Profiling of terminating ribosomes reveals translational control at stop codons
- Division of Nutritional Sciences, Cornell University, United States
- Laboratory of Molecular Modelling and Bioinformatics (LAMMB), Department of Physical and Biological Sciences, Campus Sete Lagoas, Universidade Federal de São João Del Rei, Brazil
Figures
Figure 1 with 1 supplement
Characterizing terminating ribosomes using Ribo-seq.
(A) Aggregation plots of mean Ribo-seq reads around the start (left) and stop (right) codons. Both 5′ end (green) and 3′ end (orange) of footprint reads (29 nt) were used for mapping. For the bottom panels, the line plots show the distribution of footprints with different lengths, whereas the bar plots show the size distribution of initiating (left) and terminating (right) ribosome footprints relative to elongation ribosomes. (B) Aggregation plots of mean Ribo-seq reads around the stop codon. The reads were stratified by the length, followed by mapping using the 5′ end of footprint reads. (C) A histogram plot of ribosome pausing index at individual stop codons. The top shows the enriched sequence motif of mRNAs with strong ribosome pausing at stop codons. (D) Aggregation plots of mean Ribo-seq reads around the stop codon. ‘High’ and ‘Low’ refer to mRNAs with differential pausing indexes shown in (C). The 3′ UTR read density was highlighted in the insert.
Figure 1—figure supplement 1
Characterizing terminating ribosomes.
(A) HEK293 cells were treated with cycloheximide (CHX, 100 μg/mL) for varied times followed by Ribo-seq. Aggregation plots show the distribution of mean reads across the transcriptome aligned at start and stop codons. (B) A representative mRNA (RPS2) with reads obtained from ribo-seq in cells as (A). The 5’ end of reads were used for mapping. (C) The ratio of ribosome density in 3’ UTR over the density in coding sequence (CDS). The mRNAs were stratified into different groups based on the identify of stop codons and the nucleotide immediately after the stop codon. The mRNAs were stratified into different groups based on the identify of stop codons and the nucleotide immediately after the stop codon. (D) Relative fractions of Ribo-seq reads mapped to different reading frames. Reads in CDS and 3’ UTR were shown.
Figure 2 with 1 supplement
Characterizing terminating ribosomes using eRF1-seq.
(A) The left panel shows the schematic of eRF1-seq procedures. The right top panel shows the aggregation plots of Ribo-seq (green) and eRF1-seq (orange). The right bottom panel shows a representative mRNA (RPS2) with reads obtained from Ribo-seq and eRF1-seq. The 5′ end of reads was used for mapping. (B) Heatmaps of individual mRNAs with reads obtained from Ribo-seq (right) and eRF1-seq (left). (C) Aggregation plots of mean eRF1-seq reads around the stop codon. The reads were stratified by the length, followed by mapping using the 5′ end of footprint reads. (D) Comparison of codon frequencies upstream of stop codons between mRNAs with and without eRF1 peaks. The right panel shows the enriched sequence motif for mRNAs with eRF1 peaks at stop codons.
Figure 2—figure supplement 1
Developing eRF1-seq to capture terminating ribosomes.
(A) HEK293 cells with or without crosslinking by formaldehyde were subjected to eRF1 immunoprecipitation followed by western blotting using antibodies indicated. Representative results of three independent experiments are shown. (B) A scatter plot shows the correlation of read counts between two biological replicates of eRF1-seq (Rho = 0.89). (C) Comparison of 3’ UTR length (left) and folding free energy (right) between mRNAs with and without eRF1 peaks at the stop codon. (D) Aggregation plots of ribosome density using Ribo-seq and eRF1-seq data sets on mRNAs with different stop codons. (E) The enriched sequence motif preceding different stop codons on mRNAs with eRF1 peaks.
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Figure 2—figure supplement 1—source data 1
PDF file containing original western blots for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Uncropped western blots shown in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
eRF1-seq reveals prevailing termination sites.
