TTP 家族蛋白在果蠅細胞與老鼠前脂肪細胞的標的RNA和功能分析

Identification of mRNA targets and functional characterization of tristetraprolin (TTP) family proteins in Drosophila and mouse preadipocytes

楊文瑄(Wen-Hsuan Yang)

指導教授 ： 張瀞仁

國立臺灣大學/生命科學院/生化科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00020

摘要

訊息核醣核酸 (mRNA)在細胞內受到嚴密調控。其中一種轉錄後調控是透過降解相關因子結合到位於mRNA三端未轉譯區 (3' untranslated region, 3'UTR)上的多腺嘌呤及尿嘧啶序列 (AU-rich element, ARE)來促進降解。TTP (tristetraprolin)家族蛋白可以透過鋅指區域 (tandem CCCH zinc finger)結合到ARE，造成mRNA易被降解。其在老鼠內有四個成員：分別是TTP，ZFP36L1，ZFP36L2和ZFP36L3；而在果蠅只有一個：DTIS11。為了探討TTP家族蛋白的功能作用，我們分別利用果蠅細胞及老鼠前脂肪細胞來研究。第一部分，發現TTP的新標的mRNA：eyes absent (eya)。利用核醣核酸交互作用沉降試驗 (RNA pull-down)以及螢光素酶報導基因分析 (luciferase reporter assay)，發現DTIS11會利用鋅指區域結合到eya mRNA並造成其降解。同時也發現表現DTIS11會降低細胞存活率。相同的結論我們也在人類乳癌細胞 (MCF7)得到: TTP可以使得eya homologue 2 (EYA2) mRNA降解並影響到細胞存活率，這可能是由於細胞凋亡。在第二部分的實驗，發現ZFP36L1和ZFP36L2對調節脂肪細胞分化可能扮演不同時期調控的角色。類似TTP的作用，ZFP36L1和ZFP36L2都可以結合到去磷酸酶Mkp-1 mRNA 3'UTR上並造成其去穩定性。利用慢病毒載體 (lentiviral vector)方式來抑制ZFP36L1或ZFP36L2表現，發現唯有抑制ZFP36L1會造成一些極早期基因 (immediate early genes)的核醣核酸表現量上升，其中包括Mkp-1 mRNA，同時並發現ERK活性降低以及抑制脂肪分化。於是我們提出TTP家族蛋白會在不同時期相互調控Mkp-1 mRNA進而影響到脂肪分化。綜合上述，本研究結果指出TTP家族蛋白會透過調控不同的標的訊息核醣核酸來影響到細胞的活性或是脂肪細胞的分化。

關鍵字

轉譯後調控機制 ； 鋅指蛋白36 ； 眼缺陷基因 ； 去磷酸酶鋅指蛋白36類型1

並列摘要

The turnover of AU-rich element (ARE)-containing mRNAs is post-transcriptionally regulated by one or more RNA-binding proteins, such as tristetraprolin (TTP) family proteins. In rodent, there are four proteins belonging to TTP family: TTP (TIS11, ZFP36), ZFP36L1 (TIS11b, BRF-1), ZFP36L2 (TIS11d, BRF-2), and ZFP36L3. In Drosophila, only one TTP family protein has been reported (DTIS11). TTP family proteins have two tandem CCCH zinc finger (TZF) domains that bind to ARE of target mRNAs and then cause the mRNAs to be destabilized by recruitment of 5' and 3' mRNA degradation complexes. To identify their mRNA targets and the functional effect of TTP family proteins, the overexpression and knockdown were performed in Drosophila cells and mouse preadipocytes, respectively. The eyes absent (eya) transcript is one of many ARE-containing mRNAs in Drosophila. RNA pull-down and luciferase reporter analyses demonstrated that the DTIS11 RNA-binding domain is required for DTIS11 to bind the eya 3' UTR and reduce levels of eya mRNA. Moreover, ectopic expression of DTIS11 in Drosophila Schneider 2 (S2) cells decreased levels of eya mRNA and reduced cell viability. Consistent with these results, TTP proteins overexpressed in MCF7 human breast cancer cells were associated with eya homologue 2 (EYA2) mRNA, and caused a decrease in EYA2 mRNA stability and cell viability which may be caused by apoptosis. Our results demonstrated that eya mRNA is a novel target of TTP protein. In the second part of the thesis, the functional analyses of ZFP36L1 and ZFP36L2 in regulating mitogen-activated protein kinase (MAPK) phosphatase-1 (Mkp-1) mRNA and adipogenesis were investigated. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of Mkp-1 mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Knockdown of ZFP36L1, but not ZFP36L2, increased basal levels of immediate early genes including Mkp-1 mRNA and decreased ERK activation. We also found that knockdown of constitutive expression of ZFP36L1 and ZFP36L2 would inhibit and slightly enhance differentiation of mouse 3T3-L1 preadipocyte, respectively. These results suggested ZFP36L1 and ZFP36L2 may regulate the expression of ARE-containing mRNA and thus adipogenesis differently. Collectively, our findings indicate that TTP family proteins modulate cell viability or cell differentiation through destabilizing some mRNA targets.

並列關鍵字

post-transcriptional regulation ； tristetraprolin (TTP) ； eyes absent (eya) ； MAPK phosphatase-1 (MKP-1) ； ZFP36L1

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