Analysis of arginine methylation of an RNA binding protein SEREBP1 and localization of SERBP1 in stress granules and nucleoli

• Main Authors: Yu-Jen Lee, 李侑蓁
• Other Authors: 陳凌雲
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/3397px

博士 === 中山醫學大學 === 生化暨生物科技研究所 === 102 === 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.