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.
Chen
Jia
J
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.
Zhao
Xiao-Nan
XN
Yang
Li
L
Hu
Guang-Jing
GJ
Lu
Ming
M
Xiong
Ying
Y
Yang
Xin-Ying
XY
Chang
Catherine C Y
CC
Song
Bao-Liang
BL
Chang
Ta-Yuan
TY
Li
Bo-Liang
BL
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