DataSheet_1_The differential expression and effects of Drosha on metamorphosis of Japanese flounder (paralichthys olivaceus).zip

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.

Drosha (Drosha)对于从其前体分子及小干扰RNA（small interfering RNAs, siRNAs）生成成熟微RNA（microRNAs, miRNAs）的过程至关重要。尽管目前已对多数脊椎动物的Drosha完成了充分表征，但牙鲆的Drosha基因仍未被成功鉴定。本研究成功克隆了牙鲆的Drosha基因，其全长为4122个核苷酸（nucleotides, nt），包含14 nt的5'非编码区（5'-untranslated region, UTR）、230 nt的3'非编码区（3'-UTR），以及一段长度为3879 nt的开放阅读框（open reading frame, ORF），该开放阅读框可编码一条含1292个氨基酸的多肽。随后，本研究对多个物种的Drosha基因的保守性及系统发育演化特征进行了分析。实时定量聚合酶链反应结果显示，Drosha信使RNA（mRNA）在脑组织中呈高表达，而肌肉组织中的mRNA表达量亦较其他多数组织更低。Drosha在调控牙鲆发育与代谢过程中发挥关键作用，其mRNA在牙鲆变态发育与生长阶段的孵化后36天（days post-hatching, dph）时呈现较高表达水平。经甲状腺激素（thyroid hormone, TH）处理后，Drosha的表达提前上调，并在孵化后23天达到表达峰值。为进一步探究Drosha在牙鲆变态发育中的作用，本研究在牙鲆胚胎细胞（flounder embryonic cells, FEC）中开展了Drosha的小干扰RNA敲低实验。结果表明，特异性靶向Drosha的siRNA可显著下调Drosha mRNA以及miR-1、miR-133、miR-17、miR-214、miR-181a、miR-181b的表达水平，而初级微RNA（primary miRNA, pri-miRNA）的表达量与阴性对照组（negative control, NC）无显著差异。上述结果表明，Drosha可通过加工生成成熟miRNAs，在牙鲆的发育与变态过程中发挥关键作用，为后续深入研究Drosha基因在牙鲆发育中的调控功能提供了重要参考依据。