Human eIF2A has a minimal role in translation initiation and in uORF-mediated translational control in HeLa cells

  1. Mykola Roiuk
  2. Marilena Neff
  3. Aurelio A Teleman  Is a corresponding author
  1. German Cancer Research Center (DKFZ) Heidelberg, Germany
  2. Faculty of Medicine, Heidelberg University, Germany
  3. Faculty of Biosciences, Heidelberg University, Germany
7 figures, 1 table and 8 additional files

Figures

Figure 1 with 1 supplement
eIF2A has minimum effect on cell proliferation and global translation.

(A) Validation that eIF2A knockout cells have no eIF2A protein by immunoblotting. (B) Two independent eIF2A knockout HeLa cell lines have no proliferation defect, assayed by CellTiter Glo. Error bars: standard deviation. Significance by ordinary one-way ANOVA. (C–D) eIF2A knockout HeLa cells have no detectable change in global translation rates compared to control cells. (C) The translation rate was measured by immunoblotting to detect O-propargyl-puromycin (OPP) incorporated by metabolic labeling (left), and normalized to total protein amount assayed by Ponceau S (right). Three independent replicates are quantified in panel (D). CHX = cycloheximide treated sample to completely block global translation (negative control). Error bars: standard deviation. Significance by ordinary one-way ANOVA. (E–F) Polysome profiles of eIF2AKO cells show little to no difference to profiles from control cells. Lysates from either control or eIF2AKO HeLa cells were separated on a sucrose gradient. One representative graph is shown in panel E. The polysome/80 S ratio of three independent replicates is shown in panel F. Error bars: standard deviation. Significance by ordinary one-way ANOVA.

Figure 1—figure supplement 1
Loss of eIF2A does not perturb cell proliferation and global translation.

(A) Genotyping of eIF2AKO cell lines. Two eIF2A knockout lines were generated by targeting two separate coding exons. Shown is the resulting mutation at the DNA level, and a schematic of the predicted protein product. (B) mRNA levels of eIF2A, quantified by qRT-PCR, are reduced in the eIF2AKO lines. Error bars: standard deviation. Significance by ANOVA with Dunnett’s multiple comparison test. (C–D) Subcellular localization of eIF2A, detected by immunoblotting cytosolic and nuclear fractions or whole cell lysate (WCL), does not change upon treatment with tunicamycin (TM). Cells were treated with DMSO or Tunicamycin (1 µg/ml) for 2 hr. (C). Representative immunoblot (C) of three independent replicates quantified in (D). Nuclear and cytosolic fractions were lysed in equal volumes to reflect the relative contribution of the two compartments in a cell. Error bars: deviation. Significance by multiple unpaired, t-test. (E) Overexpressed FLAG-eIF2A shows predominantly cytoplasmic localization, assessed by immunofluorescent staining with anti-FLAG antibodies. Cells transfected with plasmid expressing eIF2A-FLAG were treated for 1 hr either with DMSO or 100 µM Sodium arsenite (SA). (F–G) eIF2A levels in HeLa cells are not affected by poly (I:C) (1 µg/ml), LPC (1 µg/ml), or tunicamycin (1 µg/ml) for 3 hr. Phospho-p38 signal is used as a positive control for the treatment. Representative immunoblot (F) or three independent replicates quantified in (G). Error bars: standard deviation. Significance by ANOVA with Dunnett’s multiple comparison test. (H–I) Global protein translation levels in HeLa cells do not change upon Flag-eIF2A overexpression, assessed by immunoblot against OPP (left) and normalized to total protein amount assayed by Ponceau S (right). Representative blots in (H), of four independent replicates quantified in (I). Error bars: standard deviation. Significance by unpaired, two-sided, t-test. All panels: ns = not significant.

Figure 2 with 2 supplements
Ribosome profiling of eIF2A-KO lines finds little impact of eIF2A on translation.

(A) Ribosome profiling identifies a handful of mRNAs sensitive to eIF2A depletion. Scatter plot of log2(fold change of Translation Efficiency eIF2AKO/control) versus significance. Significant candidates with log2(fold change) < –1 are shown in red. Significance was estimated with the Wald test performed by the DESeq2 package. p-values are adjusted for multiple comparisons.(B–D) Western blot validation of ribosome profiling results. Among the tested candidates, only CCND3 shows decreased protein levels in one eIF2AKO clone. Representative blot in (B), of triplicates quantified in (C). mRNA levels of the corresponding transcripts are quantified and shown in (D). Significance by Dunnett’s multiple comparison test ANOVA. error bar = st. dev., ns = not significant, *p<0.05, **p<0.01. (E) Luciferase reporters harboring 5’ UTRs of eIF2A-dependent transcripts do not show strong changes in expression upon loss of eIF2A. Reporters carrying the 5’ UTRs of the indicated candidate genes were cloned upstream of Renilla Luciferase (RLuc) and co-transfected with a Firefly Luciferase (FLuc) normalization control. The negative control RLuc reporter and the FLuc normalization control carry the 5'UTR of Lamin B1 (LMNB1). Significance by Dunnett’s multiple comparison test ANOVA. error bar = st. dev., ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 2—figure supplement 1
Ribosome profiling of control and eIF2AKO HeLa cells.

