Juvenile hormone-regulated alternative splicing of the taiman gene primes the ecdysteroid response in adult mosquitoes

Abstract

Juvenile hormone (JH) regulates many aspects of insect development and reproduction. In some processes, JH plays a critical role in defining the action of the steroid hormone 20-hydroxyecdysone (20E). In Aedes aegypti mosquitoes, JH prepares newly emerged female adults to become competent to synthesize vitellogenin in response to 20E after blood ingestion. The molecular basis of this competence is still not well understood. Here, we report that JH regulates pre-mRNA splicing of the taiman gene, which encodes a key transcriptional regulator required for both JH- and 20E-controlled gene expression. JH stimulated the production of the Taiman isoforms A/B, while reducing the levels of the isoforms C/D, in the fat body after adult eclosion. The appearance of the A/B isoforms in maturing mosquitoes was accompanied by acquisition of the competence to respond to 20E. Depletion of the A/B isoforms, by inhibiting the alternative splicing or by isoform-specific RNA interference, considerably diminished the 20E-induced gene expression after a blood meal and substantially impaired oocyte development. In accordance with this observation, further studies indicated that in the presence of 20E, the Taiman A/B isoforms showed much stronger interactions with the 20E receptor complex than the Taiman C/D isoforms. In contrast, all four isoforms displayed similar capabilities of forming active JH receptor complexes with the methoprene-tolerant protein (Met). This study suggested that JH confers the competence to newly emerged female mosquitoes by regulating mRNA splicing to generate the Taiman isoforms that are essential for the vitellogenic 20E response.

Keywords: RNA splicing; gene expression; insect hormone; signal transduction.

Fig. 1.

JH regulates the phosphorylation of mosquito SRSF proteins in the fat body after eclosion. (A) Phosphorylation of SRSFs in female adults at 0, 12, 24, 36, 48, and 72 h PE was detected by immunoblotting. Equal amounts of proteins were loaded on SDS/PAGE and were blotted with an antibody (Clone 1H4; EMD Millipore) that binds to a phosphoepitope in SR proteins (p-SR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Phosphorylation of SRSFs was regulated by JH in the fat body of newly emerged mosquitoes. Fat bodies were isolated from female mosquitoes at 30 min PE and were cultured in vitro in the presence of JH-III (1 μM) for 0, 1, 2, 4, and 8 h. The phosphorylation of SRSFs was monitored by immunoblotting. (C) RTK/PI3K/Akt pathway was required for the JH-regulated SRSF phosphorylation. In vitro-cultured fat bodies were preincubated with genistein, U73122, wortmannin, and MK2206 (the specific inhibitors of RTK, PLC, PI3K, and Akt, respectively) for 1 h, followed by incubation with JH-III (1 μM) for 4 h. Equal amounts of proteins from each treatment were used for immunoblotting analysis. (D) SRPK and Clk were primarily responsible for the JH-regulated phosphorylation of SRSFs. SRPIN340, TG003, and harmine were used in the pretreatment to inactivate SRPK, Clk, and DYRK, respectively. The phosphorylation of SRSFs in the cultured fat bodies was assessed by immunoblotting after incubation with JH-III.

Fig. 2.

JH regulates the splicing of A. aegypti taiman. (A) Scheme of the AaTai isoforms. The constitutive exons are shown as solid blue boxes. E12 (purple) and E13 (orange) are alternatively spliced in the isoforms. The bHLH, PAS-A, and PAS-B domains are located in the N-terminal end of AaTai proteins. Green arrows point to the region where dsRNA was designed to knock down all AaTai isoforms. Black arrows indicate the locations of the primers used in RT-PCR. (B) JH induces the inclusion of E13 to produce the isoforms AaTai-A and AaTai-B. Fat bodies from female mosquitoes at 30 min PE were cultured in vitro in the presence of JH-III (1 μM). The mRNA levels of individual AaTai isoforms were assessed using RT-PCR with the primers shown in A. The RpS7 gene was used as an internal control. (C) RTK/PI3K/Akt pathway is required for the JH-regulated alternative splicing of AaTai. In vitro-cultured fat bodies were preincubated with genistein, U73122, wortmannin, MK2206, SRPIN340, TG003, or harmine for 1 h, followed by incubation with JH-III (1 μM) for 4 h.

Fig. 3.

Expression of AaTai-A and AaTai-B in the previtellogenic fat body coincides with the acquisition of the competence for the 20E response. (A) Expression profiles of the AaTai isoforms in the fat body of adult female mosquitoes were examined by RT-PCR at the indicated time points. (B) AaTai isoform proteins were detected by immunoblotting with an antibody for the common regions of all isoforms. The predicted molecular masses of isoforms A–D are 191 kDa, 186 kDa, 175 kDa, and 170 kDa, respectively. (C) Visualization of AaTai-A and AaTai-B in the fat body by whole-mount immunohistochemistry with an antibody for AaTaiE13. Nonspecific rabbit IgG was used as a negative control. Nuclei of the fat body cells were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). The red bar denotes 20 μm. (D) In vitro culture of the fat body dissected from the RNAi mosquitoes that failed to form AaTai-A and AaTai-B. DsRNAs for AaSRPK and AaClk were injected into newly emerged mosquitoes, and the dsRNA corresponding to the GFP gene (dsGFP) was used as a control. Fat bodies were isolated at 96 h PE from the RNAi mosquitoes and cultured in the presence of 20E (1 μM) or ethanol for 8 h. The mRNA transcripts of AaTai-A and AaTai-B in the cultured fat bodies were examined by RT-PCR. (E) Expression of the 20E-response genes AaE74B, AaE75A, and AaVg in the cultured fat bodies, where the formation of AaTai-A and AaTai-B was blocked by the depletion of AaSRPK or AaClk. The qRT-PCR results are the mean ± SD of three independent experiments. Statistical significance is shown (**P < 0.01).

