鐵營養對於大鼠肌肉和肝臟tRNA硫醇鹼基修飾之影響

Effects of Iron Status on tRNA Thio-modification in Muscle and Liver of Rat

余明真(Ming-Chen Yu)

指導教授 ： 蕭寧馨

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01609

摘要

Transfer RNA (tRNA) 含豐富的鹼基修飾，目前已知達 100 多種，常見的修飾位置在 tRNA 第 34 號位置 (wobble) 和第 37 號位置，真核生物 tRNAGlu(UUC)、tRNAGln(UUG)、tRNALys(UUU) 和 tRNAArg(UCU) 上的 34 號 Uridine 鹼基 (U34) 常被修飾為 5-methyl-2- thiouridine (xm5s2U, x=為任何取代型式) 的衍生物，主要功能是使 tRNA 反密碼子與 mRNA 密碼子正確辨識與結合，缺乏修飾的核苷酸會導致轉譯效能降低且增加轉譯錯誤，進而影響基因表現。前人以鐵螯合劑 DFO 模擬大鼠肌肉細胞 (L6) 內缺鐵情況，缺鐵會減少細胞質 tRNA 硫鹼基修飾作用，但目前對大鼠肌肉和肝臟 tRNA 總鹼基修飾情形仍未完全了解。因此本研究利用高效能液相層析法 (HPLC) 分析大鼠肝臟和肌肉 tRNA 總鹼基修飾和 mcm5s2U 硫鹼基修飾情形，並進一步以 Northern blot 方法探討鐵營養影響大鼠細胞質含硫修飾 tRNAGlu(UUC)、tRNALys(UUU) 和 tRNAArg(UCU) 的修飾程度與表現量，以動物實驗進行分析，利用大鼠血紅素 (Hb) 再生法，大鼠經耗鐵期三周後，補充不同濃度硫酸亞鐵兩週，結束後根據 Hb 濃度高低將動物分成五組 4-5 g/dL、6-7 g/dL、8-9 g/dL、10-11 g/dL、≥ 12 g/dL。從 HPLC 分析結果得知，肌肉以 Hb 濃度低下 (4-5 g/dL) 組別之總 tRNA 硫醇鹼基 mcm5s2U 含量較 Hb 濃度高者 (≥ 12 g/dL) 減少將近 60%，此修飾隨著 Hb 濃度降低有減少之趨勢；其他類型的修飾鹼基則沒有顯著影響；對肝臟組織皆沒有顯著影響。在肌肉細胞質中 tRNAGlu(UUC)、tRNALys(UUU) 和 tRNAArg(UCU) 表現量，以 Hb 濃度最低 (4-5 g/dL) 比最高 (≥ 12 g/dL) 分別顯著減少 65%、45%、52%。在肌肉細胞質中 tRNALys、tRNAArg 和tRNAGlu 硫醇修飾量，以 Hb 濃度最低 (4-5 g/dL) 比最高組 (≥ 12 g/dL) 分別顯著減少 20%、24%、26%；對肝臟組織皆沒有顯著影響。本研究初步證實動物缺鐵性貧血時，會導致大鼠肌肉組織細胞質 tRNA 表現量及硫醇修飾化程度降低，以及總 tRNA 之硫鹼基 mcm5s2U 含量，且隨著鐵營養狀況良好時 tRNA 硫醇鹼基的修飾會隨之上升，而在其他常見的修飾鹼基則沒有受此影響而改變，表示鐵營養對肌肉組織 tRNA 硫醇鹼基 mcm5s2U 和細胞質中 tRNA 硫醇修飾作用具有組織專一性影響。

關鍵字

tRNA ； 硫醇修飾 ； mcm5s2U ； 高效能液相層析法 (HPLC)

並列摘要

Transfer RNA (tRNA) are the most highly modified RNAs. To date, more than 100 species of tRNA modification have been reported. The majority of these modifications in tRNA have been found at the anticodon first (wobble) position 34, and at position 37 3’-adjacent to the anticodon. In eukaryotes, the wobble base of the tRNAs for Glu, Gln and Lys are universally modified to 5-methyl-2-thiouridine derivatives (xm5s2U), such as 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in cytoplasmic tRNAs. The 2-thio modification xm5s2U plays a critical role in protein synthesis, efficient codon recognition and preventing misreading. Lack of xm5s2U modification in the mutant mitochondrial tRNALys from myoclonus epilepsy associated with ragged red fibers (MERRF), results in a marked defect in whole mitochondrial translation. In yeast, the mitochondrial cysteine desulfurase NFS1 is involved in the 2-thio modification of both mitochondrial and cytoplasmic tRNAs. In a rat model, the homologous protein IscS is reduced.

In this study, we investigate the effects of iron deficiency anemia on the expression and thio-modification of tRNA in both liver and skeletal muscle. Male weanling Wistar rats were rendered to different degree of iron deficiency anemia in 5 wks using a dietary iron depletion-and-repletion method. At the end of study, rats were divided into 5 groups by their final hemoglobin levels: 4-5 g/dL, 6-7 g/dL, 8-9 g/dL, 10-11 g/dL, and ≥ 12 g/dL. Small molecule RNAs were extracted from liver and gastrocnemius muscles. Total tRNAs for Glu, Lys, Arg were measured with DIG-labeled probe and Northern blotting. Thio-modified tRNAs for these amino acids were measured similarly but with APM (N-acryloylamino) phenylmercuric chloride)-Northern blotting. Furthermore, nucleosides prepared from total tRNAs were analyzed by HPLC, and mcm5s2U were quantified using 8BrG as an internal standard. In the cytosol of the muscle, the expression of total tRNAs for Lys, Glu and Lys were related to hemoglobin levels in a dose-response fashion, and they were significantly reduced by 50% in the anemic rats (by one-way ANOVA and Duncan’s multiple range test at p < 0.05). The level of thio-modified tRNA for Lys were reduced by 20% in rats of Hb < 5 g/dL, while that for Arg and Glu were reduced by 20% in rats of Hb < 10 g/dL. The level of mcm5s2U also decreases with decreasing hemoglobin level in a dose-dependent fashion,

Our results confirms that iron deficiency anemia has a tissue-specific effects on thio-modified tRNA, that total and thio-modification of tRNAs for Lys, Arg and Glu, are significantly reduced in the muscle but not in the liver.

並列關鍵字

tRNA ； thio-modification ； HPLC ； mcm5s2U ； iron depletion-repletion

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