(A) Reproducibility between replicates of Ribosome profiling and total-mRNA libraries is shown. Three biological replicates were generated for control and eIF2AKO HeLa cells each. The Pearson’s coefficient (r) is shown for each compared pair. (B–C) Metagene profiles of footprints aligned to either the start codon (B) or the stop codon (C) of all main Open Reading Frames, for control and eIF2AKO HeLa cells. ‘Smooth’ curves were generated by averaging read counts with the sliding window of 3 nt. The dotted lines indicated the standard deviation between three replicates.

Figure 2—figure supplement 2
Loss of eIF2A does not affect translation of multiple different types of reporters.

(A) Luciferase reporters harboring 5’ UTRs of transcripts predicted to be eIF2A-dependent from ribosome footprinting do not show significantly reduced translation upon siRNA-mediated knockdown of eIF2A. The 5’ UTRs of the indicated genes were cloned upstream of Renilla Luciferase (RLuc) and co-transfected with a Firefly Luciferase (FLuc) normalization control reporter. Negative control RLuc reporter and the FLuc normalization control carry the 5'UTR of lamin B1 (LMNB1). Significance by multiple unpaired t-tests, ns = not significant. Error bars represent standard deviation. (B) Western blot control for efficiency of siRNA-mediated knockdown of eIF2A. (C) Transcripts shown in Figure 2A were reanalyzed with anota2seq package (Oertlin et al., 2019). Scatter plot of log2 fold-change of total RNA eIF2AKO/ control (x-axis) versus log2 fold-change of footprints eIF2AKO/ control (y-axis) is shown. Significant changes are detected in mRNA levels, while loss of eIF2A does not perturb translation. (D) Transfection of luciferase reporters designed to place the ribosome directly on top of the initiation AUG do not show reduced translation in eIF2AKO cells compared to controls. A 5’UTR containing the EMCV IRES or a short 5’ UTR of only 12 nt was cloned upstream of Renilla Luciferase (RLuc) and co-transfected with a FLuc normalization control. Negative control RLuc reporter and the FLuc normalization control carry the 5'UTR of Lamin B1 (LMNB1). Significance by ANOVA with Dunnett’s multiple comparison test. error bar = st. dev., ns = not significant.

Figure 3 with 1 supplement
eIF2A has little or effect on uORF translation.

(A) Synthetic reporters harboring uORFs with different start codons and initiation contexts do not show dependence on eIF2A. The sequence context of the uORF start codons is indicated: either AUG or a near-cognate start codon (GTG, TTG, CTG) was used. Significance by Dunnett’s multiple comparison test ANOVA, error bar = st. dev. ns = not significant. (B) Validation that HEK293T-H2-Kb eIF2AKO cells have no eIF2A protein by immunoblotting. (C) Schematic diagram illustrating the setup to simultaneously detect a small peptide produced by a uORF and fluorescent mNeonGreen encoded by the main ORF. The short peptide SIINFEKL is presented on the cell surface by MHC-I and detected using a monoclonal antibody. (D) eIF2A knockout does not cause a drop in uORF translation. In the graph to the right, the percent of uORF-positive cells relative to all mNeonGreen cells is quantified. Significance by unpaired, two-sided, t-test. ns = not significant.

Figure 3—figure supplement 1
Knockout of eIF2A has no effect on uORF translation.

(A–B) Metagene profiles of footprints relative to either the start codon (A) or stop codon (B) of uORFs transcriptome-wide, for control or eIF2AKO HeLa cells. ‘Smooth’ curves were generated by averaging footprint counts with a sliding window of 3 nt. The dotted lines indicated the standard deviation between three replicates. (C) eIF2A has little impact genome-wide on translation of mRNAs with uORFs. Comparison of the change in translation efficiency between eIF2AKO and control HeLa cells for three different groups of mRNAs: all transcripts, transcripts with AUG-initiated uORFs, or transcripts with uORFs that start with a near-cognate codon. Significance by ANOVA with Dunnett’s multiple comparison test. ns = not significant, ***p<0.001. (D) Activity of synthetic reporters harboring uORFs with different start codons and initiation contexts is not altered upon eIF2A-overexpression. The sequence context of the uORF start codons is indicated: either AUG or a near-cognate start codon (GTG, TTG, CTG) was used. Overexpression of eIF2A was validated by western blot against the FLAG-tag. Significance by Dunnett’s multiple comparison test ANOVA, error bar = st. dev. ns = not significant. (E) Proliferation of eIF2AKO or control HEK293T-H2-Kb cells by CellTiter Glo. Error bars: standard deviation. Significance by unpaired, two-sided, t-test. ns = not significant. (F–G) Global cellular translation, assayed via polysome profiles, shows little difference between eIF2AKO and control HEK293T-H2-Kb cells. Lysates from either eIF2AKO or control HEK293T-H2-Kb cells were separated on sucrose gradients. One representative graph is shown in (F). The polysome/80 S ratio of three independent replicates is shown in (G). Error bars: standard deviation. Significance by unpaired, two-sided, t-test. ns = not significant.

