microRNA miR-1 藉由直接抑制目標基因 seryl-tRNA synthetase 以調控斑馬魚快肌之肌動蛋白排列

The microRNA miR-1 regulates fast-twitch muscle actin organization through silencing the target gene seryl-tRNA synthetase in zebrafish embryos

楊欣蓉(Hsin-Jung Yang)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10693

摘要

微型核醣核酸 (MicroRNA, miRNA) 為短片段 (~22 nt) 之非轉譯核醣核酸 (noncoding RNAs, ncRNAs)，其藉由保守性種子序列 (conservative seed sequence )與目標基因之 3’ 端非轉譯區 (3’- untranslated region, 3’UTR) 結合，於轉錄後層級 (post-transcriptional level) 調控基因的表現。 microRNA-1 (miR-1) 為肌肉專一型微型核醣核酸 (muscle-specific miRNA)，雖然已知其可專一地表現於心肌與骨骼肌，但是參與在軀幹部骨骼肌的分子調控機制尚不清楚。我們先前利用 Labeled microRNA pull-down (LAMP) assay system (Hsu et al., 2009) 篩選在 whole-cell extracts 中可與 miR-1 結合的 mRNA 片段，再以 microarray 進行比對分析及確認。結果發現在斑馬魚中 miR-1 可能與 seryl-tRNA synthetase (sars) 的3’ 端非轉譯區結合。於是本研究中，我們利用全胚胎原位雜交實驗 (WISH) 初步證實 miR-1 與 sars 在胚胎發育早期 18-24 hpf 之軀幹部肌節中具有同位性的表現，且至發育晚期 24-48 hpf 形成軀幹部肌肉時，也一起在快肌中同位性表現。進一步利用 Luciferase reporter assay system 於 COS-1 cells 與斑馬魚胚胎中證實於 in vitro 與 in vivo 系統中，miR-1 均可透過 sars-3’UTR 抑制報導基因的表現，其抑制能力分別為90.6±1.4 % 與 64.6±4.74%。並利用西方浸漬法證實 miR-1 可抑制 Sars 蛋白質的表現。另一方面，我們發現在胚胎中，若過量表現 miR-1 時，會造成胚胎體節萎縮及分節不明顯；而注射 sars 轉譯抑制劑 (morpholino oligonucleotides, MO) 降低 Sars 蛋白質表現時，則產生類似過量表現 miR-1 所造成之缺失。若於胚胎中同時注射 sars mRNA 與 miR-1，則可降低 miR-1 過量表現所造成的缺失比例。我們進一步發現於胚胎中過量表現 sars、降低 miR-1 以及降低 sars 表現時，均會造成斑馬魚快肌之肌動蛋白排列紊亂與活動力降低。綜合以上證據，我們認為 sars 為 miR-1 下游調控的基因，且 miR-1 可藉由直接抑制目標基因 sars ，來調控斑馬魚快肌之肌動蛋白排列，進而影響斑馬魚軀幹部快肌的形成與維持。

關鍵字

微型核醣核酸 ； 快肌

並列摘要

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.

並列關鍵字

microRNA ； miR-1 ； fast-twitch muscle

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