TSG101蛋白質之穩定性受其剪接變異體作用並藉此影響Epstein-Barr病毒晚期基因的轉錄

Stabilization of TSG101 protein by its splice variant and the impact on Epstein-Barr viral late gene transcription

蔡慧慧(Huey-Huey Chua)

指導教授 ： 蔡錦華

國立臺灣大學/醫學院/微生物學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02071

摘要

於細胞癌化過程中，其前體信使核糖核酸 (pre-mRNA)之剪接(splicing) 常喪失了正常精確的自我調節能力，以致出現異常剪接 (aberrant splicing)現象；Tumor susceptibility gene 101 (TSG101) pre-mRNA之剪接即是此過程中常受影響的範例之一。在許多種癌組織細胞中，TSG101 mRNA的表現，除了其全長的TSG101 mRNA外，亦可同時偵測到其它截形(truncated)的TSG101 mRNA同質體 (isoform)；尤其是剪除TSG101核苷酸第154至1054 之mRNA (於後稱為TSG101-247)，更是TSG101諸多異常剪接產物中出現頻率最高的一員。本研究發現鼻咽癌病理組織亦不例外，我們在其中也偵查到TSG101-247的表現。獨特的是，TSG101-247在鼻咽癌細胞株中的表現與EBV的感染息息相關，尤其是EBV進入溶裂期(lytic cycle)時，TSG101-247在該細胞中的表現量較EBV滞留在潛伏期(latent state)者更勝一籌。由基因轉染試驗顯示，EBV 的病毒蛋白質EBNA1，Zta 與Rta會干預TSG101 mRNA的剪接並促成TSG101-247產生。
在功能上，我們發現TSG101-247可增強TSG101蛋白質的穩定性(stability)，其中關鍵機制在於TSG101-247可抑制TSG101的多次泛素化(polyubiquitination)，進而使TSG101不被蛋白解體(proteasome)分解。TSG101-247此功能需待其轉譯成蛋白質後方能執行，因若將TSG101-247 的啟動密碼子(start codon) AUG突變成AAA後，TSG101-247無法轉譯成蛋白質，其穩定TSG101的能力也隨既消失。再者，若將TSG101-247蛋白質上的離氨基酸基(Lysine residue, K)置換成精氨酸(Arginine, R)，使其無法與泛素(ubiquitin, Ub)結合，如此也會導致TSG101-247穩定TSG101的能力喪失。可見TSG101-247蛋白質與Ub的結合能力攸關其是否能穩定TSG101。深入分析，我們發現TSG101-247實與TSG101競爭結合TSG101專屬的E3 Ub ligase，稱TSG101-associated ligase (Tal)。一旦TSG101-247結合了Tal之後，所引發的結果與TSG101相似---TSG101-247會被多次泛素化。經由此途徑，TSG101-247不僅增強了TSG101蛋白質的穩定性，同時亦強化了TSG101在細胞中的機能活性。因表現TSG101-247的細胞，其中TSG101抑制雄性荷爾蒙受體(androgen receptor)活化基因表現以及TSG101刺激癌細胞生長的能力均顯著增強。針對EBV而言，我們發現TSG101-247透過強化TSG101蛋白質的穩定性亦可增進EBV晚期基因(late gene)的表現。
有鑒於此，我們開始探究EBV是否利用TSG101來協助其發展溶裂期並產出子代。採用微小核糖核酸干擾(small interfering RNA, siRNA)策略剔除細胞中內質性TSG101，隨後再利用化學藥物激發EBV溶裂期之進行。透過微陣列(microarray)實驗法發現多種EBV晚期基因，會因TSG101的缺乏而表現受到阻滯，包括︰ BcLF1，BDLF3，BILF2，BLLF1及BLRF2等病毒基因。進一步，由reporter gene assay實驗結果獲悉此五個晚期基因的活化主要皆受EBV溶裂期的轉活化子Rta調控。經TSG101 siRNA 減除TSG101後，Rta活化此五個晚期基因的能力便顯著下降。然而，若增加細胞中TSG101的表現量， Rta啟動這些晚期基因表現的能力則隨之遞增。其中的作用機制可能是Rta透過增加TSG101蛋白質在核中的表現量，增大了其與TSG101蛋白質在核中的結合機會。Rta藉此結合使其順利地裝載在EBV晚期基因的啟動子(promoter)上，進而活化了EBV晚期基因的表現。若TSG101此時被siRNA剔除，則Rta結合到EBV晚期基因 啟動子上的能力將嚴重受損，病毒的晚期基因產物因此而不再表現，病毒粒子的合成也告失敗。
總而言之，於本研究我們首要提出TSG101的異常剪接產物TSG101-247具備了穩定 TSG101蛋白質的獨特功能，並藉此增進TSG101在細胞中的機能活性。另，本研究亦揭發了TSG101在Rta活化EB 病毒晚期基因上的重要貢獻。此結果顯示，DNA病毒與RNA病毒一樣都會奪取細胞中的TSG101來協助他們繁衍子代。唯二者利用TSG101促成病毒粒子產生的方式可能不一樣。DNA病毒如EBV利用TSG101協助其表現晚期基因進而產生病毒顆粒。然而，enveloped RNA病毒則藉用TSG101運送其晚期基因產物至細胞膜表面以便組裝成病毒粒子。

