2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38635The Muscleblind (Mbnl) is a family of proteins that participate in the regulation of tissue-specific alternative splicing. They bind to RNA through a conserved CCCH zinc finger domain. Three paralogs (Mbnl1, 2, and 3) have been identified in mammalian, each promotes inclusion or exclusion of specific exons on different genes. Misregulation of Mbnl activity in human is associated with hereditary disorders mediated by RNA pathogenesis, most importantly myotonic dystrophy (DM). The muscleblind is required for terminal differentiation of muscle and photoreceptors in Drosophila. In addition, it is suggested that Mbnl may participate in the differentiation of several cell types, including neurons, adipocytes and blood. Alternative splicing is an important mechanism to generate protein diversity, of which the control is spatially and temporally coupled during development. In order to investigate the functions of Mbnl proteins in early vertebrate development, we propose to use zebrafish, an excellent vertebrate model for embryologic and genetic analysis, as a model system in this study. We have first cloned the three paralogous mbnl genes in zebrafish. Characterization of the structural features and analyses of the expression patterns of these genes have also been performed. In this study, further experiments are proposed to fulfill the following specific aims: (1) To investigate the roles of specific Mbnl protein in early zebrafish development, by using the antisense morpholino knockdown, rescue and overexpression strategies followed by phenotype analyses; (2) To identify the potential downstream splicing targets regulated by specific Mbnl proteins, by examining the splicing patterns of genes known to be misregulated in DM and identification of unknown targets by proteomic analysis; (3) To identify the potential upstream regulators of the Mbnl activity, by analyzing the mef2 pathway and the other potential transcriptional regulators; (4) To elucidate the mechanism of down-regulation of vinculin by Mbnl; (5) To evaluate the protein-protein interactions of Mbnl proteins.Muscleblind (Mbnl)是一參與組織特異性選擇性剪輯調控的蛋白質家族。它們藉由一個演化保留的CCCH鋅指區域結合RNA。在哺乳類已有三個同源基因(Mbnl1, 2和3)被選殖出來，它們可個別促進不同基因上特定外顯子的保留或排除。Mbnl活性的異常調節在人類已被證實與遺傳疾病-特別是RNA致病機轉的強直型肌肉萎縮症-有關。在果蠅中，muscleblind對肌肉及光感受器的末端分化是必需的。此外，其它研究也指出Mbnl可能參與包括神經元、脂肪細胞、及血液等數種不同種類細胞的分化。選擇性剪輯是用來產生蛋白質多樣性的一種重要機制，此機制在生物發育過程的時空中被嚴密的調控。為了探討Mbnl蛋白質在早期脊椎動物發育中的功能，我們擬以斑馬魚作為模式系統。斑馬魚目前已經成為研究胚胎發育和遺傳分析的一個極優異的脊椎動物模式。為開啟這個研究，我們首先選殖了斑馬魚的三個mbnl同源基因，並且分析其結構特徵及其基因表現型式。在此計畫中，我們提出進一步的實驗以達成下列的具體目標：(1) 研究特定的Mbnl蛋白質在早期斑馬魚發育扮演的角色，藉由反意morpholino基因抑制、基因救援及基因過量表現等策略，分析其改變的表型；(2) 找出特定的Mbnl蛋白質其可能的下游剪輯標的，先檢查已知在強直型肌肉萎縮症異常調控的基因，再以蛋白質體學分析探討未知的標的；(3)尋找調節Mbnl活性的可能上游分子，藉由分析Mef2訊息途徑及其它可能的轉錄調控因子；(4)闡明Mbnl對vinculin基因表現下降的調控機制；(5)探討Mbnl與本身或其他蛋白質之間的作用關係。Muscleblindmbnl選擇性剪輯斑馬魚發育基因抑制muscleblindmbnlalternative splicingzebrafishdevelopmentknockdownProject