The differential expression and effects of Drosha on metamorphosis of Japanese flounder (paralichthys olivaceus)

Drosha is critical for producing mature microRNAs (miRNAs) from their precursor molecules and small interfering RNAs (siRNAs). Although Drosha has been well characterized in most vertebrate species, identifying the Japanese flounder Drosha has remained elusive. In this study, we cloned the Japanese flounder Drosha gene, which has 4,122 nucleotides (nt), including a 5'-untranslated region (UTR) of 14 nt, a 3'-UTR of 230 nt, and a 3,879 nt open reading frame (ORF) encodes a 1,292 amino acid polypeptide. Then, we analyzed the conservation and phylogenetic evolution of Drosha in some species. Real-time quantitative PCR revealed that Drosha mRNA is highly expressed in the brain, and a lower amount of mRNA was also found in muscle than in other tissues. Drosha plays a vital role in controlling flounder development and metabolism, and its mRNA levels were relatively high at 36 dph (days post-hatching) in the period of metamorphosis and growth of the Japanese flounder. Drosha expression showed in advance with a peak at 23 dph following TH (thyroid hormone) treatment. To further investigate the role of Drosha in metamorphosis, we performed siRNA knockdown of Drosha in flounder embryonic cells (FEC) cells. The result shows that the Drosha-specific siRNA significantly down-regulated Drosha mRNA and miR-1,133,17,214,181a,181b levels, while primary miRNA (pri-miRNA) levels were not significantly different from negative control (NC). These results suggest that Drosha plays a vital role in Japanese flounder development and metamorphosis through processing to produce mature miRNAs, providing essential information for further studying of the part of the Drosha gene in the Japanese flounder development.