Abstract
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 AUG1397-1399 and GGC1274-1276 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 GGC1274-1276 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-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop1355-1384 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 GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 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.
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Acknowledgements
This work was supported by grants from the National Basic Research Program of China (No. 2002CB513003, 2006CB0D1100) and the Hi-Tech Research and Development Program of China (No. 2007AA09Z400), Foundations NNSC (No. 30571057) and SSTC (No. 07JC14061) to Bo-Liang Li, Bao-Liang Song and Ying Xiong, and NIH grant HL 36709 to Ta Yuan Chang. We thank Dr Kang-Cheng Ruan and our colleagues Lei Lei, Jia-Jia Xu, and Qin Li for helpful discussion and technical assistance during the course of this study. We thank Prof Akio Nomoto for the plasmid pC1B that contains the HCV IRES.
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Chen, J., Zhao, XN., Yang, L. et al. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform. Cell Res 18, 921–936 (2008). https://doi.org/10.1038/cr.2008.66
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DOI: https://doi.org/10.1038/cr.2008.66
