Protein N-arginine methylation in lymphoblastoid cell

• Main Authors: Lin Chia-Hui, 林佳慧
• Other Authors: Li Chuan
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/29975574088358447374

碩士 === 中山醫學院 === 醫學研究所 === 87 === N-arginine methylation in RNA binding proteins with arginine and glycine rich RGG motifs is known to be the major protein methylation in cells. Analysis of methyl-accepting polypeptides in AdOx-treated lymphoblastoid cells by SDS-PAGE and fluorography showed that many polypeptides between 29,000 and 90,000 Da were methylated by the endogenous methyltransferase. A few polypeptides could be methylated to a higher extent upon the addition of yeast GST-RMT1 fusion protein. A peptide (GGRGRGGGF) could compete for the majority of the methyl-accepting protein substrates in the AdOx-treated lymphoblastoid cell extracts, whether or not exogenous yeast RMT1 was included in the reaction. The results indicated that the protein methyl acceptors in lymphoblastoid cells share similar RGG motifs and that arginine residues should be the site of methylation. Then we fractionated the lymphoblastoid cell extracts to nucleus, ribosome and cytosolic fractions to further locate the substrates and the methyltransferase. In the presence of exogenous RMT1, hypomethylated methylaccepting polypeptides of a wide range of molecular weights were present in all three fractions. In the absence of exogenous methyltransferase, the methylaccepting polypeptides in the ribosomal fraction were heavily methylated while less polypeptides in the nucleus and cytosolic fractions could be methylated. Majority of the radioactivity on the methylaccepting polypeptides in the ribosomal fraction could be competed by recombinant fibrillarin (a nucleolar RGG protein) in a concentration-dependent manner, indicating the methylation should be specific to the RGG containing proteins. The strength of activation of fibrillarin methylation by enzymes in the three fractions was cytosolic > ribosome > nucleus fraction. Finally, we demonstrated that the majority of the protein methylation in different subcellular fraction of lymphoblastoid cell appeared to be on the arginine residues by amino acid analysis. Further analysis of the specific methylaccepting substrates and the regulation of the distribution and the activity of arginine methyltransferase would be crucial for the understanding of the modification widely present in eukaryotic RGG protein.