FIGURE 3.
5′-Leader of the CHOP mRNA contains an uORF and is sufficient for translational control in response to eIF2∼P. A, top panel, 5′-RACE was carried out for endogenous CHOP and CHOP-Luc using RNA prepared from wild-type MEF cells expressing the PTK-CHOP-Luc reporter, which were treated with the ER stress agent thapsigargin (+) or no stress agent (−). DNA products were separated and visualized by electrophoresis using a 1.2% agarose gel, with markers of the indicated size in base pairs represented on the right. A, bottom panel, sequence of the 5′-leader of the mouse CHOP mRNA is presented with the boxes indicating the uORF and the coding region of the CHOP-luciferase fusion. Residues mutated in the analysis of CHOP translational control are indicated below the box. The bold arrow indicates the transcription start site of the CHOP gene as determined by sequencing of the 5′-RACE products. A stem-loop structure or 120-bp segment was inserted at the indicated position upstream of the uORF. B, polypeptide sequences encoded by the uORF in the CHOP mRNAs from different vertebrates. The uORF polypeptide sequences were from representative cDNAs derived from the indicated CHOP orthologs, including human (GenBankTM accession number BC003637), mouse (BC013718), rat (BC100664), hamster (M29238), pig (AK346731), bear (GW278660), cow (BC122721), sheep (DY499855), dog (DN431044), frog Xenopus tropicalis (BC153679), and fish Danio rerio (BC134052). The number of polypeptide residues encoded by each uORF is listed following the sequences. Residues conserved among the uORF sequences are listed in the consensus, with invariant residues in capital letters and those conserved in lowercase letters. C, CHOP translational control in response to ER stress was measured by a Dual-Luciferase assay. The PTK-CHOP-Luc reporter and a Renilla luciferase plasmid, which served as an internal control, were transfected into wild-type MEF cells (WT) or A/A cells expressing eIF2α-S51A, and treated with thapsigargin (Stress), or no stress. The PTK-CHOP-Luc reporter contains cDNA sequences corresponding to the entire 5′-leader of the CHOP mRNA, which is illustrated along with the luciferase reporter gene. Three independent experiments were carried out for each measurement, and relative values are represented as histograms for each, with the S.D. indicated. In parallel, the levels of the CHOP-Luc mRNA were measured by qRT-PCR, and relative values of the reporter transcripts were presented as histograms, with error bars representing the S.D.