人類 ENDOU-1 與同源基因斑馬魚 Endouc 皆會減弱內質網壓力中由人類 uORFchop所主導抑制轉譯的能力

Human ENDOU-1 and Its Ortholog Zebrafish Endouc Attenuates Human uORFchop- mediated Translational Inhibition during ER stress

郁芸(Yun Yu)

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(2016年)

https://doi.org/10.6342/NTU201602551

摘要

當細胞受到Endoplasmic reticulum (ER) stress時，C/EBP homologous protein (CHOP) 對於細胞存活與凋亡扮演重要的角色，而CHOP mRNA的轉譯主要受到其5’UTR上的upstream open reading frame (uORFchop) 所抑制。當細胞受到ER stress時，CHOP mRNA會因為uORFchop抑制功能喪失而被轉譯出來。但對於ER stress造成uORFchop喪失轉譯抑制能力的分子機制仍不清楚。本實驗室先前利用由CMV promoter驅動下游帶有人類uORFchop (huORFchop)及綠色螢光蛋白(green fluorescent protein,GFP) cDNA之基因構築的轉殖斑馬魚品系huORFZ作為平台，因為當它受到ER stress時，便會引發huORFchop喪失功能而促使GFP表現。前人利用雷射顯微切割器(Laser microdissection) 的方式收集逆境後表現於huORFZ的GFP-(+) 細胞，接著以GFP-(-) 細胞作為比較基礎，建立microarray資料庫。當中，本實驗室篩選到一個相關斑馬魚基因endouc。然而，人類參與在調控 huORFchop 的基因仍不清楚。為了證實人類也具有在huORFchop擔任轉譯抑制的基因，首先我以生資軟體比對氨基酸序列，獲知斑馬魚 Endouc 與人類的 ENDOU同樣都含有endoribonucleases能力的 XendoU domain。接著，利用RNase activity的實驗突顯了人類 ENDOU與斑馬魚 Endouc 一樣都會透過其endoribonuclease的能力把長片段的huORFchop mRNA序列切割為小片段。而在in vivo 方面，斑馬魚胚胎若大量表現人類 ENDOU mRNA可以降低因斑馬魚 endouc knockdown 所造成胚胎型態異常的比例，顯示人類 ENDOU的功能可以彌補斑馬魚 endouc 的缺失。In vitro 方面，在HEK293T大量表現人類 ENDOU會使磷酸化 eIF2α 及CHOP蛋白質的表現上升卻不會有 ER stress 產生，所以大量表現人類 ENDOU並不是誘發 ER stress而產生的副作用。而大量表現人類 ENDOU 基因發現會促使5’-UTR帶有 huORFchop 的luciferase 活性上升，這結果表示人類 ENDOU會壓制huORFchop的轉譯抑制能力，開啟下游基因的轉譯。進一步地為了證實人類 ENDOU 其 endoribonuclease的能力與huORFchop 轉譯抑制能力的關係，我構築了一失去 endoribonuclease 活性的突變株 ENDOU-1H285A ，而大量表現 ENDOU-1H285A會使磷酸化 eIF2α 及CHOP蛋白質的表現量都減少，顯示人類 ENDOU其 endoribonuclease的活性會影響 huORFchop 轉譯抑制的能力。最後， polysome profiling assay 的結果顯示，人類 ENDOU在正常情況下會與translation相關的蛋白質結合在polysomes上；而在ER stress的情況下，ENDOU則會離開與 polysomes 的結合反而以 free form形式游離在細胞中。
綜合上述實驗結果，本研究發現 (1)人類 ENDOU 與斑馬魚 Endouc具有功能上的保守性；(2) 兩者藉由 endoribonuclease 的能力會對 huORFchop mRNA 之結構進行切割而壓制了 huORFchop 抑制轉譯的能力；(3)人類 ENDOU 會與translation 相關蛋白結合在polysomes，直到 ER stress時ENDOU會利用它具有的endoribonuclease活性切割 huORFchop mRNA的5’-UTR 序列而脫離polysomes，以重新開啟下游 CHOP 的轉譯。因而我們提出這一個新發現的機制。

