Translation Regulation of the Glutamyl-prolyl-tRNA Synthetase Gene EPRS through Bypass of Upstream Open Reading Frames with Noncanonical Initiation Codons

Abstract

In the integrated stress response, phosphorylation of eIF2α (eIF2α-P) reduces protein synthesis while concomitantly promoting preferential translation of specific transcripts associated with stress adaptation. Translation of the glutamyl-prolyl-tRNA synthetase gene EPRS is enhanced in response to eIF2α-P. To identify the underlying mechanism of translation control, we employed biochemical approaches to determine the regulatory features by which upstream ORFs (uORFs) direct downstream translation control and expression of the EPRS coding region. Our findings reveal that translation of two inhibitory uORFs encoded by noncanonical CUG and UUG initiation codons in the EPRS mRNA 5'-leader serve to dampen levels of translation initiation at the EPRS coding region. By a mechanism suggested to involve increased translation initiation stringency during stress-induced eIF2α-P, we observed facilitated ribosome bypass of these uORFs, allowing for increased translation of the EPRS coding region. Importantly, EPRS protein expression is enhanced through this preferential translation mechanism in response to multiple known activators of eIF2α-P and likely serves to facilitate stress adaptation in response to a variety of cellular stresses. The rules presented here for the regulated ribosome bypass of noncanonical initiation codons in the EPRS 5'-leader add complexity into the nature of uORF-mediated translation control mechanisms during eIF2α-P and additionally illustrate the roles that previously unexamined uORFs with noncanonical initiation codons can play in modulating gene expression.

Trial registration: ClinicalTrials.gov NCT00064142.

Keywords: EPRS; aminoacyl tRNA synthetase; endoplasmic reticulum stress (ER stress); eukaryotic initiation factor 2 (eIF2); integrated stress response; stress response; translation control; translation initiation; uORF.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

EPRS translational expression is increased in response to eIF2α-P.A, WT MEF cells were treated with thapsigargin for 6 h or left untreated. Lysates were collected and separated by sucrose gradient centrifugation followed by analysis of polysome profiles at 254 nm. Total RNA was isolated from sucrose fractions, and the percentage of total ATF4 and EPRS mRNA was determined by qRT-PCR. Profiles and ATF4 and EPRS mRNA polysome shifts are representative of at least three independent biological experiments. B, WT and A/A MEF cells were treated with thapsigargin for up to 9 h or left untreated. Protein lysates were processed, and levels of EPRS, eIF2α-P, eIF2α total, and β-actin were measured by immunoblot. C, total RNA was collected from WT and A/A MEF cells treated with thapsigargin for 6 h or left untreated, and the relative levels of EPRS mRNA were measured using qRT-PCR. D, representation of EPRS 5′-leader. The uORFs in the 5′-leader of the EPRS mRNA are illustrated by the colored boxes, with the initiation codon(s) for each uORF listed. The green box indicates the CDS for EPRS. Ribosomes above UUG1 and CUG2 indicate those start codons that have been suggested to facilitate translation initiation in previous ribosome profiling studies (13, 14). TG, thapsigargin; NS, not significant.

FIGURE 2.

The 5′-leader of the EPRS mRNA directs preferential translation. A, top panel, a 5′-RACE was conducted for EPRS using WT MEFs treated with thapsigargin for 6 h or left untreated. Total RNA and cDNA were prepared, and DNA products were separated by gel electrophoresis, with markers for the indicted base pair sizes listed on the left. Bottom panel, nucleotide representation of the EPRS 5′-leader in lowercase letters, with uppercase letters representing the 5′-linker added during the 5′-RACE procedure and the beginning of the CDS of the EPRS-Luc fusion. Colored boxes represent the EPRS uORFs, with uORF1 in blue, uORF2 in yellow, and uORF3 in purple. Start codons for each uORF are indicated above the colored boxes. The coding region of the EPRS-Luc fusion is illustrated by the green box. The transcription start site is indicated with an arrow, and the location of the stem loop insertion is illustrated. B, the PTK-EPRS-Luc construct and a Renilla luciferase reporter were co-transfected into WT or A/A MEFs and treated with thapsigargin for 6 h or left untreated. EPRS 5′-leader mediated translation control was measured via Dual-Luciferase assay, and the corresponding EPRS-Luc mRNA was measured by qRT-PCR. The PTK-EPRS-Luc construct contains the cDNA sequence corresponding to the EPRS 5′-leader fused to the luciferase reporter gene with both the EPRS uORFs and the CDS of the EPRS-Luc fusion indicated with colored boxes that are the analogues to those indicated in Fig. 2A. TG, thapsigargin; Luc, luciferase.

