关键词: 酵母双杂交系统, 哺乳动物双杂交系统 , R916W突变, ADAR1蛋白

Abstract: Objective: Mutations in the ADAR1 gene cause dyschromatosis symmetrica hereditaria (DSH), a rare autosomal dominant skin disorder. We have shown that nonsense and frame-shift mutations in the ADAR1 gene result in nonsense-mediated mRNA decay thereby leading to ADAR1 haploinsufficiency. The present study was designed to detect protein-protein interaction between the wild type ADAR1 (ADAR1^WT) and its R916W mutant (ADAR1^R916W) using yeast and mammalian two-hybrid systems, and to gain some insight into the molecular mechanism by which the missense mutations in the ADAR1 gene cause DSH. Methods: The full length normal and mutant ADAR1 cDNA clones were obtained from the peripheral lymphocytes from a DSH patient with R916W missense mutation by RTPCR. The cDNA fragments were cloned into the bait and prey vectors in the Matchmaker^TM GAL4 yeast two-hybrid system 3 and the CheckMate^TM mammalian two-hybrid system, respectively. The two-hybrid experiments were performed by following the manufac-turer's protocols. Results: Yeast cells co-transformed with plasmids pGADT7-ADAR1^WT and pGBKT7-ADAR1^WT or plasmids pGADT7-ADAR1^WT and pGBKT7-ADAR1^R916W failed to form colonies on SD/-Trp/-Leu/-His/-Ade plates, indicating a lack of protein-protein interaction. HeLa cells co-transfected with plasmids pACT-ADAR1^WT, pBIND-ADAR1^WT and pG5luc showed a significant increase in luciferase activity (P＜0.05). Whereas cells co-transfected with pACT-ADAR1^WT, pBIND-ADAR1^R916W and pG5luc exhibited a considerable decrease in luciferase activity (P＜0.05), retaining 66% of the activity compared with the wild-type counterpart. Conclusion: ADAR1^R916W displayed decreased interaction with ADAR1^WT in mammalian cells, as compared with the ADAR1^WT-ADAR1^WT interaction.

Key words: ADAR1 protein, R916W mutant, Yeast two-hybrid system, Mammalian two-hybrid system