The Masc-PSI complex directly induces male-type doublesex splicing in silkworms

Abstract

The WZ sex determination system is found in a diverse range of animals, including lepidopteran insects. In the silkworm Bombyx mori, the masculinizing protein Masculinizer (BmMasc) induces the production of the male-type Bombyx mori doublesex (Bmdsx^M), which is the master genetic switch of B. mori sex differentiation. However, the molecular mechanism through which BmMasc transduces the masculinizing signal to the Bmdsx^M production remains unknown. Here, we show that BmMasc physically interacts with Bombyx mori P-element somatic inhibitor (BmPSI), a RNA binding protein required for Bmdsx^M expression. Knockdown experiments indicate that BmPSI is essential for the masculinizing activity of BmMasc. RNA immunoprecipitation experiments also reveal that BmMasc-containing protein complex is associated with female-specific regions of Bmdsx pre-mRNA. Taken together, our findings show that the BmMasc-BmPSI protein complex binds to female-specific Bmdsx regions, inducing exon skipping, and thereby promoting Bmdsx^M expression in B. mori males.

Fig. 1. Identification of BmPSI as the BmMasc-interacting protein.

A LC-MS/MS analysis of the immunoprecipitates obtained using anti-GFP nanobody-conjugated magnetic agarose beads from BmMasc-GFP-, BmMascΔNLS-GFP-, or GFP-transfected BmN-4 cells. The dots indicate the proteins detected in this analysis (773 proteins). BmPSI is indicated by red. The potential proteins involved in RNA splicing are shown in blue (see Table S1). The quantitative data are shown on a log-log scale. The x-axis represents the abundance ratio log₂(BmMasc-GFP)/(GFP), and the y-axis represents the abundance ratio log₂(BmMascΔNLS-GFP)/(GFP). B Co-immunoprecipitation experiments using BmN-4 cells transfected with BmMasc-GFP or GFP. The cell lysate (Lysate) and immunoprecipitates with anti-GFP nanobody agarose beads (IPed w/ GFP-Trap) were immunoblotted using an anti-GFP or anti-BmPSI antibody. The BmPSI bands are indicated by a bracket. Note that BmPSI is detected as two bands presumably due to its modifications. Similar results were obtained in two independent experiments (n = 2, biological replicates). Source data are provided as Supplementary Data 1.

Fig. 2. Identification of protein domains that mediate the interaction between BmMasc and BmPSI.

A Structure of BmPSI-mCherry derivatives. The KH domains and AB motifs in BmPSI, glycine linker, and mCherry are shown. B Co-immunoprecipitation experiments using BmN-4 cells co-transfected with BmMasc-GFP and BmPSI-mCherry, N-BmPSI-mCherry (N-BmPSI), C-BmPSI-mCherry (C-BmPSI), or mCherry. The immunoprecipitates with anti-RFP nanobody agarose beads were immunoblotted using an anti-mCherry or anti-GFP antibody. The BmMasc-GFP bands are indicated by an arrowhead. Non-specific bands are indicated by asterisks. Similar results were obtained in two independent experiments (n = 2, biological replicates). C Co-immunoprecipitation experiments using BmN-4 cells co-transfected with BmMasc-GFP and C-BmPSI-mCherry (C-BmPSI), dAB1-C-BmPSI-mCherry (dAB1-C), dAB2-C-BmPSI-mCherry (dAB2-C), or mCherry. The immunoprecipitates with anti-RFP nanobody agarose beads were immunoblotted using an anti-mCherry or anti-GFP antibody. The BmMasc-GFP bands are indicated by an arrowhead. Similar results were obtained in two independent experiments (n = 2, biological replicates). D Structure of BmMasc-GFP derivatives. The CCCH zinc finger domains and masculinizing domain in BmMasc, glycine linker, and GFP are shown. In CS-BmMasc-GFP, the functionally important cysteine residues (Cys-301 and Cys-304) in the masculinizing domain are replaced by serine. E Co-immunoprecipitation experiments using BmN-4 cells transfected with BmMasc-GFP, dzf1-BmMasc-GFP (dzf1), dzf2-BmMasc-GFP (dzf2), or GFP. The immunoprecipitates with anti-GFP nanobody agarose beads were immunoblotted using an anti-GFP or anti-BmPSI antibody. Non-specific bands are indicated by an asterisk. Similar results were obtained in two independent experiments (n = 2, biological replicates). F Expression of Bmdsx^M in BmN-4 cells transfected with BmMasc-GFP or CS-BmMasc-GFP (CS). The Bmdsx^M levels were estimated by RT-qPCR. The data are the means of three independent experiments (n = 3, biological replicates). Adjusted p values from Tukey’s multiple comparisons tests are shown. G Co-immunoprecipitation experiments using BmN-4 cells transfected with BmMasc-GFP or CS-BmMasc-GFP (CS). The immunoprecipitates with anti-GFP nanobody agarose beads were immunoblotted using an anti-GFP or anti-BmPSI antibody. Non-specific bands are indicated by an asterisk. Similar results were obtained in two independent experiments (n = 2, biological replicates). Source data are provided as Supplementary Data 1.

