tRNA抑制子对转录因子NKX2.5提前终止密码子的通读

doi:10.16288/j.yczz.14-410

遗传 ›› 2015, Vol. 37 ›› Issue (4): 367-373.doi: 10.16288/j.yczz.14-410

tRNA抑制子对转录因子NKX2.5提前终止密码子的通读

欧阳平¹,刘远航²,黄志刚³,齐小娟¹,倪倩¹,刘亚萍¹,宋仁生⁴,李涛¹,吴柱国⁴

1. 广东医学院广东省医学分子诊断重点实验室,东莞 523808;
2. 内蒙古呼和浩特市第一医院急诊科,呼和浩特 010050;
3. 广东医学院公共卫生学院流行病与卫生统计学教研室,东莞 523808;
4. 广东医学院第二临床医学院,东莞 523808

收稿日期:2014-11-24 出版日期:2015-04-20 发布日期:2015-03-12
通讯作者: 吴柱国,博士,教授/主任医师,硕士生导师,研究方向：临床心血管病学。E-mail: wugdmc@126.com E-mail:ouyang_ping@126.com
作者简介:欧阳平,博士,助理研究员,研究方向：医学遗传学。E-mail: ouyang_ping@126.com
基金资助:
国家青年基金项目(编号：81200082),广东省医学科研基金项目(编号：B2012272)和广东医学院博士启动基金项目(编号：B2011019)资助

Readthrough on transcription factor NKX2.5 premature stop codon by tRNA suppressors

Ping Ouyang¹,Yuanhang Liu²,Zhigang Huang³,Xiaojuan Qi¹,Qian Ni¹,Yaping Liu¹,Rensheng Song⁴,Tao Li¹,Zhuguo Wu⁴

1. Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China;
2. Department of Emergency, Inner Mongolia Hohhot No.1 Hospital, Hohhot 010050, China;
3. Department of Epidemiology, School of Public Health, Guangdong Medical College, Dongguan 523808, China;
4. The Second Clinical Collage, Guangdong Medical College, Dongguan 523808, China

Received:2014-11-24 Online:2015-04-20 Published:2015-03-12

摘要/Abstract

摘要： 人类NKX2.5基因(NK2 homeobox 5,NKX2.5)提前终止密码子(Premature termination codon,PTC)突变会引起房间隔缺损、房室传导阻滞等先天性心脏病。目前,已报道的NKX2.5 PTC突变有8个(E109X、Q149X、Q170X、Q187X、Q198X、Y256X、Y259X和C264X)。为了检测tRNA抑制子是否对PTC突变诱导通读产生有功能的全长蛋白,文章将8个NKX2.5 PTC突变克隆到pcDNA3.1(-)载体,将NKX2.5全长和E109X、Q149X及C264X克隆到pEGFP-N1载体,形成NKX2.5-EGFP融合质粒。将NKX2.5-EGFP与对应的tRNA抑制子质粒分别或共转染后观察绿色荧光数量定性判断tRNA抑制子是否诱导通读。Western blotting检测通读后全长蛋白和截短蛋白表达并计算通读效率。Real-time PCR检测NKX2.5下游重要调控基因Cx43 mRNA的表达判断通读后蛋白功能。结果表明,文章成功构建了8个基于pcDNA3.1(-)的NKX2.5表达质粒、4个基于pEGFP-N1的质粒;tRNA抑制子tRNA am能有效通读Q149X、Q170X、Q187X和Q198X,且对后三者的通读效率均在50%以上;tRNA op能有效通读C264X,通读效率约50%左右;tRNA oc不能通读NKX2.5 PTC突变;各通读后样本Cx43 mRNA相对表达量增加7%~41.7%;tRNA am和tRNA op能有效通读NKX2.5 PTC突变,产生具有功能的全长蛋白,但tRNA抑制子对细胞的其他影响还不明确,有待于进一步观察。

关键词: tRNA抑制子, NKX2.5, 提前终止密码子, 通读, 先天性心脏病

Abstract: Human NKX2.5 (NK2 homeobox 5) premature stop codon (PTC) mutations cause congenital heart diseases such as atrial septal defect and atrioventricular block. At present, eight NKX2.5 PTC mutations were reported as E109X, Q149X, Q170X, Q187X, Q198X, Y256X, Y259X and C264X. To observe the ability of tRNA suppressors to read through NKX2.5 PTC mutations and produce functional full-length proteins, eight NKX2.5 PTC mutations were cloned into pcDNA3.1(-) vectors and four fragments (wild-type NKX2.5, E109X, Q149X and C264X) were cloned in pEGFP-N1 vectors to acquire NKX2.5-EGFP fusing plasmids. After transfection of NKX2.5-EGFP with or without corresponding tRNA suppressor into HeLa cells, the quantity of EGFP was measured to confirm the readthrough ability of the PTCs. NKX2.5 full-length and truncated protein expression levels were examined by Western blotting and the readthrough efficiency of tRNA suppressors on the PTCs was calculated respectively. The activity of NKX2.5 full-length and truncated protein was confirmed on NKX2.5 target gene-Cx43 mRNA level measured by Real-time PCR. Three tRNA suppressors were used: tRNA am, tRNA oc and tRNA op. tRNA am could suppress UAG-containing PTCs Q149X, Q170X, Q187X, Q198X and the readthrough efficiency for the latter three was above 50%. tRNA op could suppress UGA-containing PTC C264X with ~50% readthrough efficiency. tRNA oc failed to read through NKX2.5 PTC mutations. The relative Cx43 mRNA level in all readthrough samples was increased to 7%-41.7%. In conclusion, tRNA am and tRNA op could suppress NKX2.5 PTCs and induce functional protein expression. However, the effects of tRNA suppressors on cellular function are not clear yet, warranting further researches.

Key words: tRNA suppressors, NKX2.5, premature stop codon (PTC), readthrough, congenital heart disease

欧阳平,刘远航,黄志刚,齐小娟,倪倩,刘亚萍,宋仁生,李涛,吴柱国. tRNA抑制子对转录因子NKX2.5提前终止密码子的通读[J]. 遗传, 2015, 37(4): 367-373.

Ping Ouyang,Yuanhang Liu,Zhigang Huang,Xiaojuan Qi,Qian Ni,Yaping Liu,Rensheng Song,Tao Li,Zhuguo Wu. Readthrough on transcription factor NKX2.5 premature stop codon by tRNA suppressors[J]. HEREDITAS(Beijing), 2015, 37(4): 367-373.

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tRNA抑制子对转录因子NKX2.5提前终止密码子的通读

Readthrough on transcription factor NKX2.5 premature stop codon by tRNA suppressors

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