes. However, the detailed mechanisms are not fully understood. We previously identified an antisense lncRNA, Bmdsx-AS1, that is involved in pre-mRNA splicing of the sex-determining gene Bmdsx in the silkworm. In this study, we analyzed the changes in the male external genitalia of transgenic overexpressed Bmdsx-AS1 silkworm lines and analyzed downstream and upstream responses. We found that Bmdsx-AS1 transgenic silkworms, compared with wild type, showed more claspers in the male external genitalia. Quantitative real-time PCR (qPCR) results indicated that overexpression of Bmdsx-AS1 decreased the expression of genes in the EGFR signaling pathway. Knockdown of Bmdsx-AS1 increased the activity of the EGFR pathway. Through promoter prediction, promoter truncation and electrophoretic mobility shift assay (EMSA) analyses, we found that the protein encoded by the Hox gene BmAbd-B specifically binds to the promoter of Bmdsx-AS1. Moreover, overexpression of BmAbd-B in the silkworm BmE cell line indicated that BmAbd-B negatively regulates the mRNA expression of Bmdsx-AS1. Our study provides insights into the regulatory mechanism of the lncRNA in the silkworm.

A Novel Splice Variant of the Masculinizing Gene Masc with piRNA-Cleavage-Site Defect Functions in Female External Genital Development in the Silkworm, Bombyx mori.

In the silkworm, the sex-determination primary signal Fem controls sex differentiation by specific binding of Fem-derived piRNA to the cleavage site in Masc mRNA, thus inhibiting Masc protein production in the female. In this study, we identified a novel splicing isoform of Masc, named Masc-S, which lacks the intact sequence of the cleavage site, encoding a C-terminal truncated protein. Results of RT-PCR showed that Masc-S was expressed in both sexes. Over-expression of Masc-S and Masc in female-specific cell lines showed that Masc-S could be translated against the Fem-piRNA cut. By RNA-protein pull-down, LC/MS/MS, and EMSA, we identified a protein BmEXU that specifically binds to an exclusive RNA sequence in Masc compared to Masc-S. Knockdown of Masc-S resulted in abnormal morphology in female external genital and increased expression of the Hox gene Abd-B, which similarly occurred by Bmexu RNAi. These results suggest that the splice variant Masc-S against Fem-piRNA plays an important role in female external genital development, of which function is opposite to that of full-length Masc. Our study provides new insights into the regulatory mechanism of sex determination in the silkworm.