Volume 18, No 9, Sep 2008

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 18 Issue 9, September 2008: 921-936

ORIGINAL ARTICLES

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

¹State 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

²Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756, USA Correspondence: Bo-Liang Li(blli@sibs.ac.cn )

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 (2008) 18:921-936. doi: 10.1038/cr.2008.66; published online 10 June 2008

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