Mechanisms By Which Iron Status Regulates mRNA Translation In Skeletal Muscle Of Rats

• Main Authors: Chang, Yuying, 張佑瑛
• Other Authors: Liew, Yihfong
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/13606996254973968016

碩士 === 輔仁大學 === 營養科學系 === 100 === Mechanisms by which iron status regulates mRNA translation in skeletal muscle of rats Abstract Our previous study demonstrated that cytosol thiouridine-containing tRNAs expression and it’s tion-modification were specifically decreased in iron-dificient skeletal muscle. Because the nucleotide modification of tRNA may relate to protein synthesis, hence in this study we investigated the regulation of dietary iron intake on mRNA translation in skeletal muscle. It is to understand the relationship between thio-midification of thiouridine-containing tRNAs and the mRNA translation in the muscles of iron deficency rats. In this study, male weanling Wistar rats were divided into four group by weight：control (AI) , moderate iron dificient (MID) , severe iron dificient (ID) and severe iron dificient paired-fed (IPF) (n=6). The rats were euthanized after feeding with AIN93G formula four weeks. Blood samples were collected to measure iron status and one of the skeletal muscle measured the cytosol ER strss marker, eIF2α/PERK phosphorylation pathway, the phosphorylation of p-eEF2 and relation protein of mTOR signaling by Western blotting. Another of the skeletal muscles measured the charged- tRNA and uncharged-tRNA expression by acid extraction and Northern blotting. The results showed in ID rats the hematological indices of iron status (hemoglobin, serum iron and transferrin saturation) decreased about 62%, 79% and 82% , respectively. Nevertheless, the TIBC (total iron binding capacity) increased 1.1 fold compared with control group. This indicated ID rats have severe iron-deficiency anemia. The expression of p-mTOR, p-S6 and p-4EBP1 proteins in cytosol of ID rat’s muscle were not affected. However, the ER stress marker GRP78 (known as Bip) and it’s downsteam target p-PERK, p-eIF2α and eIF2α protein siginificantly increased 74%, 75%, 41% and 43% in muscles of ID rats. In addition, the expression of phosphorylation of p-eEF2 also siginificantly increased 69% in muscle of ID rats. In the moderate iron-deficient group which fed 10 mg Fe/kg diet, the hemoglobin declined to 19% of control group, while the GRP78, eIF2α, the phosphorylation of p-PERK, p-eIF2α, p-eEF2, p-mTOR, p-S6 and p-4E-BP protein levels were not affected. Furthermore, we analysed the total RNA by acid extraction and acid electrophoresis which could effectively differentiate cytosol charged-tRNALys(UUU) and uncharged-tRNALys(UUU). No matter the expresion of charged-tRNALys(UUU), uncharged-tRNALys(UUU) or total tRNALys(UUU) were not siginificantly different in iron-deficient group, but the mean experssion respectively decreased 28%, 27% and 28%. However, the other cytosol tRNA and mitochondrial tRNALys(UUU) and tRNAGlu(UUC) all could not be discriminated charged or uncharged form tRNA. We speculated that the other cytosol and mitochondrial tRNA excepte cytosol tRNALys(UUU) expressed the uncharged-tRNA in skeletal muscles of the rat. These results indicated severe iron dificiency may not have any sigicificant influence on tRNA aminoacylation. In conclusion, our study demonstrated that severe iron dificiency induced the ER stress and then activated the endoplasmic reticulum kinase PERK, leading to phosphorylation of p-eIF2α and p-eEF2 to inhibit initiation and elongation of mRNA translation and reducded the rate of protein synthesis. Key words：mRNA translation , mTOR signaling , tRNA , eEF2, eIF2α/PERK pathway