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Title: | RBM4.1參與降低human chop uORF序列所主導的轉譯抑制的能力 RBM4.1 is involved in repressing the translation inhibition mediated by human chop uORF sequence |
Authors: | Yu-Fan Chueh 闕郁帆 |
Advisor: | 蔡懷楨 |
Keyword: | 內質網壓力,內質網相關壓力,缺氧,chop,upstream open reading frame, ER stress,ER-associated stress,Hypoxia,chop,upstream open reading frame, |
Publication Year : | 2014 |
Degree: | 碩士 |
Abstract: | 在in vitro的情況下,已知細胞受到外界壓力刺激,如hypoxia,會促使內質網壓力(Endoplasmic reticulum stress, ER stress)產生,以應對外界壓力。此外,ER stress也發現和人類的神經疾病,如帕金森氏症或阿茲海默症等有所相關。但是ER stress在疾病或發育過程中的具體角色並不明朗,因此探討ER stress在in vivo中的調控是一個急待探討的領域。其中在ER stress的下游基因,DNA damage-inducible transcript 3, 又被稱作C/EBP homologous protein (CHOP),其5’ 端的untranslated region(5’ UTR)中的upstream open reading frame (uORF)卻主導了本身基因轉譯的調控。然而對於ER stress如何造成chop uORF主導轉譯抑制能力的失效,其分子機制仍舊不清楚。因此本研究利用帶有人類chop uORF (huORFchop)與GFP reporter gene的基因轉殖斑馬魚品系(huORFZ)作為實驗動物,經hypoxia處理後會誘導huORF的translational inhibition功能失效,而促使GFP轉譯而產生螢光。並且表現在中樞神經系統。藉由Fluorescence-activated cell sorting將細胞群體區分為GFP(+)和GFP(-),再用microarray偵測這兩個細胞群體中的transcriptome上的變化量偵測。結果顯示,significant up的基因有1295個,而significant down有3018個。接著,我們利用The Database for Annotation, Visualization and Integrated Discovery的分析軟體,將microarray中有著顯著下降差異的基因進行功能性分群後,以RNA binding群組作為本研究對象。我們鎖定其中的兩個基因,celf2和rbm4.1,因為其基因表現量(microarray中的A值)大於8以上;並且這兩個基因皆擁有對mRNA轉譯調控的功能。若用whole mount in situ hybridization偵測這兩個基因在胚胎受到ER stress和在正常狀態的表現相比,發現celf2表現量下降符合microarray結果而rbm4.1表現量上昇和microarray不同。而在in vitro transcription後,將celf2和rbm4.1 mRNA分別注射至huORFZ胚胎內以增加mRNA的表現,結果(1)注射celf2的胚胎有GFP表現,但與施加stress後的胚胎GFP表現率並沒有減少,表示celf2可能沒有減弱huORFchop轉譯抑制的功能。(2)注射rbm4.1的胚胎在沒有施加stress下,卻會在眼睛和(或)軀幹轉譯出GFP,表示過量表現rbm4.1會降低了huORFchop轉譯抑制的能力。因此我們推測rbm4.1基因可能參與在ER stress時,huORFchop序列失去轉譯抑制能力而開啟下游coding sequence的功能。 In vitro, for example, hypoxia, cells response endoplasmic reticulum stress(ER stress) to compromise the unbalance homeostasis. In vivo, ER stress has an important role with human neurodegenerative disease, for example, Alzheimer’s and Parkinson’s. However, it’s not clear about the mechanism in disease or development affecting by ER stress in vivo. The downstream gene in unfolded protein response of ER stress, chop, is regulated by its own uORF of 5’UTR. However, the regulation mechanism of the uORFchop translation inhibition is not clear. This research, we use a transgenic zebrafish line, called huORFZ carried with human chop uORF and a GFP reporter gene, to selection the candidate gene to regulation the uORFchop by inducing hypoxia to create ER stress. Using hypoxia, it’s proved that the function of uORFchop translation inhibition is inactivated and translates GFP at central nerve system. By embryos cell suspension and Fluorescence-activated cell sorting (FACS), we collect two kinds of cell population, GFP+ and GFP- cells to run microarray. By comparing the transcriptome of these two groups with a microarray, we could select the candidate gene for regulation the uORFchop. There is 1295 genes for significant up and 3018 for down in microarray. Next, we use the analysis software, the Database for Annotation, Visualization and Integrated Discovery (DAVID), to analyze the significant down expressed genes of the microarray described above. By selecting the RNA binding chart, the gene annotation clustering result by DAVID, we conclude two potential gene, celf2 and rbm4.1 because these two genes’ the average intensity number (A) of microarray are higher than 8. And these two genes have the ability for regulating specific mRNA translation. In WISH, we find celf2 has a down expression pattern but rbm4.1 is opposed comparing with the no hypoxia induced embryos. In the end, we use microinjection to overexpress these two genes in huORFZ. In 96 hpf, we find (1) celf2 expresses a weak pattern of GFP in huORFZ but these injected embryos gets the same GFP express ratio with controls. Therefore we suggest that celf2 may not participate in inhibition of huORFchop translation inhibition. (2) rbm4.1 overexpression in huORFZs expresses GFP at eyes and(or) trunk with no any stress. This result shows overdose of rbm4.1 can depress the translation inhibition ability of huORFchop. In conclusion, we suggest that rbm4.1 has a role in regulating the huORFchop translation inhibition during ER stress. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18671 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 分子與細胞生物學研究所 |
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