Figure 4 with 4 supplements
eIF2A has a minor impact on translation during the integrated stress response.

(A) Loss of eIF2A does not blunt induction of target genes of the integrated stress response. Reporters carrying the 5’ UTRs of the indicated candidate genes were co-transfected with a Firefly Luciferase (FLuc) normalization control reporter into eIF2AKO and control HeLa cells and treated for 16 hr either with DMSO or 1 µg/ml tunicamycin (TM). Significance by Dunnett’s multiple comparison test ANOVA. error bar = st. dev., ns = not significant, *p<0.05, ***p<0.001 (B) Ribosome profiling identifies 12 mRNAs that are significantly induced upon tunicamycin treatment (1 ug/ml) in control cells but not eIF2AKO cells. Scatter plot of log2(fold change) of Translation Efficiency TM/DMSO for control cells on the x-axis versus eIF2AKO cells on the y-axis. mRNAs that are statistically significantly induced with log2(fold change)>1 in control cells but not in eIF2AKO cells are shown in yellow and marked by gene name. Significance was estimated with the Wald test performed by DESeq2 thepackage. p-values are adjusted for multiple comparisons. (C) Transfection of luciferase reporters harboring 5’ UTRs of eIF2A-dependent transcripts does not show impaired induction in eIF2AKO cells upon tunicamycin treatment. 5’ UTRs of eIF2A-dependent transcripts from panel B were cloned upstream of Renilla luciferase and co-transfected with a FLuc normalization control reporter into control or EIF2AKO HeLa cells with subsequent treatment for 16 hr either with DMSO or 1 μg/ml TM. Significance by Dunnett’s multiple comparison test ANOVA. error bar = st. dev., ns = not significant, *p<0.05.

Figure 4—figure supplement 1
The integrated stress response suppresses translation equally well in control and eIF2AKO HeLa cells.

(A–B) Polysome profiles of eIF2AKO versus control HeLa cell lines show hardly any differences upon stress caused either with (A) 1 µg/ml tunicamycin (TM) for 16 hr or (B) 100 µM sodium arsenite (SA) for 1 hr. Profiles were aligned by the height of the 80 S peak. (C–D) eIF2A knockout cells form stress granules to the same degree as the parental control HeLa cell line. Control or eIF2AKO HeLa cells were treated for 2 hr with 100 µM sodium arsenite and stained for G3BP1. (C) Representative images. (D) The number of stress granules, normalized to the number of nuclei in each field of view is shown. Significance by ANOVA with Dunnett’s multiple comparison test. ns = not significant. (E) eIF2AKO and control HeLa cells phosphorylate eIF2α to the same degree in response to tunicamycin (1 µg/mL for 16 hr). ATF-4 is used as a marker for downstream activation of the integrated stress response.

Figure 4—figure supplement 2
Ribosome profiling of tunicamycin-treated eIF2AKO and control HeLa cells.

(A) Reproducibility between replicates of Ribosome profiling or total-mRNA libraries from eIF2AKO or control HeLa cells treated with tunicamycin. Two biological replicates were generated for each genotype. The Pearson’s coefficient (r) is shown for each comparison. (B) Ribosome profiling identifies zero transcripts affected by eIF2A knockout in tunicamycin-treated conditions. Scatter plot of log2(fold change) of Translation Efficiency comparing eIF2AKO to control HeLa cells, both treated with tunicamycin, on the x-axis, versus significance on the y-axis. Significant candidates with log2(fold change) < –1 are shown in red. Significance was estimated with the Wald test performed by the DESeq2 package. p-values are adjusted for multiple comparisons. (C–D) Metagene profiles of footprints aligned to either the start codon (C) or the stop codon (D) of all main Open Reading Frames, for control and eIF2AKO HeLa cells, both treated with tunicamycin. ‘Smooth’ curves were generated by averaging read counts with the sliding window of 3 nt. The dotted lines indicate standard deviation between three replicates.

Figure 4—figure supplement 3
Translation of uORF-bearing transcripts is not affected upon loss of eIF2A in tunicamycin-treated cells.

(A–B) Metagene profiles of footprints relative to either the start codon (A) or stop codon (B) of uORFs transcriptome-wide, for control or eIF2AKO HeLa cells, both treated with tunicamycin. ‘Smooth’ curves were generated by averaging footprint counts with a sliding window of 3 nt. The dotted lines indicated the standard deviation between three replicates. (C) eIF2A has little impact genome-wide on translation of mRNAs with uORFs in cells treated with tunicamycin. Comparison of the change in translation efficiency between eIF2AKO and control HeLa cells, both treated with tunicamycin, for three different groups of mRNAs: all transcripts, transcripts with AUG-initiated uORFs, or transcripts with uORFs that start with a near-cognate codon. Significance by ANOVA with Dunnett’s multiple comparison test. ns = not significant. (D) Synthetic reporters harboring uORFs with different start codons and initiation contexts do not show dependence on eIF2A also when the integrated stress response is activated, thereby suppressing eIF2 function. Either control or eIF2AKO HeLa cells were transfected with the indicated reporters and then treated with 1 µg/mL tunicamycin for 16 hr prior to assaying luciferase activity. The sequence context of the uORF start codons is indicated: either AUG or a near-cognate start codon (GTG, TTG, CTG) was used. Significance by Dunnett’s multiple comparison test ANOVA, error bar = st. dev. ns = not significant. *p<0.05, **p<0.01.

