淋巴母細胞中蛋白精胺酸甲基化之研究

目前已經知道N-arginine methylation是細胞中最主要蛋白甲基化的形式，會發生在RNA binding protein的RGG motifs上。為了想了解發生在淋巴母細胞內的甲基化，我們藉由SDS-PAGE和fluorography分析以AdOx處理、淋巴母細胞中的甲基接受蛋白顯示：有許多多胜被內生性甲基轉移所催化，少數多胜會在加入yeast GST-RMT1後，訊號變得更強。而不論yeast GST-RMT1是否參與，這些甲基接受蛋白的甲基化會被peptide GGRGRGGGF特異性的競爭掉，這個結果顯示在淋巴母細胞中的甲基接受蛋白具有相似的RGG motifs，且甲基化可能發生在arginine residues。在接下來的研究中，我們將淋巴母細胞分為nucleus、ribosome and cytosolic fractions以進一步觀察甲基接受蛋白以及甲基轉移的分佈。實驗發現：在不同的fraction中都有外生性RMT1的甲基接受蛋白存在，而如果甲基化反應中不外加外生性RMT1，也就是純粹觀察內生性甲基轉移的活性時發現：被甲基化的多胜存在ribosomal fraction中要比nucleus and cytosolic fractions來得多，而ribosomal fraction中多胜的甲基化可被recombinant fibrillarin (核仁RGG蛋白) 競爭掉。當我們以fibrillarin作為受質加入反應中發現：酵素活性在cytosolic fraction中最強，其次是ribosomal fraction 、nucleus fraction。最後胺基酸分析的實驗則更直接的顯示發生在淋巴母細胞內的甲基化主要是精胺酸的甲基化。未來如果更進一步分析特定的甲基接受蛋白以及甲基轉移的分佈與調控，將能夠幫助我們瞭解甲基化為何普遍存在真核細胞的RGG protein中。

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.