Fig. 4.

Blocking the formation of AaTai-A and AaTai-B by the knockdown of AaSRPK or AaClk arrests ovarian development. DsRNAs for AaSRPK and AaClk were injected into female mosquitoes within 30 min PE. DsRNA for the GFP gene (dsGFP) was used as a control. The knockdown of AaSRPK and AaClk was validated at 96 h PE (A) and 24 h PBM (B) using qRT-PCR. The results are presented as the mean ± SD of three independent experiments (**P < 0.01). The expression of AaTai isoforms was examined using RT-PCR at 96 h PE (C) and 24 h PBM (D). Noninj, noninjected. (E and F) Images of representative ovaries that were isolated from the Noninj and dsRNA-injected female mosquitoes at 96 h PE and 24 h PBM. (Scale bars: 500 µm.) (G and H) Average lengths of primary follicles at 96 h PE and 24 h PBM. The sizes were measured using the Leica Application Suite (v4.5). Thirty individuals in each group were used, and the results are shown as the mean ± SD. (I) Expression of the JH response genes AaKr-h1 and AAEL002576 was examined by qRT-PCR in the fat body at 96 h PE. qRT-PCR results are presented as a fold change compared with the dsGFP-injected sample (P > 0.05). (J) Expression of the 20E response genes AaE74B, AaE75A, and AaVg was measured by qRT-PCR at 24 h PBM. Statistical significance is shown (**P < 0.01).

Fig. 5.

RNAi-mediated knockdown of AaTai-A and AaTai-B impedes ovarian development and ecdysteroid response. DsRNAs for the AaTai common region (dsTaiCore), AaTai-E12 (dsTaiE12), and AaTai-E13 (dsTaiE13) were injected into adult female mosquitoes within 30 min PE. DsGFP was used as a control. (A and B) Ovaries isolated from each group of mosquitoes at 96 h PE and 24 h PBM. Non-inj, noninjected. (Scale bars: 500 µm.) (C and D) Average lengths of primary follicles were measured at 96 h PE and 24 h PBM. Thirty individuals were measured for each group, and the results are presented as the mean ± SD. (E) Expression of the JH response genes AaKr-h1 and AAEL002576 in the fat body at 96 h PE. qRT-PCR results are presented as a fold change compared with the dsGFP-injected sample. Results are the mean ± SD of three independent experiments. (F) Expression of AaE74B, AaE75A, and AaVg at 24 h PBM. qRT-PCR results are the mean ± SD of three independent experiments. Statistical significance between the groups is shown (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 6.

AaTai-A and AaTai-B potentiate transcriptional activation by the AaEcR–AaUSP complex in response to 20E. (A) Drosophila L57 cells were transfected with the 4× JHRE-Luc reporter construct and the expression plasmids of AaMet and individual AaTai isoforms. A Renilla luciferase reporter construct was cotransfected in each well. Transfected cells were treated with JH-III (1 μM) or ethanol and then were lysed for measuring the luciferase activity. (B) L57 cells were transfected with the hsp27-EcRE-Luc reporter construct and the expression plasmids of AaEcR, AaUSP, and each AaTai isoform. Transfected cells were incubated with 20E (1 μM) or ethanol. Results are presented as the ratio of firefly/Renilla luciferase activity and are reported as the mean ± SD of three independent experiments.

Fig. 7.

Binding of individual AaTai isoforms to the AaEcR–AaUSP complex and AaMet. (A) Protein interaction between AaTai and AaEcR/AaUSP. Drosophila L57 cells were transfected with the expression plasmids for the V5-tagged AaTai isoforms, together with the expression vectors for AaEcR and AaUSP. The transfected cells were treated with 20E (1 μM) or ethanol. AaEcR in the cell extracts was precipitated by anti-AaEcR antibody, and the pellets were analyzed by immunoblotting using anti-V5 antibody. Input [10% of the amount of cell lysate used for the immunoprecipitation (IP)] was analyzed using anti-V5, anti-AaEcR, anti-AaUSP, and anti-GAPDH antibodies. (B) Coimmunoprecipitation of AaTai isoforms with AaMet. Drosophila L57 cells were transfected with the expression plasmids for the V5-tagged AaTai isoforms and for AaMet. The transfected cells were treated with JH-III (1 μM) or ethanol. Immunoprecipitation was performed using an anti-AaMet antibody, and Western blotting was performed using anti-V5 antibody. Input was analyzed using anti-V5, anti-AaMet, and anti-GAPDH antibodies.