Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

Materials and Reagents

1. Agilent Bio SEC-5 HPLC column (7.8 × 300 mm, 5 μm particles) 1,000 Å (Agilent, catalog number: 5190-2536) or 2,000 Å (Agilent, catalog number: 5190-2541)

2. Vials (e.g., Agilent, catalog number: 5184-3550)

3. Caps (e.g., