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. 2020 Jun 26:11:636.
doi: 10.3389/fgene.2020.00636. eCollection 2020.

Poly(A) Binding Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster

Takumi Kamiyama  1 , Wei Sun  2   3 , Naoki Tani  4 , Akira Nakamura  4 , Ryusuke Niwa  3
Affiliations

Poly(A) Binding Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster

Takumi Kamiyama et al. Front Genet. .

Abstract

Steroid hormone signaling contributes to the development of multicellular organisms. In insects, ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E), promote molting and metamorphosis. Ecdysone is biosynthesized in the prothoracic gland (PG), via several steps catalyzed by ecdysteroidogenic enzymes that are encoded by Halloween genes. The spatio-temporal expression pattern of ecdysteroidogenic genes is strictly controlled, resulting in a proper fluctuation of the 20E titer during insect development. However, their transcriptional regulatory mechanism is still elusive. A previous study has found that the polyadenylated tail [poly(A)] deadenylation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) regulates the expression of spookier (spok), which encodes one of the ecdysteroidogenic enzymes in the fruit fly Drosophila melanogaster. Based on this finding, we speculated whether any other poly(A)-related protein also regulates spok expression. In this study, we reported that poly(A) binding protein (Pabp) is involved in spok expression by regulating nuclear localization of the transcription factor molting defective (Mld). When pabp was knocked down specifically in the PG by transgenic RNAi, both spok mRNA and Spok protein levels were significantly reduced. In addition, the spok promoter-driven green fluorescence protein (GFP) signal was also reduced in the pabp-RNAi PG, suggesting that Pabp is involved in the transcriptional regulation of spok. We next examined which transcription factors are responsible for Pabp-dependent transcriptional regulation. Among the transcription factors acting in the PG, we primarily focused on the zinc-finger transcription factor Mld, as Mld is essential for spok transcription. Mld was localized in the nucleus of the control PG cells, while Mld abnormally accumulated in the cytoplasm of pabp-RNAi PG cells. In contrast, pabp-RNAi did not affect the nuclear localization of other transcription factors, including ventral vein lacking (Vvl) and POU domain motif 3 (Pdm3), in PG cells. From these results, we propose that Pabp regulates subcellular localization in the PG, specifically of the transcription factor Mld, in the context of ecdysone biosynthesis.

Keywords: Halloween gene; ecdysone biosynthesis; insect development; nuclear localization; poly(A) binding protein.

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Figures

Figure 1
Figure 1
Pabp function is required in the PG for larval development. Phenotypes caused by PG-specific pabp-RNAi at 132 h AEL. (A) Whole bodies of the control and RNAi larvae. Scale bar: 5 mm (B) Magnified intensity of Figure 1A. Scale bar: 2 mm. (Left) phm>dicer2, + larvae molted in the third larval instar as judged by the branched morphology of the anterior tracheal pits (yellow arrowheads), typical features of third instar larvae. (Right) phm>dicer2, pabp-IR larvae raised at the second larval instar and exhibit singular insertions of anterior tracheal pits. (C) The developmental progression of phm>dicer2, + (N = 41) and phm>dicer2, and pabp-IR (N = 49) animals at 132 h AEL. L2: second instar larva, L3: third instar larva.
Figure 2
Figure 2
Expression analysis of ecdysteroidogenic genes. All photos are single-plane confocal images. (A) Relative expression levels of seven ecdysteroidogenic genes in phm>dicer2, pabp-IR at 60 h AEL compared to controls (phm>dicer2, +) based on the quantitative reverse transcription-polymerase chain reaction (qRT-PCR; N = 4). RNA was isolated from the whole bodies of the second instar larvae. (B) Immunostaining of the PG cells from phm>dicer2, + and phm>dicer2, and pabp-IR second instar larvae at 60 h AEL with antibodies against Phm (magenta) and Spok (green). (C) Quantifications of fluorescence intensities of Phm and Spok in the PG (each N = 3). Error bars represent standard deviations. *** and n.s. indicate p < 0.001 and non-significance (p > 0.05), respectively, by Student’s t-test. (D) Fluorescence images of the PG cells from phm>dicer2, + and phm>dicer2, pabp-IR larvae with spok enhancer/promoter-driven nuclear localized-GFP construct (spok>GFP) at 60 h AEL. (E) Quantification of GFP fluorescence intensity in the PG nuclei (N = 4). The bar plots are drawn in the same manner as (C). PG cells are immunostained with anti-Phm antibody (magenta). Scale bar: 20 μm.
Figure 3
Figure 3
Evaluation of newly-generated anti-molting defective (Mld) antibody. Magnified image of prothoracic gland (PG) cells. All photos are single-plane confocal images. Tissues were stained with anti-Mld antibody (magenta) and 4',6-diamidino-2-phenylindole (DAPI; blue). Representative nuclei are outlined by dashed lines. Mld signals were detected in the nuclei of the PG cells of phm>dicer2, + (yellow arrow) at 36 h after egg laying (AEL). In contrast, the signal disappeared in phm>dicer2 and pabp-IR. Scale bar: 10 μm.
Figure 4
Figure 4
Subcellular localization of transcription factors in the PG cells. All photos are single-plane confocal images. (A,C,E) Immunostaining of the PG cells from phm>dicer2, + and phm>dicer2, pabp-IR second instar larvae at 60 h AEL with the antibodies against Mld (green), POU domain motif 3 (Pdm3; magenta), Ventral vein lacking (Vvl; magenta), respectively. Dashed lines indicate the shape of nuclei. (A) Nuclei are stained by TOPRO3 (magenta). Bottom panels are single channels of Mld (grayscale). Scale bar: 5 μm. (B,D,F) Quantifications of fluorescence intensities of Mld (N = 5), Pdm3 (N = 3), and Vvl (N = 3) in the PG nuclei. Error bars represent standard deviations. ***, ** and n.s. indicate p < 0.001, p < 0.005 and non-significance (p > 0.005), respectively, by Student’s t-test.
Figure 5
Figure 5
Model of the Pabp function contributing nuclear localization of Mld for spok transcription.
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