FIGURE 3.

Preferential translation of EPRS features two uORFs with noncanonical initiation codons. The full-length EPRS uORFs were each individually fused in-frame to the luciferase CDS and were transcriptionally expressed from a TK promoter for generation of PTK-CUG123 uORF-Luc, PTK-UUG1 uORF-Luc, and PTK-UUG2 uORF-Luc. WT and mutant versions of the uORF-Luc fusions were transfected into WT MEF cells, and uORF translation control was measured by Dual-Luciferase assay, and the corresponding CUG123 uORF-Luc, UUG1 uORF-Luc, and UUG2 uORF-Luc mRNA levels were measured by qRT-PCR. WT versions of each luciferase fusion are illustrated by the green boxes. Mutant versions of PTK-CUG123 uORF-Luc include mutations of the CUG initiation codons, as represented by ΔCUG1, ΔCUG2, and ΔCUG3, and optimization of the start codon for CUG2 to an AUG in strong Kozak consensus sequence (ACCAUGG), as represented by CUG2 to optimized AUG. Mutant versions of PTK-UUG1 uORF-Luc include mutation of the UUG initiation codon, as represented by ΔUUG1, and optimization of the UUG start codon to an AUG in a strong Kozak consensus sequence (ACCAUGG), as represented by UUG1 to optimized AUG. Mutation of PTK-UUG2 uORF-Luc includes mutation of the UUG initiation codon, as represented by ΔUUG2. Loss of the indicated initiation codon in the uORF-Luc fusion is illustrated by the gray boxes, and the optimized initiation codon in the uORF-Luc fusion is illustrated in orange. Relative values are represented as histograms for each with the S.D. indicated. Luc, luciferase.

FIGURE 4.

EPRS translation control involves bypass of an uORF with a noncanonical CUG initiation codon. WT and mutant versions of PTK-EPRS-Luc constructs were transfected into WT MEF cells, treated for 6 h or left untreated, and measured using a Dual-Luciferase assay, and the relative levels of the corresponding EPRS-Luc mRNAs were measured by qRT-PCR. Mutant versions of PTK-EPRS-Luc include a stem loop insertion and mutation of the CUG initiation codons individually or together, as represented by ΔCUG1, ΔCUG2, and ΔCUG3. Losses of the initiation codons CUG1, CUG2, and CUG3 are indicated in the EPRS-Luc fusion that is indicated by the gray boxes. Optimization of the CUG2 initiation codon to an AUG in optimal Kozak consensus sequence (ACCAUGG) is represented as CUG to optimized AUG (orange box). Relative values are represented as histograms for each with the S.D. indicated. TG, thapsigargin; Luc, luciferase.

FIGURE 5.

