RNA結合蛋白SERBP1精胺酸甲基化之分析並探討其存在於壓力顆粒與核仁之現象

蛋白質精胺酸甲基化為一重要的轉譯後修飾，調控細胞內多種生理反應，如：訊息傳遞、轉錄調節、RNA運輸與蛋白質之間的交互作用等。此修飾反應透過蛋白質精胺酸甲基轉移酶 (PRMT) 之作用，將甲基由甲基提供者S-adenosyl-L- methionine (SAM) 轉移至甲基接受蛋白的精胺酸上。實驗室以甲基接受蛋白常見區域GAR motif進行比對，找到當時仍未有研究之ㄧopen reading frame (accession number：AL080119) SERBP1 (SERPINE1 mRNA binding protein 1)，此蛋白上帶有Arg-rich、RG-rich及RGG box區域，此三區域可能為精胺酸甲基轉移酶作用位置；而影響蛋白進入細胞核內的Nuclear localization signal (NLS) 區域則與RG-rich區域有重疊的情形。 首先針對SERBP1蛋白的精胺酸甲基化修飾來探討。由本研究結果發現，SERBP1會受到間接抑制甲基轉移酶藥物AdOx影響而造成蛋白的不對稱精胺酸甲基化程度降低，其中間RG-rich與C端RGG box區域為甲基化的接受區域。透過免疫沉澱發現，SERBP1會與甲基轉移酶PRMT1結合，其結合能力會受到蛋白上的甲基化程度而影響。在in vitro狀況下，重組蛋白SERBP1可受到PRMT1的甲基催化；利用siRNA減少細胞內PRMT1的表現量也可觀察到SERBP1的甲基化程度減少，代表SERBP1蛋白的確為PRMT1的受質蛋白。由免疫螢光染色觀察，內生性SERBP1蛋白主要表現在細胞質中。當細胞呈現低度甲基化時，SERBP1蛋白表現由原本在細胞質表現為主，而變成細胞核內增加或蛋白會平均分布於細胞內，且細胞核中有明顯顆粒狀結構形成；分析不同片段蛋白的表現情形，發現中間RG-rich區域會影響其在細胞核的表現，N端及C端區域可能與蛋白在細胞核輸出的過程有關。 進一步探討SERBP1與壓力顆粒之間的關係。由免疫沉澱實驗結果發現，在正常狀況下SERBP1會與部分已知的壓力顆粒相關蛋白 (如：FMRP、hnRNP A1等) 有結合關係，此結合是需要RNA的參與協助。觀察在壓力狀況下SERBP1於細胞內的表現位置，發現全長SERBP1蛋白會與壓力顆粒標的蛋白TIA-1共同存在於細胞質的壓力顆粒內；其他不同片段的SERBP1蛋白雖都會形成壓力顆粒，但其形成能力仍低於全長蛋白；也觀察到SERBP1蛋白在細胞核內的分布增加，且核仁有堆積的情形。比較不同甲基化程度的SERBP1與壓力顆粒形成之關係，發現當在移除壓力來源後，低度甲基化的SERBP1所形成的壓力顆粒其保留比例會較正常高，且細胞核內的分布也會因低度甲基化影響而造成保留比例增加。綜合以上實驗結果，可推論出SERBP1蛋白為精胺酸甲基轉移酶PRMT1的受質蛋白，此蛋白上的精胺酸甲基化程度，不僅會影響到在細胞內的表現情形，也影響在受到外界壓力刺激下其形成壓力顆粒的能力及在細胞核內堆積之情形。

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 groups from the methyl group donor S-adenosyl-L-methionine to themethyl group recipients. Methylarginines are often found in arginine and glycine rich motif or arginine rich domain.SERBP1 (SERPINE1 mRNA binding protein 1) implicated in tumor progression through its putative involvement in the plaminogen activator protease cascade, is an RNA binding protein containing an RG-rich domain and an RGG box domain that might be methylated by protein arginine N-methyltransferases (PRMTs). Asymmetric dimethylarginine (aDMA) was detected in SERBP1 and an indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) significantly reduced the methylation signals. Arginines in the middle RG and C-terminal RGG region of SERBP1 are methylated based on the analyses of different deletion constructs. The predominant type I protein arginine methyltransferase PRMT1 co-immunoprecipitated with SERBP1 and the level of bound PRMT1 decreased upon the addition of AdOx. Recombinant PRMT1 methylated SERBP1 and knockdown of PRMT1 significantly reduced the aDMA level of SERBP1, indicating that SERBP1 is specifically methylated by PRMT1. Immunofluorescent analyses of endogenous SERBP1 showed predominant cytoplasmic localization of SERBP1. Treatment of AdOx or PRMT1 siRNA increased the nuclear localization of SERBP1. Analyses of different deletions indicated that the middle RG region is important for the nuclear localization while both N- and C- terminus are required for nuclear export. Low methylation of the C-terminal RGG region also favors nuclear localization. We show that under normal growth condition without stress, SERBP1 interacts with some arginine methylated and stress granule-associated proteins such as hnRNPA1, FMRP, FXR1 in an RNA-dependent manner. We also show that after arsenite treatment, a fraction of full-length SERBP1 protein co-localizes with the typical stress granule marker TIA-1in the cytoplasmic SGs. Truncated SERBP1 with the N-terminal, middle RG or C-terminal deletions, or else any single domain segment containing the N-terminal, middle or C-terminal region, was recruited to SGs upon arsenite treatment but with reduced efficiency. Besides, upon arsenite treatment, diffuse cytoplasmic localization of SERBP1 was shifted to nuclear-dominant and concentrated nucleolar distribution. Similar distribution was observed when cells were treated with a methylation inhibitor adenosine periodate (AdOx) and was also detected in the N- or C-terminal domain deletions and all three single domain fragments even without stress induction. We further demonstrate that AdOx treatment delays the association/dissociation of SERBP1 with SGs. Hypomethylation retains SERBP1 in the nucleus/nucleolus regardless of arsenite treatment. Our study indicates that arginine methylation is correlated with the recruitment of SERBP to as well as its retention in stress granules and nucleoli. Our study is the first report of an RNA binding protein that can be shifted simultaneously to cytoplasmic SGs and nucleoli, two RNP-concentrated subcellular compartments, upon stress.