Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC
- School of Life Sciences, University of Dundee, United Kingdom
- University of Sydney, Australia
- University of Edinburgh, United Kingdom
Figures
Figure 1 with 5 supplements
Assignment of the peaks separated by a single 2,000 Å SEC column.
(a) The HeLa cell lysate containing 100 μg of RNA was separated into 22 fractions by ultracentrifugation with a 10–45% sucrose density gradient with continuous monitoring of absorbance at 254 nm. (b)…
Figure 1—figure supplement 1
Comparison of the polysome profile from the lysate prepared either by Triton X-100 or CHAPS.
The lysate prepared either by Triton X-100 or CHAPS from HeLa cells was analyzed by ultracentrifugation with 10–45% sucrose density gradient. The absorbance at 254 nm was monitored continuously. The …
Figure 1—figure supplement 2
Comparison of the UV chromatograms of polysome profiles for three different pore size columns.
The lysate extracted from HCT116 p53+/+ cells was injected onto three different pore size (300, 1,000, or 2,000 Å) SEC columns. The retention time is indicated on x-axis and the UV absorbance of 260 …
Figure 1—figure supplement 3
UV chromatogram of polysome profiles by sequential SEC columns.
HeLa cell lysate containing either 80 μg of RNA (a) or 50 μg of RNA (b) was analysed by SEC with the sequential columns; that is (a) two 2,000 Å columns or (b) a 2,000 Å column and a 1,000 Å column, …
Figure 1—figure supplement 4
Comparison of the separation profile by the flow rate of 0.2, 0.5, 0.8, or 1.0 ml/ min.
HeLa cell lysate containing 20 μg of RNAs was injected onto the 2,000 Å SEC column and the separation profiles of ribosomes were compared among four different flow rate. Green dash line indicated …
Figure 1—figure supplement 5
Comparison of Ribo Mega-SEC chromatograms for the lysates from different cell lines.
The equal amount of the lysate from HeLa, U2OS, HCT116 p53+/+, and HCT116 p53-/- cells was individually injected onto the 2,000 Å SEC column. The retention time is indicated on x-axis and the UV …
Figure 2 with 3 supplements
Ribo Mega-SEC chromatograms of polysomes and free ribosomal subunits.
(a) The UV chromatogram of HeLa cell lysates either untreated or treated with 30 mM EDTA (EDTA-treated) is shown. The line is the mean profile and the surrounding ribbon shows the standard deviation …
Figure 2—figure supplement 1
Polysome profile of untreated or EDTA-treated cell lysates by SDG analysis.
HeLa cell lysate containing 100 μg of RNA treated with or without EDTA was separated into 21 fractions by ultracentrifugation with a 10–45% sucrose density gradient. The absorbance at 254 nm was …
Figure 2—figure supplement 2
Ribo Mega-SEC chromatogram and fractions collected (Figure 2A).
The UV chromatogram of HeLa cell lysate either untreated or treated with 30 mM EDTA (EDTA-treated) from one of three biological replicates was shown. 48 fractions numbered at the top of chromatogram …
Figure 2—figure supplement 3
Western blotting with anti-eS10 antibody across the fractions separated from EDTA-treated cell lysates.
HeLa cell lysate treated with EDTA was separated into 24 fractions by Ribo Mega-SEC and the fractions were analyzed by western blotting with anti-eS10 antibody. Input: 20 μg of protein was loaded.
Figure 3 with 2 supplements
Stability of polysomes isolated by Ribo Mega-SEC.
(a) Fractions analyzed by the subsequent SDG analysis are numbered and highlighted in colors (yellow, blue, and green) in the Ribo Mega-SEC profile of HeLa cell lysate. Fractions analyzed by the …
Figure 3—figure supplement 1
Comparison of polysome profiles of lysates extracted either by normal salt or by high-salt containing buffer.
HeLa cell lysate extracted by normal salt or by high-salt containing buffer was analyzed by SDG (a) or by SEC (b).
Figure 3—figure supplement 2
Ribo Mega-SEC profile for in vitro puromycylation (Figure 3D).
10 fractions from polysomes to 60S subunits highlighted in green were collected by the flow rate of 0.5 ml/min of Ribo Mega-SEC HPLC run and subjected to in vitro puromycylation. The retention time …
Figure 4 with 1 supplement
Reproducibility of Ribo Mega-SEC and SDG analysis.