Figure 4—figure supplement 4
Expression of initiation factors reported to possess tRNAiMet binding activities.

(A) Heat map showing the log2 fold change of translation efficiency (eIF2AKO/control cells) of different initiation factors reported to possess binding capacity to tRNAiMet. Changes in DMSO and tunicamycin-treated samples are shown.

Author response image 1

(A-B) Average reads occupancy on the eIF4G2 (ENST0000339995) transcript in DMSO treated (panel A, n=3) or tunicamycin treated samples (panel B, n=2) derived from either control (black) or eIF2A-KO (red) HeLa cells. Reads counts were normalized to sequencing depth and averaged between either 3 (DMSO-treated) or 2 (tunicamycin-treated) replicates. Graphs were then smoothened with a sliding window of 3 nt. (C-D) The total number of reads mapping to the eIF4G2 CDS, normalized to library sequencing depth per replica was quantified. No significant difference between control and eIF2A-KO cells was observed in either DMSO treated (panel C) or tunicamycin treated (panel D) cells. Significance by unpaired, two-sided, t-test. ns = not significant.

Author response image 2

(A-B) Average reads occupancy on the PEG10 (ENST00000482108) transcript in DMSO treated (panel A, n=3) or tunicamycin treated samples (panel B, n=2) derived from either control (black) or eIF2A-KO (red) HeLa cells are shown. Reads counts were normalized to sequencing depth and averaged between either 3 (DMSO-treated) or 2 (tunicamycin-treated) replicates. Graphs were then smoothened with a sliding window of 3 nt. (C-D) The ratio of reads mapping to the ORF upstream of the slippery site to reads mapping to the predicted extended protein downstream to the slippery site is shown. Reads counts were normalized to the sequencing depth. Neither DMSO treated samples (panel C) nor tunicamycin treated samples (panel D) had a significant difference between control and eIF2A-KO cells. Significance by unpaired, two-sided, t-test. ns = not significant.

