ORIGINAL ARTICLE

Cell Research (2008): 1-16
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RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform

Jia Chen^1,*, Xiao-Nan Zhao^1,*, Li Yang¹, Guang-Jing Hu¹, Ming Lu¹, Ying Xiong¹, Xin-Ying Yang¹, Catherine CY Chang², Bao-Liang Song¹, Ta-Yuan Chang² and Bo-Liang Li¹

Correspondence: Bo-Liang Li Tel: +86-21-5492-1278; Fax: +86-21-5492-1279 E-mail: blli@sibs.ac.cn *These two authors contributed equally to this work.

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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.

Cell Research advance online publication 10 June 2008; doi: 10.1038/cr.2008.66

Keywords: human ACAT1 isoform, chimeric human ACAT1 mRNA, interchromosomal region, RNA secondary structure, internal ribosome entry site