訊息RNA去頭蓋蛋白質1a磷酸化之功能分析

Functional Characterization of Phosphorylation of Decapping Protein 1a (Dcp1a)

高靖涵(Ching-Han Kao)

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(2008年)

https://doi.org/10.6342/NTU.2008.02028

摘要

訊息核醣核酸降解的調控在基因的表現中是非常重要的一環。由於在去除3’端的長poly(A)序列 (deadenylation)後，去頭蓋的動作會將標的訊息核醣核酸5’端的頭蓋移除，製造出m7GDP和在5’端帶有單一個磷酸，而會受到Xrn1進行5’到3’端的exonucleolytic降解的受質，所以，去頭蓋在訊息核醣核酸代謝過程扮演著相當重要的角色。去頭蓋蛋白質1 (Dcp1)和去頭蓋蛋白質2 (Dcp2)會形成去頭蓋複合物，當中並包含其他和降解相關的因子，像是Tristetraprolin (TTP)。TTP是在3T3-L1前脂肪細胞在受到誘發之後，所表現的立即早期基因，並會與人類去頭蓋蛋白質1a形成在ARE傳達降解路徑中作用的複合物。因此，本研究想要了解在前脂肪細胞的分化過程中，老鼠去頭蓋蛋白1a參與在有絲分裂細胞株落擴張的訊息核醣核酸降解(mitotic clonal expansion)的可能性。去頭蓋蛋白1a在3T3-L1早期分化時期會受到磷酸化，而這活性很可能是受到ERK訊息路徑的調控。老鼠去頭蓋蛋白1a主要會存在於細胞質中，並且它在細胞中的位置是不會受到磷酸化的影響的。而大量表現的去頭蓋蛋白1a和去頭蓋蛋白2 也被發現會聚集在細胞質中的特定裂解體中。在in vitro 去頭蓋的實驗，去頭蓋蛋白2展現強烈的去頭蓋活性，另一方面，去頭蓋蛋白1a表現了些微的去頭蓋活性。進一步的實驗發現，處於高度磷酸化狀態的去頭蓋蛋白1a在高劑量時，可以增強去頭蓋蛋白2的去頭蓋活性。這方面在之後會需要更多的分析來了解磷酸化在對於去頭蓋蛋白1a在3T3-L1細胞分化早期進行去頭蓋的動作或增進Dcp2之活性時所扮演的角色。

關鍵字

去頭蓋 ； 訊息核醣核酸降解 ； 去頭蓋蛋白1a ； 特定裂解體 ； 前脂肪細胞分化

並列摘要

Regulation of mRNA decay is a critical step in gene expression. Decapping plays a major role in mRNA turnover as it removes the 5’ cap of target mRNA after deadenylation to generate m7GDP and a 5’ monophosphate substrate that is susceptible for 5’ to 3’exonucleolytic degradation by Xrn1. Dcp1p and Dcp2p were noted to form a decapping complex and associate with other mRNA decay regulators such as Tristetraprolin (TTP). TTP is an immediate early gene stimulated upon induction in 3T3-L1 preadipocytes, and was reported to precipitate with hDcp1a and the complex functioned in the ARE-mediated decay pathway. The potential role of mouse Dcp1a during mitotic clonal expansion in preadipocyte differentiation was thus studied. Dcp1a was phosphorylated during the early differentiation of 3T3-L1 cells, and was possibly regulated by the ERK signaling pathway. Mouse Dcp1a was localized mostly in the cytoplasm, and its position was not affected by its phosphorylation status. The colocalization of overexpressed Dcp1a and Dcp2 was also observed in cytoplasmic processing bodies. In the in vitro decapping assay, mouse Dcp2 displayed strong decapping activity while Dcp1a expressed partial decapping activity. In addition, highly phosphorylated Dcp1a appeared to enhance the catalytic activity of Dcp2 in a dosage dependent manner. Further analysis would be required to reveal the effect of phosphorylation on Dcp1a’s role in decapping and enhancing the activity of Dcp2 during early differentiation of 3T3-L1 cells.

並列關鍵字

decapping ； mRNA decay ； Dcp1a ； processing bodies ； preadipocyte

參考文獻

1. Fenger-Gron, M., et al., Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. Mol Cell, 2005. 20(6): p. 905-915.

2. Lykke-Andersen, J., Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay. Mol Cell Biol, 2002. 22(23): p. 8114-8121.

3. Beelman, C.A. and R. Parker, Degradation of mRNA in eukaryotes. Cell, 1995. 81(2): p. 179-183.

4. Shatkin, A.J., Capping of eucaryotic mRNAs. Cell, 1976. 9(4 PT 2): p. 645-653.

5. Coller, J. and R. Parker, Eukaryotic mRNA decapping. Annu Rev Biochem, 2004. 73: p. 861-890.