Author response image 3

(A-B) Average read occupancy on the ATF4 (ENST00000674920) transcript in DMSO treated (n=3) or tunicamycin treated samples (n=2) derived from either control (panel A) or eIF2A-KO (panel B) HeLa cells are shown. Read counts were normalized to sequencing depth and averaged between either 3 (DMSO-treated) or 2 (tunicamycin-treated) replicates. Graphs were then smoothened with a sliding window of 3 nt. (C) Scatter plot of log2(fold change) of Translation Efficiency TM/DMSO for control cells on the xaxis versus eIF2AKO cells on the y-axis. The induction of ATF4 as well as the downstream target PPP1R15B are shown. The upregulation of HSP5A translation, the other hallmark of ER-stress induced by tunicamycin treatment is shown.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)eIF2AEnsembleENSG00000144895
Cell line (Homo sapiens)HeLaDSMZACC 57
Cell line (Homo sapiens)HeLa-eIF2A-KOThis studyThis study
Cell line (Homo sapiens)HEK293T-H2-Kbgift from Rienk Offringa labRienk Offringa lab
Cell line (Homo sapiens)HEK293T-H2-Kb-eIF2A-KOThis studyThis study
Transfected construct (human)siRNA to eIF2A (mix of all 4 was used)Horizon discoveryD-014766–01GCUCCCAGGUUACGGGUUA
Transfected construct (human)D-014766–02GAUUUGGAAUUGGGUAUUU
Transfected construct (human)D-014766–03GCAGAUAAAGUUACAAUGC
Transfected construct (human)D-014766–04CCACAAUCAGGAAACGAUA
Sequence-based reagent (oligos used to produce KO)Pair 8 sg_eIF2A 1This studyOMR064CACCGCTCACCCAAAAATACTGTCC
Sequence-based reagent (oligos used to produce KO)Pair 8 sg_eIF2A 2This studyOMR065AAACGGACAGTATTTTTGGGTGAGC
Sequence-based reagent (oligos used to produce KO)Pair 5 sg_eIF2A 1This studyOMR058CACCGAATACTAATATATGTCCATG
Sequence-based reagent (oligos used to produce KO)Pair 5 sg_eIF2A 2This studyOMR059AAACCATGGACATATATTAGTATTC
Antibodyanti-ATF-4 (D4B8) (Rabbit monoclonal)Cell Signalingcat. No #11815 Lot#6 WB (1:1000)
Antibodyanti-C-Myc (Rabbit monoclonal)Cell Signalingcat. No. #13987 Lot#6 WB (1:1000)
Antibodyanti-CCND3 (Rabbit polyclonal)invitrogencat no. #PA5-80416 Lot#UH2828593 WB (1:1000)
Antibodyanti-eIF2A (3A7A8) (mouse monoclonal)santa cruzcat. No. sc-517214 Lot#B0821 WB (1:1000)
Antibodyanti-FLAG (Rabbit polyclonal)SIGMAcat. No. F7425-.2MG Lot#0000252651 WB (1:1000)
Antibodyanti-GAPDH (Rabbit monoclonal)Cell Signalingcat. No. #2118 LOT#16 WB (1:1000)
Antibodyanti-HSP90 (Rabbit monoclonal)Cell signalingcat. No. 4877 Lot#6 WB (1:1000)
Antibodyanti-Lamin A/C (636) (mouse monoclonal)Santa Cruzcat. No. sc-7292 Lot#C0218 WB (1:1000)
Antibodyanti-NCAPH2 (Rabbit polyclonal)Proteintechcat. No. 26172–1-AP Lot#00039440 WB (1:1000)
Antibodyanti-p-p38 (Rabbit polyclonal)Cell Signalingcat. No. #9211 Lot#25 WB (1:1000)
Antibodyanti-PPFIA1 (Rabbit polyclonal)Proteintechcat. No. 14175–1-AP Lot#00005224 WB (1:1000)
Antibodyanti-puromycin (mouse monoclonal)Sigmacat. No. MABE343 Lot#3484967 WB (1:1000)
Antibodyanti-RPS6KB2 (Rabbit polyclonal)Proteintechcat. No. 26194–1-AP Lot#00040692 WB (1:1000)
Antibodyanti-TubulinSigmacat. No. T9026 LOT#0000307925 WB (1:2500)
Antibodyanti-G3BP1 (mouse monoclonal)santa cruzcat. No. sc-81940 Lot@G0617IF: 1:50
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)ACTN4This studypMR11845' UTR cloned from transcript
with id ENST00000252699
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)ARHGAP11AThis studypMR1815' UTR cloned from transcript
with id ENST00000361627
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)ATG9AThis studypMR0915' UTR cloned from transcript
with id ENST00000361242
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)CBX4This studypMR11715' UTR cloned from transcript
with id ENST00000269397
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)CCDC125This studypMR11745' UTR cloned from transcript
with id ENST00000383374
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)CCND3This studypMR9655' UTR cloned from transcript
with id ENST00000372991
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)CLPTM1LThis studypMR11785' UTR cloned from transcript
with id ENST00000337392
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)CNOT1This studypMR11825' UTR cloned from transcript
with id ENST00000317147