(a) The UV chromatograms of Ribo Mega-SEC from the three biological replicates of untreated cell lysates were showed. The retention time is indicated on the x-axis and the UV absorbance of 260 nm is …
Figure 4—figure supplement 1
The three biological replicates showing elution profiles of EDTA-treated cell lysates by Ribo Mega-SEC.
The UV chromatograms from the three biological replicates of EDTA-treated cell lysate were showed. The retention time is indicated on x-axis and the UV absorbance of 260 nm is indicated on y-axis. …
Figure 5
Ribo Mega-SEC profile with or without amino acid starvation.
(a) Sixteen fractions separated from the lysates of HeLa cells grown in EBSS, either with (+AA), or without (-AA), exogenous amino acids for 2 hr, were collected. The line is the mean profile and …
Figure 6 with 3 supplements
Proteomic analysis of Ribo Mega-SEC fractions from mouse liver tissue.
(a) Workflow for Ribo Mega-SEC analysis of mouse liver tissue and LC-MS/MS methodology. (b) Ribo Mega-SEC profile of mouse liver tissue. The collected fractions are highlighted and numbered at the …
Figure 6—figure supplement 1
The three biological replicates of the elution profile of mouse liver extract analysed by Ribo Mega-SEC.
The UV chromatograms from the three biological replicates of the extract from mouse liver tissue were showed. The retention time is indicated on x-axis and the UV absorbance of 260 nm is indicated …
Figure 6—figure supplement 2
Comparison of Ribo Mega-SEC profiles using a mobile phase with or without heparin.
Mouse liver tissue extracts were analyzed by Ribo Mega-SEC using mobile phase with or without heparin. The retention time is indicated on x-axis and the UV absorbance of 260 nm is indicated on y-axis.
Figure 6—figure supplement 3
Hierarchical clustering of protein elution profiles in the mouse liver tissue.
(a) Optimum hierarchical cluster number was calculated and the average Pearson correlation coefficient (y-axis) was plotted over a range of cluster numbers between 50 and 2,000 (x-axis). (b) All …
Figure 7
Schematic image of whole translation process highlighted with the identified proteins in mouse liver tissue.
Ribo Mega-SEC elution profiles for 40S small ribosomal subunit, EIF2alpha, EIF3 complex, EIF4F complex, PABPC1, 60S large ribosomal subunit, EF-Tu, EF-G, exon junction complex (EJC), UPF1, and eRF …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HeLa | ATCC | RRID:CVCL_0030 | Tested negative for mycoplasma |
| Cell line (human) | U2OS | ATCC | RRID:CVCL_0042 | Tested negative for mycoplasma |
| Cell line (human) | HCT116 p53 +/+ | ATCC | RRID:CVCL_0291 | Tested negative for mycoplasma |
| Cell line (human) | HCT116 p53 -/- | Horizon | RRID:CVCL_S744 | Tested negative for mycoplasma |
| Biological sample (Mus musculus) | C57BL/6J | Australian BioResources | RRID:IMSR_JAX:000664 | |
| Antibody | Anti-Ribosomal protein S10 antibody [EPR8545] | Abcam Cat# ab151550 | RRID:AB_2714147 | |
| Antibody | Ribosomal Protein L28 (A-16) antibody | Santa Cruz Biotechnology Cat# sc-14151 | RRID:AB_2181749 | |
| Antibody | RPL14/Ribosomal Protein L14 Antibody | Bethyl Cat# A305-052A | RRID:AB_2621246 | |
| Antibody | RPLP0 antibody | Abcam Cat# ab88872 | RRID:AB_2042838 | |
| Antibody | Anti-mouse IgG, HRP-linked Antibody | Cell Signaling Technology Cat# 7076 | RRID:AB_330924 | |
| Antibody | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology Cat# 7074 | RRID:AB_2099233 | |
| Antibody | Anti-Goat IgG (whole molecule) -Peroxidase antibody produced in rabbit | Sigma-Aldrich Cat# A5420 | RRID:AB_258242 |
Additional files
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Supplementary file 1
Protein level data identified in mouse liver tissue, classified by cluster.
The table summarizes the proteins identified in mouse liver tissue and includes the following data for each protein identification: protein ID, protein name, Gene name, cluster number, total iBAQ intensities from two biological replicates and individual intensities.
- https://doi.org/10.7554/eLife.36530.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.36530.024
