上海科技大学知识管理系统

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.