(A) A pie chart shows fractions of eRF1 peaks mapped to different mRNA regions. ‘Inframe’ and ‘Outframe’ refer to the positions of eRF1 peaks within coding sequence (CDS) relative to the annotated start codons. (B) The left panel shows mean eRF1-seq reads around the position of eRF1 peaks within 5′ UTR. The dot plot shows the frequency of A-site codons at eRF1 peaks. The right panel shows mean Ribo-seq reads around the position of eRF1 peaks within 5′ UTR. The bar graph shows the mean ribosome densities before (-) and after (+) the eRF1 peaks. (C) The left panel shows mean eRF1-seq reads around the position of out-of-frame eRF1 peaks within CDS. The dot plot shows the frequency of A-site codons at eRF1 peaks. The right panel shows mean Ribo-seq reads around the position of out-of-frame eRF1 peaks within CDS. The bar graph shows the mean ribosome densities before (-) and after (+) the eRF1 peaks. (D) The distribution of eRF1 peaks before the annotated stop codon. Only the out-of-frame eRF1 peaks were used for plotting. (E) A bar graph shows the HiBiT-based reporter assays in HEK293-Kb cells. HiBiT signals were measured from cells transfected with mRNA reporters bearing different sequences between GFP and HiBiT. Error bars, mean ± SEM n=3 biological replicates. ****p≤0.0001 by unpaired two-tailed t-test.
Figure 3—figure supplement 1
eRF1-seq reveals prevailing termination sites in 3‘ UTR and coding sequence (CDS).
(A) A Venn diagram shows the number of uORFs identified by Ribo-seq and eRF1-seq. (B) The left panel shows mean eRF1-seq reads around the position of eRF1 peaks within 3’ UTR. The dot plot shows the frequency of A-site codons at eRF1 peaks. The right panel shows mean Ribo-seq reads around the position of eRF1 peaks within 3’ UTR. The bar graph shows the mean ribosome densities before (-) and after (+) the eRF1 peaks. (C) The left panel shows mean eRF1-seq reads around the position of in-frame eRF1 peaks within CDS. The dot plot shows the frequency of A-site codons at eRF1 peaks. The right panel shows mean Ribo-seq reads around the position of in-frame eRF1 peaks within CDS. The bar graph shows the mean ribosome densities before (-) and after (+) the eRF1 peaks.
Figure 4 with 3 supplements
Termination pausing is influenced by sequence contexts.
(A) The upper panel shows the schematic of a massively parallel reporter assay, where the stop codon of upstream open reading frame (uORF) was replaced with a random 9-nt sequence. The uORF translation was monitored by the number of associated ribosomes separated by a sucrose gradient (M, monosome; P, polysome). The heatmap shows the ratio of monosome fraction over polysome fraction (M/P) when different codons were placed at individual positions within the 9-nt random sequence (left). The bar graph (right) shows the mean M/P ratio averaged across all positions. The stop codons UGA, UAG, and UAA are highlighted in red. (B) Similar to (A) except that the random 9-nt sequences were placed before the stop codon UAG. (C) Bar graphs show the HiBiT signals in HEK293-Kb cells transfected with mRNA reporters bearing C-rich or GA-rich sequences between the uORF and HiBiT-GFP. The upper panel shows the control without the stop codon UAG. Error bars, mean ± SEM n=3 biological replicates. *p≤0.05; **p≤0.01 by unpaired two-tailed t-test. (D) A bar graph shows the HiBiT signals in HEK293-Kb cells transfected with mRNA reporters bearing C-rich or GA-rich sequences before the GFP stop codon UAG. The downstream HiBiT sequence was inserted into different reading frames. Error bars, mean ± SEM n=3 biological replicates. **p≤0.01; ***p≤0.001 by unpaired two-tailed t-test. (E) Representative western blots of GFP and HiBiT in HEK293-Kb cells transfected with C-rich or GA-rich reporters as described in (D).
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Figure 4—source data 1
PDF file containing original western blots for Figure 4E.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig4-data1-v1.zip
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Figure 4—source data 2
Uncropped original western blots shown in Figure 4E.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig4-data2-v1.zip
Figure 4—figure supplement 1
MPRA assays reveal the sequence context of stop codons.
