Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR

Abstract

Chop is a ubiquitously expressed mammalian gene encoding a small nuclear protein related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. CHOP protein plays an important role in various cellular processes such as growth, differentiation and programmed cell death. CHOP expression is strongly increased in response to a large variety of stresses including perturbation of the endoplasmic reticulum function, DNA damage and nutrient deprivation. Multiple mechanisms including transcriptional and post-transcriptional controls are involved in the regulation of CHOP expression. We show here that the 5'UTR of the Chop transcript plays an important role in controlling the synthesis of CHOP protein. In particular, the 5'UTR contains a conserved uORF which encodes a 31 amino acid peptide that inhibits the expression of the downstream ORF. Mutational analysis of the 5' leader region and peptide coding sequences suggests that the peptide itself inhibits expression of the downstream ORF. Such results suggest a role for uORF in limiting ribosomal access to downstream initiation sites. With respect to the importance of CHOP protein in the regulation of cellular functions, the mechanisms that regulate its basal level are of considerable interest.

Figure 1

The Chop 5′UTR sequence represses translation of the downstream coding sequences. (A) NIH-3T3 cells were infected with recombinant retrovirus expressing, under the control of the CMV promoter, the tagged CHOP ORF that contained the 5′UTR of human Chop (5′UTR–9E10–CHOP, lane 3) or not [5′UTR(Δ)–9E10–CHOP; lane 2]. A control experiment was performed by infecting cells with an empty virus (Untransfected, lane 1). Cells were then incubated for 4 h with or without 2.5 µg/ml of tunicamycin. On the construct scheme, the arrow represents the transcription start site. Protein extracts were analyzed for the presence of CHOP protein and mRNA as described in Materials and Methods. (B) HeLa cells were transiently transfected with LUC constructs containing either the wild-type (5′UTR–LUC) or the deleted [5′UTR(21)–LUC] Chop 5′UTR downstream of the Chop promoter. Forty-eight hours after transfection, cells were harvested and analyzed for relative luciferase activities and relative LUC mRNA level as described in Materials and Methods. (C) HeLa cells were transiently transfected with constructs containing the CMV promoter driving a LUC construct containing or not containing the Chop 5′UTR. The CHOP 5′UTR was cloned +6 nt after the start site for transcription. The relative Luc mRNA content was not affected by the CHOP 5′UTR (t-test, n > 3, ** = P < 0.0001, NS = not significant).

Figure 2

CHOP 5′UTR contains a conserved uORF. Sequence alignment of the 5′UTR region of the human, mouse and hamster Chop transcript. (A) Nucleotide sequence, (B) amino acid sequence. The uAUG codons and the stop codon are indicated. The conserved nucleotides or amino acids are shown and the nucleotide sequence of the uORF is boxed.

Figure 3

Mutations in the uAUGs abolish the repressor effect of the 5′UTR. HeLa cells were transiently transfected with LUC constructs containing the wild-type [uAUG(+)] or mutated Chop 5′UTR downstream of the CMV promoter. The AUGs shown in Figure 2 were mutated individually (uAUG#1, uAUG2, uAUG#3) or all together [uAUG(–)]. On the construct scheme, the arrow represents the transcription start site and the open box shows the uORF. Forty-eight hours after transfection cells were trypsinised, then half was proceeded for luciferase and β-galactosidase activity assays, and the second half was used for RNA extraction and transcript quantification. Relative LUC activities and mRNA levels were determined as the ratio LUC/βGAL as described in Materials and Methods. (LUC activity: t-test, n > 5, ** = P < 0.0001, NS = not significant; LUC mRNA: t-test, n = 3, NS = not significant).

Figure 4

Translation is initiated at the uORF AUG codon. HeLa cells were transiently transfected with LUC constructs containing either (1) LUC downstream of a CMV promoter or (2) a construct containing CHOP-uORF/LUC in-frame fusion downstream of the CMV promoter. Seventy-two hours after transfection, whole cell lysates were prepared and analyzed for the luciferase protein content by western blotting as described in Materials and Methods.

Figure 5

Translational repression by the Chop uORF is impaired by elongated uORF. The constructs shown in this figure are derived from the CMV–5′UTR–LUC (1). (2) The uAUG#2 was mutated in an optimum consensus start site (gccgccaccAUGg). In the following construct, the STOPmt construct (3) was generated by mutation of the stop codon of the uORF and by the introduction of a 1 nt frame shift in order to generate an extended uORF that is not in-frame with the LUC ORF. The CHOP 5′UTR and the LUC coding sequence are presented for each construct and the uORF is boxed. Forty-eight hours after transfection, cells were harvested and analyzed for relative LUC activities and mRNA level as described in Materials and Methods.

Figure 6

Translational repression by the Chop uORF is not dependent on the intercistronic region. Expression constructs containing the 5′UTR either in a wild-type form (5) or mutated on the uAUGs (6) were used to mutate the intercistronic region. The arrows represent point mutation of the uAUGs. When present the uORF is boxed. These constructs were transfected in HeLa cells then, 48 h after transfection, cells were harvested. The relative LUC activities and mRNA levels were analyzed as described above. In plasmid ICmt, the CHOP intercistronic region was replaced by a LacZ sequence which does not contain any AUG [(3) and (4)]. The initiation context of the LUC AUG was not changed. Plasmids mutated on the AUG#3 are given as a control (1) (2). A spacer of 47 bp [(7) and (8)] or 140 bp [(9) and (10)] that does not contain AUG codon was introduced into the intercistronic region of the pCMV–5′UTR–LUC plasmid mutated or not on the uAUGs. In the constructs ΔIC, the intercistronic region was deleted [(11) and (12)]. The mutations do not affect the Kozak consensus sequence of the LUC AUG.

Figure 7

Inhibition of translation by the uORF is dependent on its peptide sequence. (A) HeLa cells were transiently transfected with LUC constructs [derived from the CMV–5′UTR–LUC(1) (2)] mutated in the uORF sequence. Forty-eight hours after transfection cells, were harvested then relative LUC activities and mRNA levels were analyzed. Plasmids contain the 5′UTR either in a wild-type form or mutated on uAUG#1 and AUG#2. The arrows represent point mutation of the uAUGs. When present, the uORF is boxed. Point mutations of the uORF coding sequence are indicated by asterisks. In the first set of constructs a stop codon was introduced at the nucleotide +52, the UGG codon is replaced by the UGA codon (3) and (4) generating a three amino acid peptide. In the second set of constructs, the sequence of the synthesized peptide was mutated by introduction of a frame shift in the nucleotide sequence (5) and (6). One base was introduced after AUG#2 and one base was withdrawn just before the stop codon. The mutation does not affect the Kozak consensus sequence of AUG#2. The mutated uORF is boxed and hatched. In the last set of constructs, we have generated ‘silent mutation’ in the uORF coding sequence, which modify the mRNA sequence but not the amino acid sequence [we have mutated 13 nt located in the last part the uORF sequence; (7) and (8)]. (B) HeLa cells were transiently co-transfected with 0.5 µg of the reporter plasmid [CMV–5′UTR–uAUG(–)–LUC] and increasing amounts (0, 0.5, 1, 2, 3 and 4 µg) of a CHOP uORF expression vector encoding the uORF peptide (pCMV–uORF). In the control experiment, this expression vector has been mutated on AUG#1 and AUG#2 [pCMV–uORF–AUG(–)]. The total amount of expression vector was kept constant by addition of empty vector (pCMV). Forty-eight hours after transfection, cells were harvested and relative LUC activities were analyzed.