Hormone-dependent activation and repression of microRNAs by the ecdysone receptor in the dengue vector mosquito Aedes aegypti

Abstract

Female mosquitoes transmit numerous devastating human diseases because they require vertebrate blood meal for egg development. MicroRNAs (miRNAs) play critical roles across multiple reproductive processes in female Aedes aegypti mosquitoes. However, how miRNAs are controlled to coordinate their activity with the demands of mosquito reproduction remains largely unknown. We report that the ecdysone receptor (EcR)-mediated 20-hydroxyecdysone (20E) signaling regulates miRNA expression in female mosquitoes. EcR RNA-interference silencing linked to small RNA-sequencing analysis reveals that EcR not only activates but also represses miRNA expression in the female mosquito fat body, a functional analog of the vertebrate liver. EcR directly represses the expression of clustered miR-275 and miR-305 before blood feeding when the 20E titer is low, whereas it activates their expression in response to the increased 20E titer after a blood meal. Furthermore, we find that SMRTER, an insect analog of the vertebrate nuclear receptor corepressors SMRT and N-CoR, interacts with EcR in a 20E-sensitive manner and is required for EcR-mediated repression of miRNA expression in Ae. aegypti mosquitoes. In addition, we demonstrate that miR-275 and miR-305 directly target glutamate semialdehyde dehydrogenase and AAEL009899, respectively, to facilitate egg development. This study reveals a mechanism for how miRNAs are controlled by the 20E signaling pathway to coordinate their activity with the demands of mosquito reproduction.

Keywords: coactivator; corepressor; small RNA; steroid hormone; vector-borne disease.

Fig. 1.

Transcriptome-wide screening of ecdysone receptor (EcR)-regulated miRNAs in the female mosquito fat body (FB). (A) Schematic representation of the study design. (B) Differentially expressed miRNAs in the FBs of iEcR females at 24 h post blood meal (PBM) compared with iLuc control. Filter condition, log₂ (iEcR/iLuc) greater than 0.5 or less than −0.5. qRT-PCR validation is highlighted by blue for down-regulation and red for up-regulation. (C and D) Time course expression profiles of iEcR–down-regulated (C) and –up-regulated (D) miRNAs in the female mosquito FB after a blood meal. Data are derived from Zhang et al. (23). Expression levels are represented by averages of normalized expression from three biological replicates.

Fig. 2.

20E activates miR-275 and miR-305 transcription in the FB. (A) Schematic diagram showing the genomic location of pre–miR-275 and pre–miR-305. (B) Time course expression profiles of pre–miR-275 and pre–miR-305 in the female mosquito FB posteclosion (PE) and PBM. The 20E titers in Ae. aegypti females after a blood meal are derived from Hagedorn et al. (22) and are shown in yellow. (C) Relative levels of pre–miR-275 and pre–miR-305 in the FBs at 6 h after injection of the indicated reagents. A 0.5-μL aliquot of injection solution containing either ethanol (solvent), ethanol and amino acids (AA), 20E (10⁻⁴ M) dissolved in ethanol, or amino acids and 10⁻⁴ M 20E (AA + 20E) was injected into female mosquitoes at 96 h PE. (D and E) Relative levels of pre–miR-275 and pre–miR-305 in the FBs. (D) FBs from 96-h PE female mosquitoes were incubated in culture solution containing either ethanol (solvent), ethanol and amino acids (AA), 20E (10⁻⁶ M) dissolved in ethanol, or amino acids and 10⁻⁶ M 20E (AA + 20E) for 6 h. (E) FBs from 96 h PE female mosquitoes were incubated in amino acids plus culture solution containing either ethanol (AA), ethanol and 10⁻³ M Chx (AA + Chx), 10⁻⁶ M 20E (AA + 20E), or 10⁻⁶ M 20E and 10⁻³ M Chx (AA + 20E + Chx) for 6 h. (B–E) Mean ± SEM from three independent experiments; n.s., not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 (independent-samples t test).

Fig. 3.

EcR represses expression of the miR-275 and miR-305 cluster at the low 20E level before blood feeding, whereas it activates its expression at the high 20E level after a blood meal. (A) Luciferase assays in S2 cells transfected with the pGL4.17 reporter plasmid carrying the miR-275/miR-305 promoter (−2002 to +98) conjugated to firefly luciferase (miR-275-Luc) and the expression vectors for EcR and/or Ultraspiracle (USP). Treatments with empty pAc5.1 expression vector served as controls. A Renilla luciferase reporter construct was cotransfected in each well as a reference. Transfected cells were exposed to 20E (10⁻⁶ M) dissolved in ethanol (Eth) or Eth alone. Data represent normalized luciferase activity (firefly/Renilla) and are shown as mean ± SEM from three independent experiments. (B and C) Relative levels of primary transcript (pri–miR-275), pre-miRNAs and mature miRNAs of miR-275 and miR-305 in the FBs. (B) FBs were dissected from iLuc, iEcR, or iUSP female mosquitoes at 96 h PE and incubated in culture solution containing 20E (10⁻⁶ M) dissolved in Eth or Eth alone for 6 h. (C) FBs were dissected from iLuc, iEcR, and iUSP female mosquitoes at 96 h PE and 24 h PBM. (D–F) Luciferase assays in S2 cells transfected with the expression vectors for EcR and USP and the desired reporter constructs. Transfected cells were exposed to 20E (10⁻⁶ M) dissolved in Eth or Eth alone. Data represent fold induction of normalized luciferase activity from EcR/USP/20E-treated cells over that of pAc5.1/Eth-treated cells. (G–I) Luciferase assays in S2 cells transfected with the expression vector for EcR and the desired reporter constructs. (G and H) Data represent percent reduction of normalized luciferase activity from EcR-treated cells compared with pAc5.1-treated cells. (I) Data represent normalized luciferase activity. (A–I) Mean ± SEM from three independent experiments; n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (independent-samples t test).

