P-element Somatic Inhibitor Protein Binding a Target Sequence in dsx Pre-mRNA Conserved in Bombyx mori and Spodoptera litura
State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Beibei, Chongqing 400715, China
Chongqing Key Laboratory of Sericultural Science, Southwest University, Chongqing 400715, China
Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing 400715, China
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(9), 2361; https://doi.org/10.3390/ijms20092361
Received: 15 April 2019 / Revised: 10 May 2019 / Accepted: 10 May 2019 / Published: 13 May 2019
(This article belongs to the Special Issue Molecular Ecology, Physiology and Biochemistry of Insects)
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.