2021-01-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/702551Musashi-1與Musashi-2是兩個相似的核糖核酸結合蛋白，皆含有RNA-recognition motif (RRM)，經過調節多種目標核糖核酸的表現，在哺乳動物中調控神經幹細胞的分裂與分化。Musashi蛋白質也經由調控許多致癌基因或維持幹細胞性狀的基因，而在多種癌症的生成中扮演重要的角色。在線蟲中，只有一個Musashi同源基因msi-1被發現，msi-1在線蟲胚胎發育及神經幹細胞中大量表現，到目前為止，線蟲MSI-1是否在後胚胎發育期在其他的類幹細胞中，如線蟲的上皮接縫幹細胞，發揮調節基因表現的功能，則仍不清楚。此外，此種與幹細胞發育分化調控相關的Musashi蛋白，是否在功能上與也和調控幹細胞發育分化的let-7微小核糖核酸有交互作用，也是未知的。在線蟲中，let-7(n2853)帶有一個G-to-A的點突變，使得let-7微小核糖核酸與其目標結合不穩定化，並使得let-7的主要目標LIN-41表現上升。LIN-41蛋白將抑制其下游基因lin-29，而lin-29的基因產物為一成蟲專一表現的轉錄因子，並且是人類EGR1 (Early Growth Response)轉錄因子的同源蛋白。此一let-7/LIN-41/LIN-29基因調控路徑調節線蟲的上皮接縫幹細胞的分裂與分化。功能降低的let-7微小核糖核酸影響接縫幹細胞的發育，並使得接縫幹細胞與生殖孔細胞之間接合不良，而使得此突變種線蟲易在進入成蟲期時因為生殖孔爆裂而死亡。我們發現，利用核糖核酸干擾技術降低msi-1基因表現，可以有效抑制let-7(n2853)突變種生殖孔爆裂的現象，並且抑制其接縫幹細胞分化不良的現象。但是在lin-29(n333) loss-of-function完全失去功能的突變種中，接縫幹細胞分化不良的現象就無法由降低msi-1基因表現來抑制。我們的初步結果顯示MSI-1核糖核酸結合蛋白可能參與線蟲的let-7/LIN-41/LIN-29基因調控路徑，去影響接縫幹細胞的發育。在此計畫中，我將利用基因剔除的方法，及並建立一套新穎的雙螢光報告子，來研究MSI-1核糖核酸結合蛋白在線蟲的let-7/LIN-41/LIN-29基因調控路徑中所扮演的角色，並藉由生化方法來研究Musashi蛋白可能在線蟲與人類細胞中影響let-7/LIN-41/LIN-29基因調控路徑的機制。我們的研究結果將有助於了解Musashi蛋白在let-7微小核糖核酸調節基因表上的新穎功能，並且建立篩選與Musashi蛋白及let-7/LIN-41/LIN-29基因調控路徑有交互作用的分子之線蟲模式生物平台。 The RNA-binding proteins (RBPs) Musashi1 and Musashi2 are two closely related RNA-recognition motif (RRM) containing proteins that regulate neural stem cell proliferation in mammals via controlling the fate of various target mRNAs. The Musashi proteins play a carcinogenic role by regulating mRNA stability and protein translation of multiple genes involved in oncogenic signaling pathways or stemness maintenance. In C. elegans, expression of the sole homolog Musashi-1 (msi-1) has been detected in embryogenesis intensely and also in multiple neuron stem cells. So far, whether the C. elegans MSI-1 could play roles in post-embryonic stem cell-like division outside the nervous system, such as those in epithelial seam cells, remains unclear. Also, whether stemness-related of Musashi proteins is linked to the well-characterized miRNA-mediated stem cell program, such as let-7 determining cell differentiation and stemness, is unknown. In C. elegans, the let-7(n2853) mutation changes the fifth G to an A in the mature let-7 miRNA, leading to destabilization of target interactions and up-regulation of its major target LIN-41. LIN41 represses its downstream target lin-29 that encodes an adult specific transcription factor, homologous to human EGR1 (Early Growth Response) family proteins. The let-7/LIN-41/LIN-29 pathway regulates terminal differentiation of stem cell-like epithelial seam cells. Dysfunction of let-7 in seam cells cause impaired attachment of the vulva to the seam and bursting of the vulva. Here, we found that msi-1(RNAi) significantly suppressed the lethal let-7(n2853) vulva bursting phenotype. Interestingly, we found that msi-1(RNAi) also suppressed abnormal terminal differentiation of stem cell-like seam cells. By contrast, msi-1(RNAi) failed to suppress the same retarded seam phenotype in lin-29(n333) loss-of-function worms.核醣核酸結合蛋白let-7 微小核醣核酸線蟲MSI-1LIN-41RNA-binding proteinlet-7 microRNAC. elegansMSI-1LIN-41