RNA結合蛋白PAIRBP1的精胺酸甲基化研究

Study of the Arginine Methylation of an RNA Binding Protein PAIRBP1

謝琬玉(Wan-Yu Hsieh)

指導教授 ： 李娟

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00125

摘要

蛋白質精胺酸甲基化在哺乳動物細胞裡是一種普遍的轉譯後修飾作用，並且參與細胞的許多活動，包括轉錄的調節、訊息的傳遞、RNA的處理和DNA的修復。精胺酸甲基轉移酶將甲基團從甲基提供者S-腺基-L-甲硫胺酸上轉移到其甲基接受者上，在精胺酸與甘胺酸豐富的區域裡或精胺酸豐富的區域裡有發現甲基化的精胺酸。第一型纖維蛋白分解酶原活化者的抑制者的mRNA結合蛋白（PAIRBP1）會結合上第一型纖維蛋白分解酶原活化者的抑制者的mRNA的AU-rich的3’-UTR，而且在結合蛋白PAIRBP1裡有精胺酸豐富區、精胺酸與甘胺酸豐富區和精胺酸-甘胺酸-甘胺酸重複序列區，在這三個區域裡的精胺酸可能會被甲基化。實驗顯示GST-PAIRBP1融合蛋白可以在in vitro被蛋白精胺酸甲基轉移酶1甲基化，並且知道GST-PAIRBP1（1-150）不會被PRMT1甲基化而GST-PAIRBP1（151-270）和GST-PAIRBP1（1-270）會被PRMT1甲基化，所以PAIRBP1蛋白可能是PRMT1的受質，而且PRMT1有可能催化精胺酸與甘胺酸豐富區裡的精胺酸形成非對稱型雙甲基精胺酸。
再者，將將含有Pairbp1各個基因片段的pFLAG-CMV-2質體轉殖進入HeLa細胞，之後再用免疫沉澱法純化各個片段的FLAG-PAIRBP1 蛋白。免疫沉澱之後所得的FLAG融合蛋白分別以辨識對稱型或非對稱型雙甲基精胺酸的抗體SYM10和ASYM24偵測，由實驗結果知道，出PAIRBP1裡有精胺酸在in vivo被甲基化，其中的1-150胺基酸裡偵測不到甲基化的訊號；151-270胺基酸裡有對稱型雙甲基精胺酸與非對稱型雙甲基精胺酸；而271-387胺基酸裡有非對稱型雙甲基的精胺酸，因此知道不只一種精胺酸甲基轉移酶催化PAIRBP1蛋白形成對稱型或非對稱型雙甲基精胺酸。

關鍵字

結合蛋白 ； 精胺酸 ； 甲基化

並列摘要

Protein arginine methylation is a common posttranslational modification in mammalian cells, and participates in many cellular processes, including transcriptional regulation, signal transduction, RNA processings, and DNA repair. Arginine methyltransferases transfer methyl group from methyl group donor S-adenosyl-L-methionine to its methyl group recipient. Methylarginines are found in arginine and glycine rich motif or arginine rich domain. Type-1 plasminogen activator inhibitor (PAI-1) mRNA binding protein (PAIRBP1) binds to the AU-rich 3’-UTR of PAI-1 mRNA. There are an Arg-rich domain, an RG-rich domain, and an RGG box in PAIRBP1. Arginines in these three domains may be methylated. We showed that GST-PAIRBP1 fusion protein could be in vitro methylated by protein arginine methyltransferase 1 (PRMT1). And GST-PAIRBP1（1-150） could not be in vitro methylated by PRMT1 but GST-PAIRBP1（151-270） and GST-PAIRBP1（1-270） could be in vitro methytlated by PRMT1. PAIRBP1 may be a substrate for PRMT1 and PRMT1 may methylate arginines in arginine and glycine rich motif.
Furthermore, we transfected independently HeLa cells with different pFLAG-CMV-2 plasmids including truncated Pairbp1 genes and different truncated FLAG-PAIRBP1 proteins were purified by immunoprecipitation. The immunoprecipitated FLAG-fusion proteins could be detected by antibodies recognizing symmetric or asymmetric di-methylarginines, SYM10 or ASYM24. We show that arginines in PAIRBP1 could be in vivo methylated. 1-150 amino acid sequence in PAIRBP1 could not be detect for methylated arginine, there are symmetric or asymmetric arginines in 151-270 amino acid sequence in PRI1BP1, and there are asymmetric arginines in 271-387 amino acid sequence in PRI1BP1. We conclude that there are not only one of protein arginine methyltransferase methylate PAIRBP1 to be symmetric or asymmetric di-methylarginines.

並列關鍵字

arginine methylation ； RNA binding protein ； PAIRBP1

參考文獻

Andreasen PA, L. K, Christensen L, Duffy MJ: The urokinase-type plasminogen activator system in cancer metastasis: a review. International Journal of Cancer Journal International Du Cancer 1997; 72:1-22.

Bains R, Miles DM, Carson RJ, Adeghe J: Hyaluronic acid increases motility/intracellular CA2+ concentration in human sperm in vitro. Archives of Andrology 2001; 47:119-125.

Bedford MT: Arginine methylation at a glance. Journal of Cell Science 2007; 120:4243-4246.

Bedford MT, Frankel A, Yaffe MB, Clarke S, Leder P, Richard S: Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. The Journal of Biological Chemistry 2000; 275:16030-16036.

Boulanger M-C, Liang C, Russell RS, Lin R, Bedford MT, Wainberg MA, Richard S: Methylation of Tat by PRMT6 regulates human immunodeficiency virus type 1 gene expression. Journal of Virology 2005; 79:124-131.