RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform.
We have previously reported that the human ACAT1 gene produces a chimeric mRNA through the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG(1397-1399) and GGC(1274-1276) as translation initiation codons to produce normal 50-kDa ACAT1 and a novel enzymatically active 56-kDa isoform, respectively, with the latter being authentically present in human cells, including human monocyte-derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC(1274-1276) codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268, 1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type, deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to the ORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop(1255-1268) from chromosome 7 and downstream stem-loop(1286-1342) from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop(1355-1384) from Chromosome 1 was dispensable. The results of experiments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC(1274-1276) codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC(1274-1276) codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACAT1 transcript.
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
Jia
J
Chen
Xiao-Nan
XN
Zhao
Li
L
Yang
Guang-Jing
GJ
Hu
Ming
M
Lu
Ying
Y
Xiong
Xin-Ying
XY
Yang
Catherine C Y
CC
Chang
Bao-Liang
BL
Song
Ta-Yuan
TY
Chang
Bo-Liang
BL
Li
D000101:Acetyl-CoA C-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D002877:Chromosomes, Human / Q000235:genetics*; D002878:Chromosomes, Human, Pair 1 / Q000235:genetics; D002897:Chromosomes, Human, Pair 7 / Q000235:genetics; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010957:Plasmids; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics; D014158:Transcription, Genetic
D003062:Codon; D020033:Protein Isoforms; D012333:RNA, Messenger; D012313:RNA; C506639:ACAT1 protein, human; D000101:Acetyl-CoA C-Acetyltransferase