title: [Readthrough on transcription factor NKX2.5 premature stop codon by tRNA suppressors].
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.
authors.affiliation: Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Ouyang
authors.affiliation: Department of Emergency, Inner Mongolia Hohhot No.1 Hospital, Hohhot 010050, China.
authors.forename: Yuan-Hang
authors.initials: YH
authors.lastname: Liu
authors.affiliation: Department of Epidemiology, School of Public Health, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Huang
authors.affiliation: Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Xiao-Juan
authors.initials: XJ
authors.lastname: Qi
authors.affiliation: Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Ni
authors.affiliation: Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Liu
authors.affiliation: The Second Clinical Collage, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Ren-Sheng
authors.initials: RS
authors.lastname: Song
authors.affiliation: Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: The Second Clinical Collage, Guangdong Medical College, Dongguan 523808, China.
authors.forename: Zhu-Guo
authors.initials: ZG
authors.lastname: Wu
mesh_terms: D018388:Codon, Terminator / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010443:Peptide Chain Termination, Translational; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D014157:Transcription Factors; D012343:RNA, Transfer