Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes
- Department of Systems Biology, Columbia University Irving Medical Center, United States
- Medical Scientist Training Program, Columbia University Irving Medical Center, United States
- Department of Chemistry, Columbia University, United States
- Department of Biochemistry & Molecular Biophysics, Columbia University Irving Medical Center, United States
- Sulzberger Columbia Genome Center, Columbia University Irving Medical Center, United States
Figures
Figure 1 with 2 supplements
PLA targeting eL22-HA and puromycylated nascent chains produces translation- and antigen-dependent signal, but does not distinguish between emetine-stalled and untreated ribosomes.
(A) Schematic depicting the ribopuromycylation assay as described by David et al., 2012. Experimental procedures used in these studies are shown below. (B) Schematic depicting our intended model of …
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Figure 1—source data 1
Statistical information pertaining to Figure 1C and Figure 1—figure supplement 2C.
Summary of replicates, cells, and statistical testing (Mann-Whitney U) for indicated treatment groups.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig1-data1-v2.docx
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Figure 1—source data 2
Raw source data for Figure 1C.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
U87 glioma cells do not express eL22-HA, and eL22-HA/Puro PLA does not distinguish between emetine-treated and untreated cells at high resolution.
(A) Representative images (20x) of eL22-HA and RiboP immunofluorescence in RiboTag and U87 glioma cells. Scale bar, 50 µm. (B) Quantification of eL22-HA/Puro PLA puncta per cell. Each dot represents …
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Figure 1—figure supplement 1—source data 1
Raw source data for Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Puromycin washout and Harringtonine run-off confirm that eL22HA/Puro PLA signal does not report on proximity of ribosomes and puromycylated nascent chains.
(A) Schematic depicting state of cells after puromycin washout. Cells are briefly treated with puromycin, washed, and allowed to continue in fresh media for 15–45 min. The vast majority of …
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Figure 1—figure supplement 2—source data 1
Raw source data for Figure 1—figure supplement 2C.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig1-figsupp2-data1-v2.xlsx
Figure 2 with 1 supplement
Regardless of pretreatment with elongation inhibitors, ribosomes and puromycylated nascent chains are not stable biochemical complexes.
(A) Sucrose gradient fractionation of polysomes from cells pretreated with emetine prior to puromycin. Top: Absorbance spectra from 15–50% linear sucrose gradient. Dashed lines indicate fractions …
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Figure 2—source data 1
Raw western blot images pertaining to Figures 2A, B and C.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig2-data1-v2.pdf
Figure 2—figure supplement 1
Pretreatment with emetine increases yield of intact ribosomes in eL22-HA immunoprecipitation, but does not enable capture of ribosomal RNA in puromycin immunoprecipitation.
(A) Regardless of puromycin treatment condition, emetine increases intact ribosome capture as shown by lower 28 s/18 s rRNA ratio (left; IP is against eL22-HA and therefore captures more free large …
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Figure 2—figure supplement 1—source data 1
Raw source data for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 3 supplements
SunTag translation reporter imaging shows that elongation inhibitor pretreatment does not prevent puromycylated nascent chain release from polysomes.
(A) Schematic depicting the SunTag translation reporter system used in this study, as described by Wu et al., 2016. Note that although the SunTag reporter construct harbors MS Coat Protein Binding …
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Figure 3—source data 1
Raw source data for Figure 3C and D.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
SunTag puncta detection, minimal photobleaching, and consistency of replicate imaging trials.
(A) Representative images of SunTag puncta quantification using TrackMate spot detection; registered spots are circled in purple. Scale bars: 10 µm for large field, 5 µm for inset. (B) Live imaging …
Figure 3—figure supplement 2
Representative images and traces of single cell SunTag imaging.
(A–D) Representative single cell raw puncta counts from each frame are plotted during live imaging with treatments as indicated. Puromycin was added at 60 s into the 8 min imaging trial (dashed red …
Figure 3—video 1
Time lapse live imaging of puromycin-induced SunTag puncta disappearance.
Representative time lapse imaging of SunTag puncta disappearing upon puromycin treatment, corresponding to Figure 3C. Cycloheximide or emetine was added 4 min before the start of imaging (5 min …
Figure 4
Structural analysis does not support a model of IgG antibodies directly contacting and recognizing puromycin within intact 80S ribosomal complexes.
(A) Schematic depicting structural modeling workflow: puromycin was positioned within the PTC of a stalled mammalian 80S ribosomal complex and the Fab fragment of a mouse IgG2a antibody was docked …
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Figure 4—source data 1
Summary of MolProbity analysis of IgG2a Fab alignments within 80S A site.
- https://cdn.elifesciences.org/articles/60048/elife-60048-fig4-data1-v2.docx