關鍵字

Epstein-Barr 病毒 ； 晚期基因 ； Rta ； 剪接 ； 轉錄 ； Tumor susceptibility gene 101 (TSG101) ； 泛素

並列摘要

The fidelity of splicing is lost in most cancers resulting in aberrant splicing of pre-mRNA. Tumor susceptibility gene 101 (TSG101) is a frequent target of these splicing defects, as evidenced by co-expression of its full-length transcript with truncated mRNA isoforms in a variety of human cancers. This study demonstrates that nasopharyngeal carcinomas (NPC) tissues express an abundance of TSG101 transcripts with nucleotides 154-1054 deleted (designated TSG101-247). In particular, Epstein-Barr virus (EBV)-infected NPC cells harbor this splice variant, and the incidence of expression of this variant increases during the EBV lytic cycle. The EBV-encoded viral proteins, EBNA1, Zta and Rta, are demonstrated as crucial factors that involved in the enhancement of TSG101 aberrant splicing. Mechanistically, TSG101-247 stabilizes TSG101 by impairing its polyubiquitination and subsequent proteasomal degradation. Mutation of the TSG101-247 start codon showed that the variant protein, and not the RNA, blocks polyubiquitination. In addition, mutation of lysine residues in TSG101-247 that particularly disrupt the conjugation of ubiquitin (Ub) moiety negates this effect, resulting in rapid turnover of TSG101 and suggesting a requirement for Ub-chain attachment in the stabilization of TSG101 mediated by TSG101-247. To explore the underlying mechanism further, we demonstrated that TSG101-247 prevents the polyubiquitination of TSG101 by binding competitively to the TSG101-associated ligase (Tal), an E3 Ub ligase. Consequently, TSG101-247 augments the functional activities of TSG101, having a negative effect on androgen receptor-induced gene activation and a positive effect on cell proliferation. Moreover, TSG101-247 enhances TSG101-induced EB viral late gene expression.
The requirement of TSG101 protein for EB virion formation has been established herein by an RNA interference assay. Results from an EBV DNA array reveal that depletion of TSG101 potently inhibits the transcription of five Rta-responsive EBV late genes, BcLF1, BDLF3, BILF2, BLLF1 and BLRF2. Rta is an EBV encoded immediate-early protein, which governs the reactivation of the viral lytic program by transactivating a cascade of lytic gene expression. TSG101 is involved in the mechanism underlying the expression of these EBV late genes mediated by Rta, seeing that depletion of TSG101 impairs Rta transactivation of these late promoters severely. Moreover, a concordant augmentation of Rta transactivating activity is observed when TSG101 is over-expressed following ectopic transfection. Mechanistically, Rta causes the TSG101 protein to accumulate principally in the nuclei, wherein both these proteins interact and are recruited onto the viral promoters. Of note, TSG101 is crucial for efficient binding of Rta onto these late promoters. As a result, cells with defective TSG101 fail to express late viral proteins, leading to a decrease in the yield of virus particles.
Altogether, we provide the first functional evidence of the splicing variant of TSG101. TSG101-247 is a novel regulator of TSG101 stability thereby facilitating role of TSG101 in cellular compartment. In addition, the contribution of TSG101 to Rta-mediated late gene activation is of great importance for completion of the EBV productive lytic cycle. These observations consolidate a role of TSG101 in the replication of EBV, a DNA virus, that differs from that for RNA viruses, where TSG101 aids mainly in the endosomal sorting of enveloped late viral proteins for assembly at the plasma membrane.

並列關鍵字

Epstein-Barr virus (EBV) ； Late gene ； Rta ； Splicing ； Transcription ； Tumor susceptibility gene 101 (TSG101) ； Ubiquitin (Ub)

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