Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene.
Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene.
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DOI：
10.1093/nar/gkq148
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发表时间：
2010-07
影响因子：
14.9
通讯作者：
Tsai HJ
中科院分区：
文献类型：
--
作者：
Hsu RJ;Lin CY;Hoi HS;Zheng SK;Lin CC;Tsai HJ
在斑马鱼myf 5基因的第一内含子+502/+835（I300）处有一个强的负性顺式元件。为了阐明这种抑制网络的分子机制，我们显微注射I300 RNA的斑马鱼单细胞胚胎，导致myf 5启动子驱动的荧光素酶活性急剧降低。在这个I300片段中，我们鉴定了位于+609/+632的内含子microRNA（miR-In 300），并发现它在较老的成熟体节中比新形成的体节中表达更高，这与斑马鱼myf 5转录本的分布呈负相关。我们证明了miR-In 300通过消除myf 5启动子活性来抑制myf 5的转录，并且我们随后鉴定了Dickkopf-3基因的长同种型（dkk 3）作为miR-In 300的靶基因。我们进一步发现，注射dkk 3-morpholinos（MO）导致体节中myf 5转录物的下调，而将myf 5 mRNA与dkk 3-MO 1共注射能够拯救单独由dkk 3-MO 1诱导的缺陷。最后，注射miR-In 300-MO增强了斑马鱼胚胎中体节中myf 5的转录和Dkk 3蛋白的水平。基于这些发现，我们得出结论，miR-In 300与其靶基因dkk 3结合，从而抑制dkk 3 mRNA的翻译，进而抑制斑马鱼myf 5启动子活性。
A strong, negative cis-element located at the first intron +502/+835 (I300) of zebrafish myf5 has been reported. To elucidate the molecular mechanism underlying this repression network, we microinjected zebrafish single-cell embryos with I300 RNA, resulting in the dramatic reduction of luciferase activity driven by the myf5 promoter. Within this I300 segment, we identified an intronic microRNA (miR-In300) located at +609/+632 and found that it was more highly expressed in the older mature somites than those newly formed, which negatively correlated with the distribution of zebrafish myf5 transcripts. We proved that miR-In300 suppressed the transcription of myf5 through abolishing myf5 promoter activity, and we subsequently identified the long isoform of the Dickkopf-3 gene (dkk3) as the target gene of miR-In300. We further found that injection of the dkk3-morpholinos (MOs) resulted in downregulation of myf5 transcripts in somites, whereas co-injection of myf5 mRNA with dkk3-MO1 enabled rescue of the defects induced by dkk3-MO1 alone. Finally, injection of miR-In300-MO enhanced both myf5 transcripts in somites and the level of Dkk3 protein in zebrafish embryos. Based on these findings, we concluded that miR-In300 binds to its target gene dkk3, which inhibits the translation of dkk3 mRNA and, in turn, suppresses zebrafish myf5 promoter activity.
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