ABSTRACT
The precise increase and decrease of hormone ecdysone are critical for accurate development in insects. Most previous works focus on transcriptional activation of ecdysone production; however, little is known about the mechanism of switching off ecdysone biosynthesis after ecdysis. Here, we showed that the precursor microRNA-14 (pre-miR-14) encodes two mature miRNAs in silkworm; both of these two mature miRNAs regulate various genes in the ecdysone-signalling pathway. Bmo-miR-14-5p targets on nine genes whereas Bmo-miR-14-3p targets on two genes in the same pathway. These two mature miRNAs increased immediately after the ecdysis, efficiently suppressing the 20-hydroxyecdysone (20E) biosynthesis, the upstream regulation, and the downstream response genes. Knocking down either of two mature miRNAs or both of them delays moult development, impairing development synchrony in antagomir-treated groups. In addition, overexpressing Bmo-miR-14-5p but not Bmo-miR-14-3p significantly affected the 20E titer and increased the moulting time variation, suggesting that Bmo-miR-14-5p, though it is less abundant, has more potent effects in development regulation than Bmo-miR-14-3p. In summary, we present evidence that a pre-miRNA encodes two mature miRNAs targeting on the same pathway, which significantly improves miRNA regulation efficiencies to programmatically switch off ecdysone biosynthesis.
Acknowledgments
We would like to thank Professor Muwang Li in Jiangsu University of Science and Technology for providing us the silkworms.
Author’s contributions
FL designed the work. KH and HX performed the bioinformatics analysis of miRNA identification, conservation analysis and target predictions. KH, HX and YS carried out the experiments of miRNA target validation, oversupply and knockdown experiments. YX and KH determined the titers of ecdysone. GS and YX conducted the experiments for gene expression. KH completed and improved the figures. FL and KH wrote the manuscript.
Availability of data and materials
3’UTR sequences for target prediction obtained by 3ʹRACE has been deposited in the NCBI database. All the accession number of mRNA sequences has been made available in additional Table S3.
Disclosure statement
No potential conflict of interest was reported by the authors.
Supplementary material
The supplemental data for this article can be accessed here.