Translational Upregulation of an Individual p21^Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress

<div><p>Multiple transcripts encode for the cell cycle inhibitor p21^Cip1. These transcripts produce identical proteins but differ in their 5’ untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through ³⁵S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5’ upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G₁/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.</p></div

GCN2-dependent translational upregulation of p21 is specific to variant 2 and mediated by the presence of 5’ uORFs.

<p>A) Partial sequence of p21 5’ UTR luciferase reporter constructs. The p21 sequence is in lowercase letters, and the luciferase sequence is in capital letters. Restriction sites used to insert the p21 5’ UTRs are underlined. The start codon for p21 is indicated in green capital letters. The start and stop codons for p21 variant 2 uORFs 1, 2, and 3 are in red, blue, and purple letters, respectively. The mutations created to disrupt the uORFs are indicated by arrows. B) Luciferase assay using p21 5’ UTR reporter constructs under leucine deprivation. Top: A schematic representation of the p21 variant 1 and 2 luciferase reporter constructs (not drawn to scale). Bottom: Luciferase activity from reporter constructs was measured in leucine-deprived GCN2^+/+ and eIF2α S51A MEFs and normalized to <i>Renilla</i> luciferase. Data are the average of three independent experiments ± S.E.M.; *p<0.05. C) Luciferase assay using mutant p21 variant 2 5’ UTR reporter constructs. Right: A schematic representation of the p21 variant 2 luciferase reporter constructs (not drawn to scale). Top left: Luciferase activity from reporter constructs was measured in leucine-deprived GCN2^+/+ MEFs. Bottom left: Relative basal translation levels of mutant p21 variant 2 reporter constructs in untreated cells. All results were normalized to <i>Renilla</i> luciferase. Data are the average of three (p21 variant 1 and p21 variant 2 ∆uORF1,2,3) or four (remaining constructs) independent experiments ± S.E.M.; *p<0.5, **p<0.01.</p

Model of p21 translational regulation.

<p>Upon amino acid deprivation, GCN2 phosphorylates eIF2α, which delays translation initiation. This results in ribosomes bypassing 5’ uORFs in the p21 variant 2 transcript and beginning translation at the p21 ORF, resulting in p21 upregulation under stress. This pathway is distinct from p53-dependent transcriptional upregulation of p21. Induction of p21 upon nutrient deprivation leads to G_<b>1</b>/S arrest and promotes cell survival.</p

p21 induction under amino acid deprivation requires GCN2 and eIF2α phosphorylation.

<p>A) The 5’ regions of the two known mouse transcript variants of p21 are shown. The start codon for p21 is indicated in green capital letters. Variant 2 contains three 5’ uORFs; the start and stop codons for each are indicated in red, blue, and purple, respectively. B) Western blot analysis of p21 induction in leucine-deprived GCN2^+/+, GCN2^-/-, and eIF2α S51A MEFs. β-actin was used as a loading control. All samples were collected at the same time and the blots were run in parallel. C) Western blot analysis of p21 induction in GCN2^+/+, GCN2^-/-, and eIF2α S51A MEFs irradiated with a dose of 4 Gy. Total eIF2α was used as a loading control. D) Western blot analysis of the reversibility of p21 induction in GCN2^+/+ MEFs. β-tubulin was used as a loading control. E) Western blot analysis of p21 induction in leucine-deprived SQ20B cells. Cells were transfected with either non-targeting siRNA (siNT) or siRNA against GCN2 (siGCN2). β-tubulin was used as a loading control. F) Western blot analysis of p27 induction in leucine-deprived GCN2^+/+, GCN2^-/-, and eIF2α S51A MEFs. β-tubulin was used as a loading control. Data information: Values below blot represent the fold change in total pixel intensity over control of p21, GCN2, or p27 normalized to the loading control for each lane.</p

p21 regulates G₁/S arrest and cell survival under conditions of amino acid deprivation.

<p>A) Western blot analysis of the MEFs used in (B) to determine p21 knockdown efficiency. Values below blot represent the fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. β-tubulin was used as a loading control. B) G_<b>1</b>/S ratio of leucine-starved GCN2^+/+ MEFs stably transfected with non-targeting shRNA (shNT) or shRNA against p21 (shp21). DNA content was measured by propidium iodide staining and flow cytometry analysis. Data are the average of three independent experiments ± S.E.M.; * p<0.05, ** p<0.01. C) Western blot analysis of the SQ20Bs used in (D) to determine p21 knockdown efficiency. Values below blot represent the fold change in total pixel intensity over control of p21 normalized to the loading control for each lane. β-tubulin was used as a loading control. D) G_<b>1</b>/S ratio of leucine-starved SQ20Bs transfected with non-targeting siRNA (siNT) or siRNA against p21 (sip21). DNA content was measured by propidium iodide staining and flow cytometry analysis. Data are the average of four (siNT) or five (sip21) independent experiments ± S.E.M.; * p<0.05, ** p<0.01. E) Clonogenic survival of control and p21 knockdown GCN2^+/+ MEFs exposed to long-term leucine starvation. Data are the average of three independent experiments ± S.E.M; *p< 0.5, **p< 0.1. F) Representative picture of control and p21 knockdown MEFs from the clonogenic survival assay described in (E) after one week of colony formation.</p

Polysome profiling reveals differences between the translational efficiencies of p21 variants 1 and 2 under nutrient stress.

<p>A)Polysome profiles of leucine-starved GCN2^+/+ MEFs over time. Lysates from GCN2^+/+ MEFs grown with or without leucine for 8, 12, 16, 20, and 24 hours were separated on 10 to 50% sucrose gradients. Polysome profiles of the gradients were generated by measuring absorbance at 254 nm. B) Time course of p21 translational efficiency as measured by mRNA association with polysomes. qPCR was performed on fractions pooled from sucrose gradients as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005212#pgen.1005212.s004" target="_blank">S4A Fig</a> p21 transcript levels in each group were normalized to total transcript. C) Time course of β-actin and ATF4 translational efficiency as measured by mRNA association with polysomes. qPCR was performed on fractions pooled from sucrose gradients as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005212#pgen.1005212.s004" target="_blank">S4A Fig</a>. Transcript levels in each group were normalized to total transcript. D) Time course of global translational efficiency in leucine-deprived GCN2^+/+ MEFs. Left: Time course of translational efficiency in leucine-deprived cells. Right: Western blot analysis of p-eIF2α in leucine-deprived GCN2^+/+ MEFs. Values below blot represent the fold change in total pixel intensity of p-eIF2α over control normalized to eIF2α.</p