Fig. 4.

The corepressor SMRTER is involved in repression of miRNA expression by EcR. (A) SMRTER is required for EcR-mediated repression. dsRNA-treated Aag2 cells were transfected with the miR-275-Luc reporter construct and the expression vector for EcR. Treatment with empty expression vector served as a control. A Renilla luciferase reporter construct was cotransfected in each well as a reference. Data represent normalized luciferase activity (firefly/Renilla). (B) Relative levels of primary transcript, pre-miRNAs, and mature miRNAs of miR-275 and miR-305 in the FBs dissected from iLuc or iSMRTER female mosquitoes at 96 h PE and incubated for 6 h in culture solution containing 20E (10⁻⁶ M) dissolved in Eth or Eth alone. (C) SMRTER interacts with EcR in a hormone-sensitive manner. (Top) Schematic representation of SMRTER domain structure and depletion constructs used for interaction assay. The two corresponding regions of the Drosophila SMRTER EcR-interacting domains (ERID1 and ERID2) are indicated by blue. (Bottom) S2 cells were transfected with the expression vectors for V5-tagged SMRTER fragments, Flag-EcR and V5-USP. Transfected cells were exposed to 20E (10⁻⁶ M) dissolved in Eth or Eth alone. Extracts were immunoprecipitated (IP) with Flag antibody or IgG, and pellets were analyzed by immunoblotting using Flag and V5 antibodies. Input (10%) was analyzed using Flag, V5, and Tubulin antibodies. Molecular masses are indicated on the Right. (D) Transcriptional repression by EcR requires histone deacetylase activity. Aag2 cells were transfected with the miR-275-Luc reporter construct and the expression vector for EcR. Transfected cells were exposed to trichostatin A (TSA) (10⁻⁵ M) dissolved in DMSO or DMSO alone (solvent). Data represent normalized luciferase activity. (A, B, and D) Mean ± SEM from three independent experiments; n.s., not significant; **P < 0.01 (independent-samples t test).

Fig. 5.

miR-275 and miR-305 promote egg development in female mosquitoes. (A) Effect of miR-275-3p and miR-305-5p depletion on ovary development observed at 24 h PBM. Ovaries were visualized using the Leica M165FC stereo microscope. (Scale bars, 1 mm.) (B) Average follicle size of ovaries isolated from wild-type (WT), Ant-Con–, Ant-275–, and Ant-305–injected mosquitoes at 24 h PBM. (C) Egg numbers deposited per female by WT, Ant-Con–, Ant-275–, and Ant-305–injected mosquitoes. (B and C) One dot represents one female mosquito. Data represent three biological replicates with 6–14 individuals in each replication. Mean ± SEM is shown. ****P < 0.0001 (nonparametric Mann–Whitney U test).

Fig. 6.

Identification of miR-275 and miR-305 target genes. (A and B) Relative mRNA levels of potential miR-275-3p (A) and miR-305-5p (B) target genes in the FBs of Ant-Con– and Ant-275– or Ant-305–treated female mosquitoes at 24 h PBM. (C) Luciferase activities in S2 cells transfected with the reporter construct containing the WT 3′-UTR of glutamate semialdehyde dehydrogenase (GSD), Ribose-phosphate pyrophosphokinase 1 (PRPS1), or AAEL009899 and the desired miRNA mimics. (D) Luciferase activities in S2 cells transfected with the mutant (Mut) reporter construct of GSD or AAEL009899 and the desired miRNA mimics. (C and D) Data represent normalized luciferase activity (Renilla/firefly). (A–D) Mean ± SEM from three independent experiments; n.s., not significant; *P < 0.05, **P < 0.01 (independent-sample t test). (E–G) GSD RNAi rescues miR-275 depletion phenotypes. Ovaries (E), average follicle size (F), and egg numbers deposited per female (G) were analyzed from WT, Ant-275–, Ant-275/iLuc–, and Ant-275/iGSD–treated mosquitos. (H–J) AAEL009899 RNAi rescues miR-305 depletion phenotypes. Ovaries (H), average follicle size (I), and egg numbers deposited per female (J) were analyzed from WT, Ant-305–, Ant-305/iLuc–, and Ant-305/iAAEL009899–treated mosquitos. (E and H) Ovaries were visualized using the Leica M165FC stereo microscope. (Scale bars, 1 mm.) (F, G, I, and J) One dot represents one female mosquito. Data represent three biological replicates with 6–14 individuals in each replication. Mean ± SEM is shown. n.s., not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001 (nonparametric Mann–Whitney U test).