| Budget Amount *help |
¥3,950,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Nonsense-mediated mRNA decay(NMD) is an mRNA quality-control mechanism that degrades aberrant mRNAs containing premature translation termination codons(PTCs) . In this study, we suppressed NMD through siRNA-mediated knockdown of 14 kinds of NMD components, including SURF complex(SMG-1, Upf-1, p130, eRF1, eRF3a, eRF3b), exon junction complex(Y14, MAGOH, eIF4A3, BtZ, RNPS1), Upf2, SMG-6, and SMG-7. We evaluated the effect of knockdown of these 14 components on the phenotype of Ullrich's disease, an autosomal recessive congenital muscular dystrophy. The patient studied, showed a homozygous frame-shift mutation with a PTC in the collagen VI α2 gene, which encodes a truncated but partially functional protein. The patient's fibroblasts showed a nearly complete loss of the triple-helical collagen VI protein and functional defects in the extracellular matrix(ECM) due to the crucial deficiency of the collagen VI α2 protein. We have shown that the siRNA-mediated knockdown of 7 NMD components, Upf1, SMG-1, p130, SMG-6, SMG-7, MAGOH, BtZ, causes significant up-regulation of the collagen VI α2 mRNA and triple helical collagen VI. Knockdown of these 7 NMD components showed no significant changes in cell cycle distribution compared to that of non-silencing siRNA-transfectants. Knockdown of SMG-1 showed mild disturbance in cell growth on the 6th day of siRNA transfection, but other 6 NMD components had no effect. Finally, knockdown of 4 NMD components(SMG-1, Upf1, p130, MAGOH) showed efficient up-regulation of the mutant triple-helical collagen VI in ECM.
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