EPRS translational control involves bypass of uORFs with noncanonical initiation codons. WT and mutant versions of PTK-EPRS-Luc constructs were transfected into WT MEF cells, treated for 6 h or left untreated, and measured using a Dual-Luciferase assay, and the corresponding EPRS-Luc mRNA levels were measured by qRT-PCR. Mutant versions of PTK-EPRS-Luc include mutation of the UUG1 and UUG2 initiation codons (ΔUUG1 and ΔUUG2), optimization of the UUG1 initiation codon to an AUG with optimal Kozak consensus sequence (UUG1 to optimized AUG), mutation of the stop codon of the UUG1 uORF to generate an overlapping out of frame uORF (UGA to UGG), combined mutation of UUG1 to an AUG with optimal Kozak consensus sequence (ACCAUGG) with the stop codon mutation (UUG1 to optimized AUG and UGA to UGG), and combined mutation of the initiation codons for UUG1 and CUG2 (ΔUUG1 and ΔCUG2). Loss of the indicated initiation codon in the EPRS-Luc fusion is illustrated by a gray box, and those involving optimization of the initiation codon or extension of the uORF are indicated by orange boxes. Relative values are represented as histograms for each with the S.D. indicated. TG, thapsigargin; Luc, luciferase.

FIGURE 6.

EPRS translation control is regulated in response to halofuginone treatment.A, GCN2+/+ and GCN2−/− MEF cells were treated with increasing concentrations of halofuginone for 6 h. Protein lysates were processed, and the levels of EPRS, eIF2α-P, eIF2α total, and β-actin were measured by immunoblot. B, WT MEF cells were treated with halofuginone for 6 h or left untreated. Lysates were collected, sheared using a 23-gauge needle, and layered on to 10–50% sucrose gradients followed by ultracentrifugation and analysis of whole lysate polysome profiles at 254 nm. C, the PTK-EPRS-Luc construct and a Renilla luciferase reporter were co-transfected into WT or A/A MEFs and treated with thapsigargin or halofuginone for 6 h or left untreated. EPRS 5′-leader mediated translation control was measured via Dual-Luciferase assay and corresponding EPRS-Luc mRNA values were measured by qRT-PCR. The relative values are represented as histograms for each with the S.D. indicated. HF, halofuginone; TG, thapsigargin.

FIGURE 7.

GCN2 confers protection against halofuginone-induced toxicity.A, equal numbers of GCN2+/+ and GCN2−/− MEFs were seeded in 96-well plates, cultured for 24 h, and treated with 12.5, 25, or 50 nm halofuginone for 6 h, followed by recovery in fresh media for 18 h. MTT activity was measured by the conversion of tetrazolium to formazan. B, model depicting gene regulation downstream of the eIF2 kinase GCN2 during halofuginone treatment. With the accumulation of uncharged tRNAPro during halofuginone treatment, activated GCN2 phosphorylates eIF2α and decreases global mRNA translation initiation. Coincident with a decrease in overall translation, mRNA encoding ATF4 is subject to preferential translation, ultimately leading to an increase in ATF4 downstream targets central to stress remediation. Also subject to preferential translation during eIF2α-P is mRNA encoding EPRS. During halofuginone treatment, EPRS is preferentially translated, and the resulting increase in its expression is suggested to quench chronic drug toxicity. HF, halofuginone.

FIGURE 8.

Model for EPRS translational control.EPRS translation control involves bypass of two inhibitory uORFs with noncanonical initiation codons. In the absence of stress, low levels of eIF2α-P, and high eIF2-GTP, ribosomes scan the 5′-leader of the EPRS mRNA and initiate translation at CUG2, encoded in uORF1, or UUG1, encoded in uORF2. uORF1 overlaps out of frame with the EPRS CDS, and translation of uORF1 results in translation termination 3′ of the start codon for EPRS. A portion of the ribosomes that translate uORF2, encoded by UUG1, terminate and are released from the EPRS mRNA. Alternatively, ribosomes can reinitiate at the downstream EPRS CDS after uORF2 translation. The presence of the CUG and UUG initiation codons allows for a portion of the scanning ribosomes to bypass the uORFs, at least in part because of their noncanonical initiation codons and instead initiate translation at the EPRS CDS during basal conditions. In the presence of stress, high levels of eIF2α-P and diminished eIF2-GTP levels are suggested to further facilitate bypass of the uORFs and allow for an increase in EPRS CDS translation and subsequent protein expression.