The microRNA miR-1 regulates fast-twitch muscle actin organization through silencing the target gene seryl-tRNA synthetase in zebrafish embryos
Date Issued
2009
Date
2009
Author(s)
Yang, Hsin-Jung
Abstract
The microRNA (miRNA) is a short (19-22nt) and endogenous non-coding RNA that silences gene expression at the post-transcriptional level by means of binding to the 3’-untranslated translated region (3’UTR) of target mRNA via a conservative seed sequence (5-8 nt) of miRNAs. miR-1, a muscle-specific miRNA, is significantly expressed in cardiac and skeletal muscle. However, the detailed molecular regulatory mechanism of miR-1 in the skeletal muscle is still unknown. Previously we screened the target mRNAs of miR-1 from whole-cell extracts of zebrafish embryos of 48 hpf by both miRNA-pull down assay (Hsu et al., 2009) and microarray analysis. We obtained the putative target gene seryl-tRNA synthetase (sars). In this study, using whole-mount in situ hybridization, we observed that the expression patterns of miR-1 and sars were co-localized in the somites during 18-24 hpf, and they were co-localized in the fast-twitch muscle of trunk after muscle formation. Then, we went further to confirm that miR-1 was able to repress the luciferase activity through binding to the 3’UTR of sars mRNAs in COS-1 cells and zebrafish embryos, respectively. Compared to the control group, the expression levels of luciferase reporter constructs harboring the 3’UTR of sars were reduced 90.6±1.4 % and 64.6±4.74%, respectively. And using Western blotting, we validated miR-1 could repress Sars protein expression. On the other hand, we observed that overexpression of miR-1 resulted in somitic atrophy and indistinct somite boundary. Interestingly, when knockdown of sars by injected with sars-MO，the defect was similar to the embryos injected with miR-1 RNA。And the miR-1-induced defect could be partially rescued by co-injection of sars mRNA. In addition, we observed that overexpression of sars, loss-of sars and loss-of miR-1 disrupted the fast-twitch muscle actin organization. Taken together, we demonstrated that sars was a direct target gene of miR-1. By directly inhibiting the expression of sars, miR-1 could regulate actin organization of zebrafish trunk fast-twitch muscle to impact on its formation and maintenance.
Subjects
microRNA
miR-1
fast-twitch muscle
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