關鍵字

斑馬魚 ； 內質網壓力 ； uORF 轉譯抑制

並列摘要

In response of endoplasmic reticulum (ER) stress, C/EBP homologous protein (CHOP) is critically involved in either cell survival or apoptosis. It has been reported that the 5’UTR of CHOP mRNA contains an upstream open reading frame (uORFchop) which inhibits the translation of downstream coding sequence (CDS) of CHOP. Yet, the uORFchop-mediated translational inhibition of CDS is abolished when ER stress is encountered, resulting in generating CHOP protein. However, underlying molecular mechanisms of uORFchop -mediated translational inhibition is not fully understood. To answer this issue, we employed the zebrafish transgenic line huORFZ, which harbors the GFP reporter fused with the human uORFchop (huORFchop) and driven by a cytomegalovirus promoter. Interestingly, the downstream reporter GFP was expressed only when huORFZ embryos were treated with ER stress. Since the endonuclease poly(U)-specific C gene (endouc) of zebrafish is proven to be involved in the huORFchop-mediated translational inhibition by my colleague, here I studied whether human ENDOU gene, which is orthologue of zebrafish endouc, might also play role on the repression of huORFchop-mediated translational inhibition. When the amino acid sequences of human ENDOU and zebrafish Endouc were compared, I found that they shared a XendoU domain, which possesses an endoribonuclease ability. Furthermore, I demonstrated that human ENDOU and zebrafish Endouc were functionally conserved because (a) both of them were able to cleave the huORFchop mRNA at uridylates located within the loop structure in a Ca2+-dependent manner; and (b) injection of human ENDOU mRNAs could rescue the occurrence of phenotypes caused by knocking down of endouc in zebrafish embryos. Next, we overexpressed the human ENDOU in HEK293T cells without giving stress, resulting in the increase levels of phosphorylated eIF2α (p-eIF2α) and CHOP proteins. Additionally, the luciferase (luc) activity driven by the huORFchop motif was extremely limited in huORFZ embryos at normal condition. However, the luc was greatly expressed if human ENDOU mRNAs were injected in those embryos even they were not treated with stresses. The line of above evidence suggested that overexpression of human ENDOU did suppress the huORFchop-translational inhibition in vitro, resulting the downstream CDS such as luc cDNA was translated. Furthermore, to demonstrate that the endoribonuclease ability of ENDOU is involved in the repression of the huORFchop-translational inhibition, I constructed a mutation form, ENDOUH285A, in which its endoribonuclease activity was impaired. Interestingly, overexpression of ENDOUH285A mRNA in HEK293T cells led to decrease the protein levels of both p-eIF2α and CHOP, suggesting that the endoribonuclease activity of human ENDOU is involved in suppressing the huORFchop-translational inhibition. Finally, I used the polyribosome profiling assay to demonstrate that, at normal condition, ENDOU was bound at polysomes; while under at ER stress, ENDOU was released from polysomes and presented as a free form by taking advantage of its endoribonuclease activity. Collectively, we conclude that, like zebrafish Endouc, human ENDOU plays role on the huORFchop-mediated translational inhibition since human ENDOU and zebrafish Endouc are functionally conserved. Thus, I hypothesize that, under stress condition, ENDOU is able to recognize specific nucleotides within huORFchop structure located at 5’ end and digest thereof mRNA, resulting in a cap-independent mRNA, which in turn, the downstream CDS of CHOP cDNA is therefore reinitiated to translate through bypassing the hindrance of huORFchop structure.

並列關鍵字

zebrafish ； ER stress ； uORFchop- mediated Translational Inhibition ； ENDOU familiy

參考文獻

胡家榕. (2015) dkey 參與內質網壓力中去除由人類 uORFchop 所主導的轉譯抑制. 國立台灣大學生命科學院分子與細胞生物研究所碩士論文。

Back SH, Schröder M, Lee K, Zang K, Kaufman RJ. (2005) ER stress signaling by regulated splicing IRE1/HAC1/XBP1. Methods, 35:395-416.

Barone MV, Crozat A, Tabaee A, Philipson L, Ron D. (1994) CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest. Genes Dev., 8:453-64.

Booth C, Koch GL. (1989) perturbation of cellular calcium induces secretion of luminal ER proteins. Cell, 59:729-37.

Bruhat A, Jousse C, Wang XZ, Ron D, Ferrara M, Fafournoux P. (1997) Amino acid limitation induces expression of CHOP, a CCAAT/Enhancer binding protein-related gene, at both transcriptional and post-transcriptional levels. J Biol Chem., 272:17588-93.