Stress-induced start codon fidelity regulates arsenite-inducible regulatory particle-associated protein (AIRAP) translation

Abstract

Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a "scanning" mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite- inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript.

FIGURE 1.

Translational induction of AIRAP. A, immunoblot of AIRAP and p97 (used as a loading control) in mouse embryonic fibroblasts (MEFs) pretreated with arsenite (25 μm) for the indicated time course. AIRAP mRNA levels from the same cells were quantified and normalized to a housekeeping gene using qPCR, and fold induction is represented in the right panel. Quantitation and statistical evaluations were obtained from three independent experiments. Comparison of Time 0 with Time 90 showed a statistical significance of t test p < 0.001. B, left panel, metabolic labeling performed in MEFs pretreated with arsenite for the indicated time course. Cell lysates were resolved by SDS-PAGE, and the autoradiogram is presented. Right panel, polysome profiles from untreated and arsenite-treated cells showing the accumulation of ribosomal subunits and monosomes in the arsenite-treated cells.

FIGURE 2.

uORF mediates AIRAP translation induction. A, organization of the 5′-UTR of the mouse AIRAP transcript and the derivative 5′ AIRAP-GFP reporters. Exons 1 and 2 of the mouse AIRAP (encoding the 5′-UTR and the initiating amino acids of AIRAP CDS) were fused in-frame to the GFP CDS and are indicated in the GFP reporter. B, autoradiogram of radiolabeled proteins after a brief labeling pulse of untreated and pretreated cells. Cells were transfected with the indicated GFP reporter and subjected to a GFP IP, resolved by SDS-PAGE. Untreated wild-type reporter quantities were set as 1 for each reporter and relative quantities are shown. All quantifications were normalized to GFP mRNA transcript levels shown in panel B. Quantitation and statistical evaluations are presented from three independent experiments. Both ATF4 and AIRAP WT reporters showed a statistical significance of t test p < 0.001 upon comparing to the non-harboring 5′-UTR GFP reporter arsenite response. C, transcript levels from cells transfected with the indicated reporter and arsenite treated as indicated were quantified and normalized to a housekeeping gene using qPCR. Quantitation and statistical evaluations were obtained from three independent experiments with error bars representing the S.D. D, RT-PCR analysis of two reporters containing the 5'-UTR of AIRAP and the GFP CDS or AIRAP CDS. The GAPDH housekeeping gene was used as an internal loading control for total mRNA. Both reporters do not show any substantial changes and a role for mRNA stability in response to a brief arsenite treatment. The same conditions were used to measure translational induction; 60 min, 50 mm).

FIGURE 3.

AIRAP translation regulation is mediated by means of leaky scanning. A, diagram presentation of the reporters used. B, autoradiogram of radiolabeled proteins after a brief labeling pulse of untreated and arsenite-pretreated 293T cells. Cells were transfected with the indicated reporter and subjected to a GFP IP resolved by SDS-PAGE. Untreated 5′ AIRAP WT reporter quantities were set as 1, and relative quantities are shown below. All quantifications were normalized to GFP mRNA transcript levels. Quantitation and statistical evaluations are present from three independent experiments.

FIGURE 4.

eIF1 regulates AIRAP translational induction. A, autoradiogram of radiolabeled proteins after a brief labeling pulse of293T cells. The cells were transfected with the indicated AIRAP reporter and with an eIF1 expressing plasmid. Quantification of the GFP or HMW GFP (AIRAP TAAmut reporter) were normalized to GFP mRNA transcript levels. Quantitation and statistical evaluations are presented from three independent experiments. B, MEF cells were transfected with an empty vector or an eIF1-expressing plasmid and were either untreated or arsenite treated as indicated. Immunoblots revealing the AIRAP and p97 content (used as a loading control) are presented.

FIGURE 5.

Arsenite-induced eIF1 phosphorylation. A, eIF1 protein half-life was determined by incubating cells in the presence of 10 μg/ml cycloheximide (CHX) for the indicated time course, after which lysates were immunoblotted for eIF1 and P97 as a control for a long lived protein. Where indicated, Velcade (10 μg/ml) was added to evaluate proteasomal-dependent degradation. B, following a two-dimensional gel separation of 293T cell lysates (treated with arsenite or thapsigargin as indicated), immunoblots toward the endogenous eIF1 are presented. Where indicated (+CIP), arsenite-treated lysate was also incubated with alkaline phosphatase to evaluate if the pI shift is due to a phosphorylation event. C, autoradiogram of radiolabeled proteins obtained from 293T cells after a brief labeling pulse performed on cells that were treated as indicated. Cells were transfected with the indicated GFP reporter, subjected to a GFP IP, and content was resolved by SDS-PAGE. Untreated quantities were set as 1 for each reporter and relative quantities are shown. All quantifications were normalized to GFP mRNA transcript levels. Quantitation and statistical evaluations are present from three independent experiments. Both arsenite and thapsigargin treatments showed a statistical significant response with t test p < 0.001 upon comparing to the non-harboring 5′-UTR GFP reporter. D, fragmented MS/MS showing the only m/z fragment with a +80 shift. The presence of the y14 ion (labeled by arrow) indicates Tyr-79 is not the phosphorylation site leaving only Thr-72 as the possible site of phosphorylation. E, 24 h following the indicated eIF1 transfection, cells were treated with arsenite, and lysates were subjected to a two-dimensional gel separation analysis. Immunoblots revealing the exogenous eIF1 are presented.

FIGURE 6.

Arsenite and eIF1 activity. A, 5′ AIRAP-GFP reporter was transfected alongside increasing amounts of the indicated eIF1 expression vector (25, 100, and 250 ng). 24 h post-transfection, cells were directly evaluated for GFP, eIF1, and PSMA1 content by immunoblot (IB). PSMA1 served as a loading control. B, SEAP reporter was introduced into 293 cells with eIF1 as indicated. SEAP levels were determined by immunoblot (left) or assayed kinetically for the reporter activity (right). * indicates a nonspecific band. C, non-transfected cells (lane 1) or transfected cells with the indicated SEAP reporter were untreated (lane 2) or arsenite-treated (lane 3) metabolically labeled, and immunoprecipitated against SEAP. * indicates a nonspecific band. Quantitation of three independent experiments is shown. Label incorporation decrease upon arsenite treatment in the kozak AUG reporter was set to 1, and relative increase in eIF1 AUG context repression is shown. D, presented is a surface structure of the human eIF1 (gray) and the rabbit 18 S RNA (yellow). The reported eIF1 residues contacting the 18 S (red) original positions in the yeast eIF1 are indicated in brackets. The phosphorylation site within eIF1 (Thr-72) is labeled in green. The figure is based on the published 4KZY PDB structural coordinates (33).