Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene.
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.
Institute of Molecular and Cellular Biology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
Ren-Jun
RJ
Hsu
Cheng-Yung
CY
Lin
Hao-Seong
HS
Hoi
Shu-Kai
SK
Zheng
Chiu-Chun
CC
Lin
Huai-Jen
HJ
Tsai
D000818:Animals; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D007438:Introns; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D019170:Somites / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
D035683:MicroRNAs; D051787:Myogenic Regulatory Factor 5; D020033:Protein Isoforms; D012333:RNA, Messenger; D029961:Zebrafish Proteins