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)IQGAP1This studypMR1685' UTR cloned from transcript
with id ENST00000268182
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)MXRA7This studypMR11735' UTR cloned from transcript
with id ENST00000355797
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)NCAPH2This studypMR11705' UTR cloned from transcript
with id ENST00000420993
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)OSBP2This studypMR6645' UTR cloned from transcript
with id ENST00000332585
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)PIGGThis studypMR0965' UTR cloned from transcript
with id ENST00000310340
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)PNPLA8This studypMR11835' UTR cloned from transcript
with id ENST00000257694
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)POLR3EThis studypMR11815' UTR cloned from transcript
with id ENST00000640588
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)PTP4A1This studypMR1965' UTR cloned from transcript
with id ENST00000626021
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)QTRT2This studypMR11805' UTR cloned from transcript
with id ENST00000281273
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)RPS6KB2This studypMR11765' UTR cloned from transcript
with id ENST00000312629
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)SCRN2This studypMR11755' UTR cloned from transcript
with id ENST00000290216
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)SLC38A1This studypMR11855' UTR cloned from transcript
with id ENST00000546893
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)SLC7A11This studypMR11795' UTR cloned from transcript
with id ENST00000280612
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)SP140LThis studypMR11775' UTR cloned from transcript
with id ENST00000415673
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)TEAD2This studypMR11725' UTR cloned from transcript
with id ENST00000311227
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)TEAD3This studypMR11695' UTR cloned from transcript
with id ENST00000338863
Sequence-based reagent (plasmids with 5' UTR of interest cloned upstream of renilla luciferase)TRAF7This studypMR9675' UTR cloned from transcript
with id ENST00000326181
Sequence-based reagent (qRT-PCR oligos)eIF2A qRT-PCRThis studyOMR202AAAGCACAGTGTTTCCAAGGG
Sequence-based reagent (qRT-PCR oligos)eIF2A qRT-PCRThis studyOMR203GCAGTAGTCCCTTGTTAGTGA
Sequence-based reagent (qRT-PCR oligos)CCND3 qRT-PCR oligoThis studyOMR412GAAGGGGCGTCTGTTCC
Sequence-based reagent (qRT-PCR oligos)CCND3 qRT-PCR oligoThis studyOMR413CAGGGAGGAGGAGCTTG
Sequence-based reagent (qRT-PCR oligos)NCAPH2 qRT-PCR oligoThis studyOMR2542CGAGTATCTGGAGGAGCTGGATCA
Sequence-based reagent (qRT-PCR oligos)NCAPH2 qRT-PCR oligoThis studyOMR2543GCCTGGTAGACGAGTGAGTAGAGG
Sequence-based reagent (qRT-PCR oligos)PPFIA1 qRT-PCR oligoThis studyOMR2546AGCAGAAAGGAATAACACCAGGCT
Sequence-based reagent (qRT-PCR oligos)PPFIA1 qRT-PCR oligoThis studyOMR2547CATCCAGAGCTTTGTGGTGTTCAA
Sequence-based reagent (qRT-PCR oligos)RPS6KB2 qRT-PCR oligoThis studyOMR2550TGGATTTGGAGACGGAGGAAGGCA
Sequence-based reagent (qRT-PCR oligos)RPS6KB2 qRT-PCR oligoThis studyOMR2551GATGCGCTCTGGGCCAACGTTCAC
Sequence-based reagent (qRT-PCR oligos)RPL13A qRT-PCR oligoThis studyOMR068CCGCCCTACGACAAGAAA
Sequence-based reagent (qRT-PCR oligos)RPL13A qRT-PCR oligoThis studyOMR069CAGGGTGGCTGTCACTGC
Sequence-based reagent (qRT-PCR oligos)GAPDH q-RT-PCRThis studyOMR496CCTTTGACGCTGGGGCT
Sequence-based reagent (qRT-PCR oligos)GAPDH q-RT-PCRThis studyOMR497GGTGGTCCAGGGGTCTT
Sequence-based reagent (oligos used to clone 5'UTR of interest)OSBP2This studyOMR1589ccggaagcttACTGGCCGCTCGGCCGCGCGCGGGTCGGCCGGCTCTccaccATGacTTCGAAccgg
Sequence-based reagent (oligos used to clone 5'UTR of interest)OSBP2This studyOMR1590ccggTTCGAAgtCATggtggAGAGCCGGCCGACCCGCGCGCGGCCGAGCGGCCAGTaagcttccgg
Sequence-based reagent (oligos used to clone 5'UTR of interest)ctg uORFThis studyOMR1877ccggaCATACctgTatTCGATAATCAACTTTGAAAAACTCtaaa
Sequence-based reagent (oligos used to clone 5'UTR of interest)ctg uORFThis studyOMR1878CCGGtttaGAGTTTTTCAAAGTTGATTATCGAatAcagGTATGt
Sequence-based reagent (oligos used to clone 5'UTR of interest)gtg uORFThis studyOMR1879ccggaTGGAgtgAaaTCGATAATCAACTTTGAAAAACTCtaaa
