MicroRNA-206 Controls Muscle Development in Zebrafish Embryos through Silencing Novel MicroRNA-206-Specific Target Genes

• Main Authors: Wen-Yen Chang, 張文彥
• Other Authors: Huai-Jen Tsai
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/51028049251583064324

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 99 === MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level by targeting the 3’-untranslated region (3’UTR) of mRNAs through a conserved seed sequence. MiR-206 is a muscle-specific miRNA and is highly expressed in skeletal muscle. Although miR-206 plays a key regulatory role in myoblast proliferation and differentiation, the detailed molecular regulatory mechanism of miR-206 in early myogenesis is still unknown. Particularly, miR-206 and miR-1 share an identical seed sequence, making it difficult to differentiate their target genes and functions. To address this issue, we employed our labeled miRNA pull-down assay (LAMP) (Hsu et al., 2009) to screen the target mRNAs specific for miR-206 from cell extracts of 16-hpf zebrafish embryos. After microarray analysis, we selected 117 high-scoring genes for further study. Using whole-mount in situ hybridization, we observed that the expression patterns of cbp/p300-interacting transactivator, Glu/Asp-rich carboxy-terminal domain 3 (cited3), and reticulon 4a (rtn4a) were co-localized with miR-206 in the fast muscle of trunk somites. Then, we demonstrated that miR-206 was able to greatly repress luciferase activity through binding to the 3’UTRs of cited3 and rtn4a mRNAs in HEK293T cells, C2C12 cells, and even zebrafish embryos. On the other hand, knockdown of endogenous miR-206 by injecting miR-206 morpholino (MO) resulted in the increase of cited3 and rtn4a transcripts. Furthermore, injection of excessive cited3 mRNA and rtn4a mRNA resulted in shorter and longer of trunk somites, respectively. Interestingly, when knockdown of endogenous miR-206, the defect was similar to the embryos injected with rtn4a mRNA, suggesting miR-206 may play a role on normal development of somites. In addition, loss-of-miR-206, overexpression-of-cited3 and overexpression-of-rtn4a disrupted the organization of actin and myosin in the fast muscle. Importantly, the regulation of miR-206 to cited3 and rtn4a is myod mRNA expression-dependent. Evidence thus far accumulated leads us to hypothesize that miR-206 controls muscle development in zebrafish embryos through silencing cited3 and rtn4a, two novel miR-206-specific target genes unrelated to miR-1.