呂勝春臺灣大學:分子醫學研究所陳明達Chen, Ming-TaMing-TaChen2010-05-042018-07-092010-05-042018-07-092009U0001-1008200917152500http://ntur.lib.ntu.edu.tw//handle/246246/178711活化轉錄因子(ATF)家族成員為受壓力所誘導而表現的蛋白。活化轉錄因子牽涉細胞的增生、存活或細胞凋亡。它們的表現受到轉錄與轉譯層次上的調控，且可能在各種壓力的刺激下被誘導而表現。目前已知某些存在於活化轉錄因子的5端非轉譯區(5’ UTR)中的上游開放閱讀框架(uORF)為其下游本身開放閱讀框架(ORF)的轉譯調控者，但有許多此類元素仍未被知道其調控機制。在本篇研究中，吾人利用雙重螢光素酶報導基因檢驗(dual-luciferase reporter assay)以探討活化轉錄因子的上游開放閱讀框架所扮演的角色：我們發現並確認了它們在一般狀況下都會抑制下游自身的開放閱讀框架之轉譯作用；然而，上游開放閱讀框架的壓抑效力會受到特定的壓力信號刺激下而減弱。我們更進一步發現了活化轉錄因子間組成特徵相同的上游開放閱讀框架，會對同樣的壓力刺激有反應。此外，我們亦發現了在紫外線的照射下，有兩種不同的訊息傳遞路徑會影響到活化轉錄因子2的上游開放閱讀框架所調控的轉譯作用。Members of the activating transcription factor (ATF) family are stress-induced proteins. ATFs are involved in cell proliferation, survival or apoptosis. Their expression is regulated at both transcriptional and translational levels and may be induced by diverse stress stimuli. Some upstream open reading frames (uORFs) in the 5’ untranslated region (5’ UTR) of the ATF mRNAs have been identified as translational regulators for their downstream open reading frames (ORFs), but the regulatory mechanisms for many of these elements remain unknown. In this thesis, I used dual- luciferase reporter assay to study the regulatory roles of uORFs on the translation of ATFs. All uORFs of ATFs repress downstream ORF translation under normal growth conditions. However, the repressive effect of uORFs can be relieved under specific stress signals. Some common features of uORFs between members of ATF family apparently respond to similar kinds of stress stimuli. On the other hand, our studies uncovered two different signaling pathways that mediate the uORFatf2-regulated translation under UV irradiation.ABBREVIATIONS……………………ii文摘要……………………iiiBSTRACT……………………ivONIENTS……………………vNTRODUCTION……………………………………………………………………1ATERIALS AND METHODS………………………………………………………5. Plasmid Construction………………………………………………………………5-1. Firefly luciferase and Renilla luciferase construct…………………………….5-2. uORFchop-Lu construct…………………………………………………………5-3. Molecular cloning of wild-type uORFatf2-Lu, uORFatf3-Lu,ORFatf4-Lu and uORFatf5-Lu………………………………………………….5-4. Molecular cloning of mutant uORFatf2-Lu, uORFatf3-Lu, uORFatf4-Lu……………6-5. Molecular cloning of dual-luciferase reporter…………………………………9. Cell Culture………………………………………………………………………..9. DNA Transfection and Chemicals Treatment…………………………………….10. Preparation of Whole Cell Extract………………………………………………..11. Luciferase Assay……………………………………………………………………11. SDS-PAGE………………………………………………………………………..12. Western bolt analysis……………………………………………………………..13ESULTS……………………………………………………………………………….15. Construction of dual-luciferase reporter system…………………………………15. The uORFs locates in the 5’ UTR of ATF genes repressranslation of downstream ORF………………………………………………….15. Under specific stress signals,he repressive effect of uORFs of ATF family can be relieved…………………………16. Functional properties of cis-elements in various uORFs…………………………18. UV-activated induces ATF2 expression through regulating p38 MAPK-nk-eIF4E and GCN2-eIF2α pathways by translational control……………………20-1. The long uORF of ATF2 is required for stress induction.……………………20-2. Signaling pathways mediate the uORFatf2-regulated translationy UV irradiation.…………………………………………………………….20-3. The p38/MAPK-Mnk-eIF4E pathway plays an essential rolen the UV-induced uORFatf2-mediated reporter expression.………………..21-4. Involvement of mTOR signalingn UV-induced uORFatf2-driven translation…………………………………..22ISCUSSION………………………………………………………………………..23EFERENCES……………………………………………………………………….26IST OF FIGURES…………………………………………………………………..31igure 1. Schematic presentation of the dual-luciferase reporter system……………31igure 2. Identification of the uORFs of CHOP, ATF2, ATF3, ATF4 and ATF5……32igure 3. uORFsatf-Lu are response to physical stress………………………………34igure 4. uORFsatf-Lu are response to chemical stress……………………………..35igure 5. uORFsatf-Lu are response to physiological stress…………………………38igure 6. uORFsatf-Lu are not response to growth factors………………………….40igure 7. Schematic presentation of the wild-type and mutantsf uORFatf2-Lu, uORFatf3-Lu and uORFatf4-Lu constructs……………….42igure 8. UV irradiation induced ulR-u12mt-Lu activity,ut not ulR-uORFatf2-Lu activity…………………………………………43igure 9. uORFatf3-Lu mutants can be response to specific stresses………………..45igure 10. uORFatf4-Lu mutants can be response to specific stresses………………46igure 11. The uORFatf2-driven luciferase expression could not inducedy stress conditions……………………………………………………..48igure 12. The pathways of p38-Mnk-eIF4E and GCN2-eIF2αere activated by UV irradiation……………………………………….50igure 13. The UV-induced ulR-u12mt-Lu expressions were repressedy p38 MAPK, Mnk, inhibitors, but not JNK inhibitor…………………53igure 14. Rapamycin repressed UV-induced uORFatf2-regulateduciferase expression……………………………………………………55igure 15. Proposed model for UV-induced signaling pathwaysor activation of uORFatf2-driven translation……………………………56IST OF TABLE……………………………………………………………………..57able 1. The fold of induction or repression of uORFchop- or uORFatf-mediateduciferase expression by treated with various kinds of stress signals orrowth factors………………………………………………………………57application/pdf841073 bytesapplication/pdfen-US活化轉錄因子上游開放閱讀框架轉錄調控逆境信號紫外線照射ATFuORFtranslational regulationstress signalUV irradiationhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178711/1/ntu-98-R95448010-1.pdf