(A) The up panel shows the schematic of a massively parallel reporter assay, where the stop codon of uORF was replaced with a random 9-nt sequences. The uORF translation was monitored by the number of associated ribosomes separated by sucrose gradient (M, monosome; P, polysome). The heatmap shows the codon frequency within the 9-nt random sequence in monosome (M) and polysome (P) fractions. (B) Bar graphs show the top 10 codons enriched in monosome (M) over polysome (P) at each reading frame. (C) The up panel shows the schematic of a massively parallel reporter assay, where a random 9-nt sequence was replaced after the uORF stop codon UAG. The uORF translation was monitored by the number of associated ribosomes separated by sucrose gradient (M, monosome; P, polysome). The heatmap shows the codon frequency within the 9-nt random sequence in monosome (M) and polysome (P) fractions. (D) A bar graph (right) shows the mean M/P ratio averaged across all positions in (C). (E) The up panel shows the schematic of a massively parallel reporter assay, where a random 9-nt sequence was replaced before the uORF stop codon UAG. The uORF translation was monitored by the number of associated ribosomes separated by sucrose gradient (M, monosome; P, polysome). The heatmap shows the codon frequency within the 9-nt random sequence in monosome (M) and polysome (P) fractions.
Figure 4—figure supplement 2
Dissecting the sequence context of stop codons using reporter assays.
(A) A bar graph shows the HiBiT signals in HEK293-Kb cells transfected with mRNA reporters bearing C-rich or GA-rich sequence before the GFP stop codon UAG. Error bars, mean ± SEM. n=3 biological replicates. **p< 0.01 by unpaired two-tailed t-test. (B) A bar graph shows the HiBiT signals in HEK293-Kb cells transfected with mRNA reporters bearing C-rich or GA-rich sequence before the GFP stop codon UAG. The downstream HiBiT sequence was inserted into different reading frames. Error bars, mean ± SEM. n=3 biological replicates. **p≤ 0.01 by unpaired two-tailed t-test. (C) A bar graph shows the HiBiT signals in HEK293-Kb cells transfected with mRNA reporters bearing C-rich or GA-rich sequence before the GFP stop codon UAG. Error bars, mean ± SEM. n=3 biological replicates. *p< 0.05; **p≤ 0.01; ***p< 0.001 by unpaired two-tailed t-test for two groups and ordinary one-way ANOVA test followed by Dunnett's multiple comparisons for more than two groups.
Figure 4—figure supplement 3
ABCE1 modulates termination pausing independent of the mRNA sequence context.
(A) Western blots of HEK293-Kb cells with or without ABCE1 knockdown. (B) Comparison of polysome profiles of cells with or without ABCE1 knockdown. (C) Aggregation plots show ribosome density around stop codons in cells with or without ABCE1 knockdown. (D) Aggregation plots of mean Ribo-seq reads around the stop codon in cells with or without ABCE1 knockdown. The reads were stratified by the length followed by mapping using the 5’ end of footprint reads. (E) A bar graph shows the HiBiT signals in HEK293-Kb cells with or without ABCE1 knockdown after transfection with mRNA reporters bearing C-rich or GA-rich sequence before the GFP stop codon UAG. Error bars, mean ± SEM. n=3 biological replicates. **p< 0.01 by unpaired two-tailed t-test.
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Figure 4—figure supplement 3—source data 1
PDF file containing the original western blots shown in Figure 4—figure supplement 3A.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Uncropped original western blots shown in Figure 4—figure supplement 3A.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig4-figsupp3-data2-v1.zip
Figure 5 with 1 supplement
The 3′ end of 18 S rRNA influences the dynamics of terminating ribosome.
(A) A cryoEM structure of mammalian initiating ribosomes (PDB: 6ZMW) showing the proximity of the 3′ terminus of 18 S rRNA and mRNA. (B) Superposition of different conformations sampled by normal mode analysis (NMA) of the region mentioned in (A). The analysis was carried out with the mRNA segment from the –10 nt to the +5 nt related to the A-site. The superposition is outlined only for the Rps26 protein (green surface), the mRNA segment (blue surface), and the last 10 nucleotides at the 3′ rRNA extremity (dark red surface), with the remaining proteins and rRNA segments at the site described only by the average structure (silver transparent surface and cartoon). The tRNA anticodon loop at the P ribosomal site is shown in dark cyan. (C) HEK293 cells were transfected with plasmids encoding 18 S rRNA WT or mutant followed by Ribo-seq. Aggregation plots show the mean reads around stop codons of mRNAs with the GA sequence motif or C-rich sequence element. (D) HEK293 cells were transfected with plasmids encoding 18 S rRNA WT or mutant followed by transfection with mRNA reporters shown in (4D). Bar graphs show the HiBiT signals in transfected cells with HiBiT at different reading frames. Error bars, mean ± SEM n=3 biological replicates. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001 by unpaired two-tailed t-test.
