The optional long 5′-untranslated region of human ACAT1 mRNAs impairs the production of ACAT1 protein by promoting its mRNA decay

• Published in: Acta biochimica et biophysica Sinica 2009-01, Vol.41 (1), p.30-41
• Main Authors: Zhao, Xiaonan, Chen, Jia, Lei, Lei, Hu, Guangjing, Xiong, Ying, Xu, Jiajia, Li, Qin, Yang, Xinying, Chang, Catherine C.Y., Song, Baoliang, Chang, Tayuan, Li, Boliang
• Format: Article
• Language: English
• Subjects: 5' Untranslated Regions; Animals; Base Sequence; Blotting, Western; CHO Cells; Codon; Cricetinae; Cricetulus; DNA Primers; Humans; Nucleic Acid Conformation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sterol O-Acyltransferase - biosynthesis; Sterol O-Acyltransferase - genetics; Sterol O-Acyltransferase - physiology
• ISSN: 1672-9145; 1745-7270
• DOI: 10.1093/abbs/gmn004

We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG1397-1399 initiation codon, and also a minor 56 kDa isoform using the upstream in-frame GGC1274-1276 initiation codon. The GGC1274-1276 codon is located at the optional long 5′-untranslated region (5′-UTR, nt 1-1396) of the mRNAs. The DNA sequences corresponding to this 5′-UTR are located in two different chromosomes, 7 and 1. In the current work, we report that the optional long 5′-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG1397-1399 codon, mainly by promoting its mRNA decay. The western blot analyses indicated that the optional long 5′-UTR potently impaired the production of different proteins initiated from the AUG1397-1399 codon, meaning that this impairing effect was not influenced by the 3′-UTR or the coding sequence of ACAT1 mRNA. The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5′-UTR dramatically reduced the contents of its linked mRNAs. Analyses of the protein to mRNA ratios showed that this 5′-UTR mainly decreased its mRNA stability rather than altering its translational efficiency. We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription. The results showed that this 5′-UTR promoted its mRNA decay. Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that, in both the cytoplasm and the nucleus of cells, the optional long 5′-UTR-linked mRNAs decayed faster than those without the link. Overall, our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.