Fig. 3. Abundance of BmPSI and BmMasc proteins in BmN-4 cells.

A Expression of Bmdsx^M in BmN-4 cells transfected with BmMasc-GFP or BmPSI. The Bmdsx^M levels were estimated by RT-qPCR. The data represents the means of three independent experiments (n = 3, biological replicates). Adjusted p values from Tukey’s multiple comparisons tests are shown. B Western blotting experiments of BmN-4 cell lysates transfected with BmMasc or empty vector. The cell lysates were immunoblotted using an anti-BmMasc or anti-actin (loading control) antibody. The BmMasc bands are indicated by an arrowhead. Similar results were obtained in two independent experiments (n = 2, biological replicates). C Western blotting experiments of BmN-4 cell lysates transfected with BmPSI or empty vector. The cell lysates were immunoblotted using an anti-BmPSI or anti-actin (loading control) antibody. Similar results were obtained in two independent experiments (n = 2, biological replicates). Source data are provided as Supplementary Data 1.

Fig. 4. BmPSI is required for BmMasc-dependent Bmdsx^M expression in BmN-4 cells.

A Western blotting of dsRNA-treated BmN-4 sid-1 cell lysates. The cell lysates were immunoblotted using an anti-BmPSI or anti-actin (loading control) antibody. Similar results were obtained in three independent experiments. B Quantification of BmPSI protein abundance in BmN-4 sid-1 cells treated with dsBmPSI-1, dsBmPSI-2, or dsLuc (control). The BmPSI protein levels were estimated by Western blotting, as shown in (A). The data are the means of three independent experiments (n = 3, biological replicates). Adjusted p values from Tukey’s multiple comparisons tests are shown. C Expression of Bmdsx^M in BmN-4 sid-1 cells treated with dsBmPSI-1, dsBmPSI-2, or dsLuc (control). The Bmdsx^M levels were estimated by RT-qPCR. The data are the means of three independent experiments (n = 3, biological replicates). Adjusted p values from Tukey’s multiple comparisons tests are shown. Source data are provided as Supplementary Data 1.

Fig. 5. Physical association between BmMasc-containing protein complex and Bmdsx pre-mRNA.

A Schematic representation of the RNA immunoprecipitation (RIP) experiments. The lysates of cells transfected with BmMasc-GFP or GFP (control) were immunoprecipitated with anti-GFP beads, and RNA fragments were purified from the immunoprecipitates and subjected to RNA-seq or RT-qPCR. B RIP-seq analysis of BmMasc-GFP- or GFP-associated RNA fragments. The x-axis indicates the genomic region of the Bmdsx gene, whereas the y-axis indicates the relative coverage in the Bmdsx region. The exon–intron boundaries are indicated by dashed lines. The region that was enriched by BmMasc-GFP-bound RNA fragments is highlighted by the red line. C RIP-qPCR analysis of BmMasc-GFP- or GFP-associated RNA fragments. The data are from two independent experiments (n = 2, biological replicates). The positions of the primers used for RIP-qPCR are shown by arrows. Source data are provided as Supplementary Data 1–3.

Fig. 6. Proposed model of BmMasc-dependent Bmdsx^M splicing.

In B. mori males, the BmMasc–BmPSI protein complex binds to the adjacent region of the female-specific exons of Bmdsx pre-mRNA and induces exon skipping, resulting in the production of Bmdsx^M. In females, BmPSI is present but BmMasc is downregulated by W-linked Fem piRNA, resulting in low-level accumulation of the masculinizing complex.