Figure 5—figure supplement 1
The putative role of 3’ terminus of 18S rRNA in termination pausing.
(A) A schematic shows the secondary structure of 18S rRNA with the 3’ end sequence highlighted. (B) Comparison of polysome profiles of cells expressing the 18S rRNA WT or mutant. (C) HEK293 cells were transfected with plasmids encoding 18S rRNA WT or mutant followed by Ribo-seq. Aggregation plots show the distribution of mean reads across the transcriptome aligned at start and stop codons. (D) Sequence alignment of the 3’ end of 18S rRNA across multiple species. (E) Relative frequency of stop codons preceded with GA-rich or C-rich seqeunces in human genome. Note the different fractions between annotated and out-of-frame stop codons.
Figure 6 with 1 supplement
Differential termination pausing in mouse tissues.
(A) Different mouse tissues were collected followed by Ribo-seq. Metagene analysis shows the distribution of mean ribosome reads across the transcriptome aligned to start and stop codons. (B) Representative western blots of different mouse tissues using the indicated antibodies. The experiment was independently repeated three times with similar results. (C) Mouse liver and testis were subjected to polysome profiling using a sucrose gradient. Representative western blots of ribosome fractions were conducted using the indicated antibodies. The experiment was independently repeated three times with similar results.
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Figure 6—source data 1
PDF file containing the original western blots shown in Figure 6B and C.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig6-data1-v1.zip
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Figure 6—source data 2
Uncropped original western blots shown in Figure 6B and C.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig6-data2-v1.zip
Figure 6—figure supplement 1
Tissue specificity of termination pausing.
(A) Different mouse tissues were collected followed by polysome profiling on sucrose gradients. (B) Mouse liver and testis were subjected to Ribo-seq. Aggregation plots show the distribution of mean reads across the common or tissue-specific transcripts aligned at start and stop codons. (C) Mouse liver and testis were subjected to Ribo-seq. Aggregation plots show the mean reads around stop codons of mRNAs with the GA sequence motif.
Figure 7 with 1 supplement
Rps26 modulates termination pausing.
(A) A cryo-EM structure of the human 48 S pre-initiation complex (PDB: 6YAL) depicted in cartoon and colored in yellow. The region at the 5′ exit of the mRNA tunnel centered in Rps26 and extended around 30 Å of this protein is highlighted in transparent surface and in dark colors. (B) Conformational sampling by normal mode analysis (NMA) of the region mentioned in (A). The analysis was carried out with the mRNA segment from the –10 nt related to the P-site to the +5 nt related to the A-site. The superposition is outlined only for the Rps26 protein (green surface), the mRNA segment (blue cartoon), and the last 10 nucleotides at the 3′ rRNA extremity (dark red cartoon), with the remaining proteins and rRNA segments at the site described only by the average structure (silver transparent surface and cartoon). The tRNA anticodon loop at the P ribosomal site is omitted on the image. (C) HEK293 cells with or without Rps26 knockdown were subjected to Ribo-seq. Aggregation plots show the mean reads around stop codons of mRNAs with the GA sequence motif or C-rich sequence element. (D) HEK293 cells with or without Rps26 knockdown were transfected with mRNA reporters shown in 4D. Bar graph shows the HiBiT signals at different reading frames upon normalization to upstream GFP levels. Error bars, mean ± SEM n=3 biological replicates. ns, nonsignificant; **p≤0.01; ***p≤0.001 by unpaired two-tailed t-test. (E) HEK293 cells with or without Rps26 overexpression were subjected to Ribo-seq. Aggregation plots show the mean reads around stop codons of mRNAs with the GA sequence motif or C-rich sequence element. (F) HEK293 cells with or without Rps26 overexpression were transfected with mRNA reporters shown in 4(D). Bar graph shows the HiBiT signals at different reading frames. Error bars, mean ± SEM n=3 biological replicates. *p≤0.05; **p≤0.01 by unpaired two-tailed t-test.
