Abstract
The steroid hormone ecdysone is the central regulator of insect metamorphosis, during which a growing, immature larva is remodeled, through pupal stages, to a reproductive adult. However, the underlying mechanisms of ecdysone-mediated metamorphosis remain to be fully elucidated. Here, we identified metamorphosis-associated microRNAs (miRNAs) and their potential targets by cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 protein in Drosophila. Interestingly, miR-8-3p targeted five Vha genes encoding distinct subunits of vacuolar H+-ATPase (V-ATPase), which has a vital role in the organellar acidification. The expression of ecdysone-responsive miR-8-3p is normally downregulated during Drosophila metamorphosis, but temporary overexpression of miR-8-3p in the whole body at the end of larval development led to defects in metamorphosis and survival, hallmarks of aberrant ecdysone signaling. In addition, miR-8-3p was expressed in the prothoracic gland (PG), which produces and releases ecdysone in response to prothoracicotropic hormone (PTTH). Notably, overexpression of miR-8-3p or knockdown of its Vha targets in the PG resulted in larger than normal, ecdysone-deficient larvae that failed to develop into the pupal stage but could be rescued by ecdysone feeding. Moreover, these animals showed defective PTTH signaling with a concomitant decrease in the expression of ecdysone biosynthetic genes. We also demonstrated that the regulatory network between the conserved miR-8-3p/miR-200 family and V-ATPase was functional in human cells. Consequently, our data indicate that the coordinated regulation of V-ATPase subunits by miR-8-3p is involved in Drosophila metamorphosis by controlling the ecdysone biosynthesis.
| Original language | English |
|---|---|
| Pages (from-to) | 6449-6465 |
| Number of pages | 17 |
| Journal | FASEB Journal |
| Volume | 34 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 2020 May 1 |
Bibliographical note
Funding Information:We thank the Bloomington Stock Center (NIH P40OD018537), the National Institute of Genetics (NIG) in Japan, the Vienna RNAi Center, and the Developmental Studies Hybridoma Bank (DSHB) for providing us with fly strains and antibodies. The antibodies developed by University of Pennsylvania and University of Colorado were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242. We also thank T. Neufeld, S. Cohen, T. Kornberg, and R. Niwa for providing fly strains or antibodies. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF‐2014R1A1A2058193 to YSL) and Korea University grants (to YSL and D.‐HL). Drosophila Drosophila
Funding Information:
We thank the Bloomington Drosophila Stock Center (NIH P40OD018537), the National Institute of Genetics (NIG) in Japan, the Vienna Drosophila RNAi Center, and the Developmental Studies Hybridoma Bank (DSHB) for providing us with fly strains and antibodies. The antibodies developed by University of Pennsylvania and University of Colorado were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242. We also thank T. Neufeld, S. Cohen, T. Kornberg, and R. Niwa for providing fly strains or antibodies. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2058193 to YSL) and Korea University grants (to YSL and D.-HL).
Publisher Copyright:
© 2020 Federation of American Societies for Experimental Biology
Keywords
- Ago1
- CLIP-seq
- V-ATPase
- ecdysone
- metamorphosis
- microRNA
ASJC Scopus subject areas
- Biotechnology
- Biochemistry
- Molecular Biology
- Genetics