斑馬魚myf5 intron I 內含之microRNA 具有專一抑制myf5 表現之能力

Abstract

Myf5 是屬於MRFs(myogenesis regulatory factors)的其中一員，在肌肉細胞的特化與分化都扮演著十分重要的角色。myf5 的活化與抑制受到嚴密的調控，因此造成myf5於體節的表現具有時間與位置的特異性。研究發現斑馬魚 myf5 的intron I (+502/+835, I300)具有抑制myf5表現的能力，然而其機制目前還不清楚。在本論文中我們發現正向的I300 RNA具有抑制myf5 promoter活性的能力，然而反向的I300 RNA則無明顯效果。並且I300 RNA所導致的抑制作用具有promoter 的專一性，僅myf5 promoter活性受到抑制，而其他promoter如myod，cytomegalovirus 和thymidine kinase 則沒有受到影響。因此我們認為I300調控是透過RNA的層次。利用生物資訊以及Northern blotting實驗，我們在I300 RNA中找到一段intronic microRNA (miR-In, +610/+632)的序列。為了研究miR-In 的功能，我們在luciferase 的3’UTR接入miR-In的target sequence，並與I300 RNA共同顯微注射到斑馬魚胚胎中，結果顯示，I300 RNA 能抑制具有target sequence 的mRNA表現，使luciferase 活性下降至25 %。大量表達I300也會影響胚胎的發育，造成個體頭部與軀幹部位發育的不正常。在whole-mount in situ 實驗中，24-hpf的斑馬魚胚胎已形成的體節myf5已經消失表現，但miR-In卻仍然存在，顯示了miR-In存在可能抑制myf5在已形成體節的表現。綜合以上證據，我們認為存在於I300的miR-In 具有抑制myf5 表現的能力，並透過抑制myf5的表現達到調控肌肉的發育。

Myf5 is one of myogenesis regulatory factors (MRFs), which plays roles in the specification and differentiation of muscular cells during myogenesis. The expression of myf5 is in a somite- and stage-specific manner under a fine-tuned control mechanism. However, the molecular mechanism of repression of myf5 is still unknown, although repressive element within intron I (+502/+835, I300) of zebrafish myf5 was reported. In this study, we microinjected the upstream region (-6300 / -1) of zebrafish myf5 fused to a luciferase reporter gene and a sense RNA corresponding for I300 into the fertilized embryos, resulting the luciferase activity was down-regulated by sense I300 RNA, but was not affected by anti-sense I300 RNA. In addition, the I300-mediated repression was promoter-specific because only myf5 promoter activity was repressed, but not for other promoters, such as myod, cytomegalovirus and thymidine kinase. These evidences demonstrate that the zebrafish myf5 repression modulated by I300 was controlled at RNA level. Using bioinformatics and Northern blotting, we identified an intronic microRNA motif (miR-In, +610/+632) within I300 fragment. In order to study the function of miR-In, we microinjected a DNA construct in which the luciferase reporter fused with 5 copies of miR-In complementary sequences at its 3’UTR region, resulting the target mRNA expression was down-regulated by sense I300 RNA: the luciferase activity was reduced down to 25 % of control. Over-expression of I300 resulted in the morphological defect of brain and somites, which photocopied the embryos injected with myf5 morpholino, a myf5-specific translation inhibitor. In 24-hpf zebrafish embryos, the myf5 transcripts were abolished at the older formed somites, but miR-In was detected prominently at these somites, indicating that miR-In played an important role in repression of myf5 during somitogenesis. Based on these evidences, we propose that miR-In is involved in muscle development through repressing the expression of myf5.