剪接變異體產物TSGΔ154-1054對TSG101 蛋白質穩定性與生物功能之影響

Abstract

腫瘤易感基因101 (Tumor susceptibility gene 101，TSG101)，首先利用老鼠纖維母細胞 (fibroblast) 進行heterozygous knockout strategy中所篩選出來，如今被認為反而與細胞癌化有著顯著的關連性。先前研究發現TSG101為細胞生長所必需，並且也在某些惡性腫瘤細胞當中可見到TSG101表現量有上升之情況。除此之外，TSG101在內體運輸、細胞質分裂、調控細胞內基因表現和病毒出芽均扮演重要的角色。在惡性腫瘤細胞當中可發現tsg101基因並不會發生基因序列上之突變，但卻會因為選擇性剪接產生不同之mRNA isoform並且更進一步也証實某些特定的isoform與腫瘤的發展有著極重要的關連性。根據本實驗室先前的研究發現，在鼻咽癌組織中可偵測到一特定選擇性剪接之產物TSGΔ154-1054，但在非惡性細胞之淋巴增生組織則不會偵測到，另外也發現在表現TSGΔ154-1054之鼻咽癌組織當中TSG101表現量也有上升的情形，因此本篇研究主要著重於TSGΔ154-1054之表現與TSG101表現之關係。利用短期轉染實驗，可見TSGΔ154-1054影響TSG101表現是透過轉譯層次而非轉錄層次。在pulse-chase和cycloheximide-chase實驗中證實TSG101表現量上升並非增加TSG101之新生合成而是延長其半衰期，除此之外，將TSGΔ154-1054之起始密碼ATG置換成AAA後，TSGΔ154-1054無法轉譯成蛋白質其延長TSG101半衰期之能力也隨之失去，証實了TSGΔ154-1054影響了TSG101表現是透過其蛋白質產物。進一步分析，TSGΔ154-1054可藉由與TSG101競爭和Tal之結合，進而防止了TSG101降解，而延長了TSG101之半衰期。最後亦證實了TSGΔ154-1054可以藉由增加TSG101的表現量促進細胞的增生和腫瘤的生成。

Tumor susceptibility gene 101 (TSG101) was originally discovered in screening potent tumor suppressor gene using heterozygous knockout strategy in mouse fibroblasts models. Rather, this gene is now being regarded with oncogenic potency. Previous studies have suggested that TSG101 is required for cell growth and proliferation, and is over-expressed in some human cancer cells. In addition, TSG101 also plays a crucial role in endosomal trafficking, cytokinesis, transcriptional regulation of cellular genes and virion budding. Although the genomic mutation of TSG101 is not founded in cancerous tissues, the alternative splicing patterns of TSG101 pre-mRNA has been identified in various human cancers. Of note, the alternative splicing isoform TSGΔ154-1054 (a deleted fragment in TSG101 nucleotides 154-1054) strongly correlated with tumor progression and advanced tumor stages. In our previous studies, the over-expression of TSGΔ154-1054 was found in nasopharyngeal carcinomas (NPC) biopsies but not in lymphoid hyperplasia (LH) control tissues. In addition, the strong correlation of over-expression of TSGΔ154-1054 and up-regulation of TSG101 protein was found in NPC biopsies. So, the relationship between over-expression of TSGΔ154-1054 and TSG101 protein expression level is the target issue in this study. In transfection assay, we found that expression of TSGΔ154-1054 enhances the expression of TSG101 at protein level but not transcrional level. In cycloheximide experiment, TSGΔ154-1054 prolongs TSG101 half-life. Data from TSGΔ154-1054 start codon mutant assay indicated that protein product of TSGΔ154-1054 is required for its ability of prolonging TSG101 half-life. Mechanistically, TSGΔ154-1054 competing with TSG101 from binding TSG101-specific E3 ubiquitin ligase (Tal) impairs the TSG101 proteasome degradation and thus prolonging TSG101 protein half-life. Of note, the biological consequences of expression of TSGΔ154-1054 are promoting cell proliferation and enhancing tumor oncogenecity. To our knowledge, this is the first study to reveal the relationship between TSG101 and its alternative splicing variant TSGΔ154-1054. Also, this is the first study to show the biological influence of alternative splicing variant TSGΔ154-1054 on cell proliferation and tumor formation via enhancement the stabilizing of its wild type TSG101 proetin.