Figure 7—figure supplement 1
The role of Rps26 in termination pausing.
(A) Western blots of HEK293 cells with or without Rps26 knockdown. (B) Comparison of polysome profiles of cells with or without Rps26 knockdown. (C) HEK293 cells with or without Rps26 knockdown were subjected to Ribo-seq. Aggregation plots show the distribution of mean reads across the transcriptome aligned at start and stop codons. (D) Western blots of HEK293 cells with or without Rps26 overexpression. (E) Comparison of polysome profiles of cells with or without Rps26 overexpression. (F) HEK293 cells with or without Rps26 overexpression were subjected to Ribo-seq. Aggregation plots show the distribution of mean reads across the transcriptome aligned at start and stop codons.
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Figure 7—figure supplement 1—source data 1
PDF file containing the original western blots shown in Figure 7—figure supplement 1A.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
Uncropped original western blots shown in Figure 7—figure supplement 1A.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig7-figsupp1-data2-v1.zip
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Figure 7—figure supplement 1—source data 3
PDF file contaiing original western blots shown in Figure 7—figure supplement 1D.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig7-figsupp1-data3-v1.zip
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Figure 7—figure supplement 1—source data 4
Uncropped original western blots shown in Figure 7—figure supplement 1D.
- https://cdn.elifesciences.org/articles/109257/elife-109257-fig7-figsupp1-data4-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Rps26 | GenBank | pcDNA3.1 (myc-His B)-Rps26 | |
| Gene (H. sapiens) | 18 S rRNA | Burman and Mauro, 2012 | pRL-CMV-18S rRNA wild type | |
| Gene (H. sapiens) | 18 S rRNA (mut) | This paper | pRL-CMV-18S rRNA mutant | |
| Cell line (H. sapiens) | HEK293-Kb | Laboratory of Jonathan Yewdell | HEK293-Kb | Cell line maintained in DMEM |
| Cell line (H. sapiens) | Lenti-X 293T | Takara Bio | Cat# 632180 | Cell line maintained in DMEM |
| Strain, strain background (E. coli) | DH5a | Thermo Fisher Scientific | Cat# 18265–017 | Strain maintained in LB |
| Transfected construct (human) | shRNA | Cellecta | DECIPHER pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro | Lentivirus production |
| Antibody | Anti-GFP antibody | Proteintech | Cat# 50430–2-AP; RRID:AB_11042881 | WB (1:1000) |
| Antibody | Anti-eRF1 antibody | Santa Cruz Biotechnology | Cat# sc-365686; RRID:AB_10843214 | WB (1:2000) |
| Antibody | Anti-eRF3 antibody | Santa Cruz Biotechnology | Cat# sc-515615 | WB (1:1000) |
| Antibody | Anti-Rps26 antibody | Proteintech | Cat# 14909–1-AP; RRID:AB_2180361 | WB (1:1000) |
| Antibody | Anti-Rpl4 antibody | Proteintech | Cat# 11302–1-AP; RRID:AB_2181909 | WB (1:1000) |
| Antibody | Anti-myc antibody | Cell Signaling | Cat# 2272 S | WB (1:1000) |
| Antibody | Anti-RACK1 antibody | BD Transduction Laboratories | Cat# 610177; RRID:AB_397576 | WB (1:1000) |
| Antibody | Anti-β-Actin antibody | Millipore Sigma | Cat# A5441; RRID:AB_476744 | WB (1:1000) |
| Antibody | Anti-Mouse IgG (Fc specific)-Peroxidase antibody | Millipore Sigma | Cat# A0168; RRID:AB_257867 | WB (1:10000) |
| Antibody | Anti-Rabbit IgG (whole molecule)-Peroxidase antibody | Millipore Sigma | Cat# A9169; RRID:AB_258434 | WB (1:10000) |
| Commercial assay or kit | QIAquick Gel Extraction Kit | Qiagen | Cat# 28706 | |
| Commercial assay or kit | Q5 Site-Directed Mutagenesis Kit | New England BioLabs | Cat# AM1344 | |