Sequence-based reagent (oligos used to clone 5'UTR of interest)gtg uORFThis studyOMR1880CCGGtttaGAGTTTTTCAAAGTTGATTATCGAatAcacGTATGt
Sequence-based reagent (oligos used to clone 5'UTR of interest)ttg uORFThis studyOMR1881ccggaTGGAttgAaaTCGATAATCAACTTTGAAAAACTCtaaa
Sequence-based reagent (oligos used to clone 5'UTR of interest)ttg uORFThis studyOMR1882CCGGtttaGAGTTTTTCAAAGTTGATTATCGAatAcaaGTATGt
Sequence-based reagent (oligos used to clone 5'UTR of interest)TRAF7This studyOMR2182ccggaagcttGGCAGCCGTCCGGGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)TRAF7This studyOMR2183ccggTTCGAAgtCATggtggGCTCTAGAGAGGCATCTACGGTCCTT
Sequence-based reagent (oligos used to clone 5'UTR of interest)TEAD2This studyOMR2504AGCttCCCACTTTTCCCAAACAAAGCTCCCGGCAACTTTCTCCCTCGCAGCGCCCCGCCCGCCCGCGGCTCCCCAGCCCCAGGCCGGGAGGCCCAGcCATGACTT
Sequence-based reagent (oligos used to clone 5'UTR of interest)TEAD2This studyOMR2505CGAAGTCATGgCTGGGCCTCCCGGCCTGGGGCTGGGGAGCCGCGGGCGGGCGGGGCGCTGCGAGGGAGAAAGTTGCCGGGAGCTTTGTTTGGGAAAAGTGGGa
Sequence-based reagent (oligos used to clone 5'UTR of interest)MXRA7This studyOMR2506agcttACTCGGCGGCC
GCGGCGCGccatgactt
Sequence-based reagent (oligos used to clone 5'UTR of interest)MXRA7This studyOMR2507cgaagtcatggCGCGCCGCGGCCGCCGAGTa
Sequence-based reagent (oligos used to clone 5'UTR of interest)CCDC125This studyOMR2508agcttGCGGCGGCAGCGGCGCACGCGCACGGAGAGGAGGCTACTTGCCAGACAGCCCATTTTTTCTTATGATAAAGACGGCATTTGGCTCccatgactt
Sequence-based reagent (oligos used to clone 5'UTR of interest)CCDC125This studyOMR2509cgaagtcatggGAGCCAAATGCCGTCTTTATCATAAGAAAAAATGGGCTGTCTGGCAAGTAGCCTCCTCTCCGTGCGCGTGCGCCGCTGCCGCCGCa
Sequence-based reagent (oligos used to clone 5'UTR of interest)SCRN2This studyOMR2510agcttGCGGCCCTGGCCAGAAGCGGAGGAGGTGGCACCCGGGACCGAGCTGGGGTCTTGGAGGAAGAGAGGccatgactt
Sequence-based reagent (oligos used to clone 5'UTR of interest)SCRN2This studyOMR2511cgaagtcatggCCTCTCTTCCTCCAAGACCCCAGCTCGGTCC
CGGGTGCCACCTCCTCCGCTTCTGGCCAGGGCCGCa
Sequence-based reagent (oligos used to clone 5'UTR of interest)RPS6KB2This studyOMR2512agcttAGTCAGTGCGCGGCCAGGTACGGGCCGACGGGCCCGCGGGGCCGGCGCCGCCccatgactt
Sequence-based reagent (oligos used to clone 5'UTR of interest)RPS6KB2This studyOMR2513cgaagtcatggGGCGGCGCCGGCCCCGCGGGCCCGTCGGCCCGTACCTGGCCGCGCACTGACTa
Sequence-based reagent (oligos used to clone 5'UTR of interest)SP140LThis studyOMR2514agcttACACTGCACGCAGGCTGGGCCGACTGGGGAGCTCATAGGCCAGGCTCTGACACCCAGGCAGGGCCTAGGGTGGGACGccatgactt
Sequence-based reagent (oligos used to clone 5'UTR of interest)SP140LThis studyOMR2515cgaagtcatggCGTCCCACCCTAGGCCCTGCCTGGGTGTCAGAGCCTGGCCTATGAGCTCCCCAGTCGGCCCAGCCTGCGTGCAGTGTa
Sequence-based reagent (oligos used to clone 5'UTR of interest)CLPTM1LThis studyOMR2516agcttGACCCGGAGCGGGAAGccatgactt
Sequence-based reagent (oligos used to clone 5'UTR of interest)CLPTM1LThis studyOMR2517cgaagtcatggCTTCCCGCTCCGGGTCa
Sequence-based reagent (oligos used to clone 5'UTR of interest)NCAPH2This studyOMR2518TAGTGAACCGTCAGATCACTAGAAGCTTGCATTTTCCTGGGCGGGAACAGCAAAATGGCGCCAGAACTAGTGGCGGGCTGAGGACGCCGTACCCCTCGGA
Sequence-based reagent (oligos used to clone 5'UTR of interest)NCAPH2This studyOMR2519CTTTCGAAGTCATGGGTCCGGGAGGGAACGGGCGGCAAAGGGACCGCAGGGCTGCCTTCCGAGGGGTACGGCGTCCTCAGCCCGCCACTAGTTCTGGCGCC
Sequence-based reagent (oligos used to clone 5'UTR of interest)CBX4This studyOMR2520AGAAGCTTAGTTGTCTGAGCGAGCGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)CBX4This studyOMR2521ACTTTCGAAGTCATGGGGCCGAGCCGGAGCG
Sequence-based reagent (oligos used to clone 5'UTR of interest)TEAD3This studyOMR2522AGAAGCTTAACACAAACTTTCCGTCCCGCTC
Sequence-based reagent (oligos used to clone 5'UTR of interest)TEAD3This studyOMR2523ACTTTCGAAGTCATGGTGTGCTGGTTGCTCTGGGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)SLC7A11This studyOMR2526TAGAAGCttGGTTTGTAATGATAGGGCGGCAG
Sequence-based reagent (oligos used to clone 5'UTR of interest)SLC7A11This studyOMR2527ccggTTCGAAgtCATggtggAGTAGGGACACACGGGGG
Sequence-based reagent (oligos used to clone 5'UTR of interest)QTRT2This studyOMR2528TAGAAGCttAGTACTCCCTGATTGGCTCTGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)QTRT2This studyOMR2529ccggTTCGAAgtCATggtggCCTAAGGGATTCTTCTAGGTCCTTTCAGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)POLR3EThis studyOMR2530TAGAAGCttACGTGTCCGCCGGAGTT
