Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene  

作　　者：Guang-Jing Hu Jia Chen Xiao-Nan Zhao Jia-JiaXu Dong-Qing Guo Ming Lu Ming Zhu Ying Xiong Qin Li Catherine CY Chang Bao-Liang Song Ta-Yuan Chang Bo-Liang Li 

机构地区：[1]State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sei-ences, Chinese Academy of Sciences, Shanghai 200031, China [2]Department of Biochemistry, Geisel School of Medicine at Dart-mouth, Hanover, NH 03 756, USA

出　　处：《Cell Research》2013年第8期1007-1024,共18页细胞研究（英文版）

摘　　要：Trans-splicing, a process involving the cleavage and joining of two separate transcripts, can expand the tran- scriptome and proteome in eukaryotes. Chimeric RNAs generated by trans-splicing are increasingly described in literatures. The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health. Certain antibiotic resistance genes, such as ampicillin resis- tance gene （Ampr）, are frequently used in recombinant plasmids. Until now, trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported. Acyl-CoA：cholesterol acyltransferase 1 （ACAT1） is a key enzyme involved in cellular cholesterol homeostasis. The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities. Here, we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exog- enous transcript encoded by the antisense strand of Ampr （asAmp） present in common Amprplasmids and the 4.3- kb endogenous ACAT1 chimeric mRNA, which is presumably processed through a prior event of interchromosomal trans-splicing. Strikingly, DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells. Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production, reveal an exogenous-endogenous trans-splicing system, in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells, and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.Trans-splicing, a process involving the cleavage and joining of two separate transcripts, can expand the tran- scriptome and proteome in eukaryotes. Chimeric RNAs generated by trans-splicing are increasingly described in literatures. The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health. Certain antibiotic resistance genes, such as ampicillin resis- tance gene （Ampr）, are frequently used in recombinant plasmids. Until now, trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported. Acyl-CoA：cholesterol acyltransferase 1 （ACAT1） is a key enzyme involved in cellular cholesterol homeostasis. The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities. Here, we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exog- enous transcript encoded by the antisense strand of Ampr （asAmp） present in common Amprplasmids and the 4.3- kb endogenous ACAT1 chimeric mRNA, which is presumably processed through a prior event of interchromosomal trans-splicing. Strikingly, DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells. Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production, reveal an exogenous-endogenous trans-splicing system, in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells, and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

关 键 词：TRANS-SPLICING chimeric RNA ampicillin resistance gene recombinant plasmid ACAT1 

分 类 号：Q26[生物学—细胞生物学] S859.796[农业科学—临床兽医学]