Control Mechanisms of Differential Translation of Hsp90 Isoforms in 9L Rat Gliosarcoma Cells Chih-Wei Lo, 1 Yuo-Sheng Chang, 1 Chih-Chung Chao, 1 Margaret Dah-Tsyr Chang, 2 Kun-Che Chang, 1 and Yiu-Kay Lai 1,3 * 1 Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC 2 Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC 3 Department of Bioresources, Da-Yeh University, Changhua 51591, Taiwan, ROC ABSTRACT Although the differential expression of heat shcok proteins, Hsp90a and Hsp90b was extensively studied in many kinds of cells, the post- transcriptional regulation of Hsp90 isoforms remains unclear. In control and GA-treated rat gliosarcoma cells, it has been reported that the translational efficiency of hsp90a is higher than hsp90b. In this study, we present evidences identifying the roles for leaky scanning and 5 0 -UTR sequence in translational regulation of Hsp90b. The result of in vitro transcription and translation (IVTT) experiment showed that hsp90a exhibited higher translation efficiency than hsp90b. Sequence analysis revealed that there is an out-of-frame downstream AUG codon in hsp90b gene. However, elimination of the downstream AUG by site-directly mutagenesis or introducing Kozak context sequence around the initiator AUG of hsp90b open reading frame increased its translational efficiency, which indicated that leaky scanning might be a possible mechanism regulating hsp90b. Furthermore, we also constructed a firefly luciferase reporter system to verify the effect of subsequent translation at the downstream out-of-frame AUG codon in 9L and A549 cells. Furthermore, it is believed that 5 0 -untranslated region (5 0 -UTR) also plays a significant role in translational control. We showed hsp90b 5 0 -UTR gives rise to the reduction of the translation efficiency in IVTT experiment. Additionally, the reductive effect of hsp90b 5 0 -UTR was further confirmed by luciferase reporter assay using truncated deletion analyses of 5 0 -UTR of hsp90b. Our results support the hypothesis that ribosome leaky scanning mechanism and 5 0 -UTR sequence acts as negative regulators in hsp90b mRNA. J. Cell. Biochem. 107: 418–427, 2009. ß 2009 Wiley-Liss, Inc. KEY WORDS: HEAT SHOCK PROTEIN 90; POST-TRANSCRIPTIONAL CONTROL; TRANSLATION EFFICIENCY; LEAKY SCANNING; 5 0 -UNTRANSLATED REGION H eat shock proteins (Hsps) belong to the stress protein superfamily, serve as molecular chaperones and play critical roles in protein folding, signal transduction, and proteasome mediated protein degradation [Neckers and Ivy, 2003; Wegele et al., 2004; Liu et al., 2007]. Hsp90, the 90 kDa molecular chaperone comprising 1–2% of cellular proteins under non-stress conditions is one of the most abundant proteins in eukaryotic cells and the major core of a so-called cytosolic molecular chaperone complex [Pearl and Prodromou, 2001; Picard, 2002]. Exposure of Hsp90 inhibitors such as geldanamycin (GA), radicicol, or their analogs generally leads to rapid release of bound client proteins and disrupts the chaperone complex [Pratt and Toft, 2003]. Hsp90 is highly conserved from prokaryotes to eukaryotes. In mammalian cells, two functionally similar Hsp90 isoforms, known as Hsp90a and Hsp90b in human (Hsp86 and Hsp84 in mouse), have been identified [Csermely et al., 1998; Picard, 2002]. These two isoforms are generally around 85% identical in amino acid sequences but encoded by separate genes [Moore et al., 1989; Chen et al., 2005]. However, the nucleotide sequences of hsp90a and hsp90b have much less similarity as compared to their translated amino acid sequences, particularly in their 5 0 - and 3 0 -untranslated regions (5 0 -UTR and 3 0 -UTR), the introns, and the 5 0 -flanking sequences [Hickey et al., 1989; Gupta, 1995]. Unlike most eukaryotic genes encoding Hsps, hsp90a and hsp90b are composed of several Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 107:418–427 (2009) 418 Grant sponsor: VGHUST; Grant numbers: 96-G2-02-5, 97-G2-4; Grant sponsor: NSC; Grant number: 97-3112-B-007- 006. *Correspondence to: Dr. Yiu-Kay Lai, Department of Bioresources, Da-Yeh University, Changhua 51591, Taiwan, ROC. E-mail: lslyk@life.nthu.edu.tw Received 2 January 2009; Accepted 17 February 2009  DOI 10.1002/jcb.22138  2009 Wiley-Liss, Inc. Published online 23 March 2009 in Wiley InterScience (www.interscience.wiley.com).