Sequence-based reagent (oligos used to clone 5'UTR of interest)POLR3EThis studyOMR2531ccggTTCGAAgtCATggtggACTAGAGGAGAGCCAGCCG
Sequence-based reagent (oligos used to clone 5'UTR of interest)CNOT1This studyOMR2532TAGAAGCttGTAGAGAAACAAGCGGAGTTAACCGA
Sequence-based reagent (oligos used to clone 5'UTR of interest)CNOT1This studyOMR2533ccggTTCGAAgtCATggtggTGCTGGTTGGGGCGGAA
Sequence-based reagent (oligos used to clone 5'UTR of interest)PNPLA8This studyOMR2536TAGAAGCttAGTGTTTGTGTTGGAAGCTCAGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)PNPLA8This studyOMR2537ccggTTCGAAgtCATggtggAACTTAAAAATCATTTATTTTCTATGACATTCTCTCACTTCTTGA
Sequence-based reagent (oligos used to clone 5'UTR of interest)ACTN4This studyOMR2538TAGAAGCttGAAGCAGCTGAAGCGGCG
Sequence-based reagent (oligos used to clone 5'UTR of interest)ACTN4This studyOMR2539ccggTTCGAAgtCATggtggTCCGCCGCCTCTCGC
Sequence-based reagent (oligos used to clone 5'UTR of interest)SLC38A1This studyOMR2540CTAgatatccaACTGACACGCAGCTTTGGTTAAA
Sequence-based reagent (oligos used to clone 5'UTR of interest)SLC38A1This studyOMR2541ccggTTCGAAgtCATggtggGATTAGAAAGTGTCTGTAGTTTGAAAATTAGTCCA
Sequence-based reagent (oligos used to clone 5'UTR of interest)short 5' UTRThis studyOMR2609CGTTTAGTGAACCGTCAGATCACCACCATGACTT
Sequence-based reagent (oligos used to clone 5'UTR of interest)short 5' UTRThis studyOMR2610CGAAGTCATGGTGGTGATCTGACGGTTCACTAAACGAGCT
Sequence-based reagent (oligos used to clone 5'UTR of interest)PIGGThis studyOMR277ccggaagcttGACGATAAGGCCTGGCG
Sequence-based reagent (oligos used to clone 5'UTR of interest)PIGGThis studyOMR278ccggTTCGAAgtCATggtggCGTGGACACGCTAGGCT
Sequence-based reagent (oligos used to clone 5'UTR of interest)ATG9AThis studyOMR293ccggaagcttGAGTGGCAGACACCCG
Sequence-based reagent (oligos used to clone 5'UTR of interest)ATG9AThis studyOMR294ccggTTCGAAgtCATggtggCACCACCGCCCCCTG
Sequence-based reagent (oligos used to clone 5'UTR of interest)IQGAP1This studyOMR472ccggaagcttGACCCCGGCAAGCC
Sequence-based reagent (oligos used to clone 5'UTR of interest)IQGAP1This studyOMR473ccggTTCGAAgtCATggtggGGCGGACGAGCCC
Sequence-based reagent (oligos used to clone 5'UTR of interest)PTP4A1This studyOMR504ccggaagcttGAGATTACTGCCAGGCACA
Sequence-based reagent (oligos used to clone 5'UTR of interest)PTP4A1This studyOMR505ccggTTCGAAgtCATggtggGTTAATTTAGTTAAAAAACACTCAATAGGGTTATGAA
Sequence-based reagent (oligos used to clone 5'UTR of interest)IFRD1This studyOMR508ccggaagcttGTTAAAACCAGACTGCACTCC
Sequence-based reagent (oligos used to clone 5'UTR of interest)IFRD1This studyOMR509ccggTTCGAAgtCATggtggCGTGGGACGCCCGG
Sequence-based reagent (oligos used to clone 5'UTR of interest)CCND3This studyOMR526ccggaagcttACCTATGCCGCGTGGG
Sequence-based reagent (oligos used to clone 5'UTR of interest)CCND3This studyOMR527CGAAGCGGCcgcATTTCACAATCATCTTTATTACAGTAGG
Sequence-based reagent (oligos used to clone 5'UTR of interest)PPP1R15BThis studyOMR549ccggaagcttATTTTGGGCTTCGCTTCC
Sequence-based reagent (oligos used to clone 5'UTR of interest)PPP1R15BThis studyOMR550ccggTTCGAAgtCATggtggACGGGATTCGGAGG
Sequence-based reagent (eIF2A Construct used for an overexpression)N-terminally Flag-tagged eIF2A in pCDNA3This studypMR007
Commercial assay or kitDual-Luciferase assay systemPromegaE1910
Commercial assay or kitCell Titer GloPromegaG7572
Commercial assay or kitNext-Seq 550 systemIllumina20024906
Commercial assay or kitNext-Flex small RNA v.4 kit protocolPerkin ElmerNOVA-5132–06
commercial assay or kitIllumina TruSeq Stranded library preparation kitIllumina20020594
Software, algorithmalgorithm to analyze ribosome profiling datalab developed software Teleman, 2025https://github.com/aurelioteleman/Teleman-Lab
Chemical compound, drugsodium arseniteSigmaS7400-100G
Chemical compound, drugO-Propargyl-puromycinEnzo Life SciencesJBS-NU-931–05
Chemical compound, drugtunicamycinSigma654380–10 MG

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  1. Mykola Roiuk
  2. Marilena Neff
  3. Aurelio A Teleman
(2025)
Human eIF2A has a minimal role in translation initiation and in uORF-mediated translational control in HeLa cells
eLife 14:RP105311.
https://doi.org/10.7554/eLife.105311.3