| Commercial assay or kit | mMESSAGE mMACHINE T7 Transcription Kit | Invitrogen | Clontech:639647 | |
| Commercial assay or kit | Poly(A) Tailing Kit | Invitrogen | Cat# AM1350 | |
| Commercial assay or kit | RNA Clean and Concentrator-25 Kit | Zymo | Cat# R1018 | |
| Commercial assay or kit | Nano-Glo HiBiT Lytic Detection System | Promega | Cat# N3040 | |
| Commercial assay or kit | Nano-Glo HiBiT Blotting System | Promega | Cat# N2410 | |
| Commercial assay or kit | SuperScript III Reverse Transcriptase | Thermo Fisher Scientific | Cat#18080–044 | |
| Commercial assay or kit | RNA Clean and Concentrator-25 Kit | Zymo | Cat# R1018 | |
| Chemical compound, drug | Lipofectamine MessengerMAX | Invitrogen | Cat# LMRNA015 | |
| Chemical compound, drug | Lipofectamine 2000 | Invitrogen | Cat# 11668–500 | |
| Chemical compound, drug | DL-Dithiothreitol | Millipore Sigma | Cat# D0632-5G | |
| Chemical compound, drug | Blotting-Grade Blocker | Bio-Rad | Cat# 1706404 | |
| Chemical compound, drug | Tween-20 | Millipore Sigma | Cat# P7949-500ML | |
| Chemical compound, drug | Triton-X100 | Millipore Sigma | Cat# T9284-100mL | |
| Chemical compound, drug | EDTA-free Protease Inhibitor Cocktail Tablets | Millipore Sigma | Cat# 11 836 170 001 | |
| Chemical compound, drug | Puromycin | Millipore Sigma | Cat# P7255 | |
| Chemical compound, drug | Cycloheximide | Millipore Sigma | Cat# C1988-1G | |
| Chemical compound, drug | TRIzol LS Reagent | Invitrogen | Cat# 10296–028 | |
| Chemical compound, drug | Nuclease-Free Water | Invitrogen | Cat# AM9932 | |
| Chemical compound, drug | SUPERase⋅In RNase Inhibitor | Invitrogen | Cat# AM2696 | |
| Chemical compound, drug | RNase I | Invitrogen | Cat# AM2295 | |
| Chemical compound, drug | 15% TBE-Urea Gels | Invitrogen | Cat# EC6885BOX | |
| Chemical compound, drug | SYBR Gold nucleic acid gel stain | Invitrogen | Cat# S-11494 | |
| Chemical compound, drug | Sodium acetate buffer solution | Millipore Sigma | Cat# S7899-500ML | |
| Chemical compound, drug | T4 Polynucleotide Kinase | New England BioLabs | Cat# M0201L | |
| Chemical compound, drug | coli Poly(A) Polymerase | New England BioLabs | Cat# M0276L | |
| Chemical compound, drug | T4 RNA Ligase 2, truncated | New England BioLabs | Cat# M0242L | |
| Chemical compound, drug | UltraPure SSC, 20 X | Invitrogen | Cat# 15557044 | |
| Chemical compound, drug | Streptavidin Magnetic Beads | New England BioLabs | Cat# S1420S | |
| Chemical compound, drug | RNaseOUT Recombinant Ribonuclease Inhibitor | Invitrogen | Cat# 10777–019 | |
| Chemical compound, drug | 8% TBE Gel | Invitrogen | Cat# EC6215BOX | |
| Chemical compound, drug | Pierce Protein A/G Agarose | Thermo Fisher Scientific | Cat# 20421 | |
| Chemical compound, drug | TRIzol LS Reagent | Invitrogen | Cat# 10296–028 | |
| Software, algorithm | GraphPad Prism | Dotmatics | RRID:SCR_002798 | |
| Software, algorithm | Snapgene | Dotmatics | RRID:SCR_015052 | |
| Software, algorithm | R | The R Project | https://www.r-project.org/ | |
| Other | Oligonucleotides | This paper | Supplementary file 1 |
Additional files
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Supplementary file 1
List of primers and shRNA targeting sequences.
- https://cdn.elifesciences.org/articles/109257/elife-109257-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109257/elife-109257-mdarchecklist1-v1.docx
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Profiling of terminating ribosomes reveals translational control at stop codons
eLife 14:RP109257.
https://doi.org/10.7554/eLife.109257.3
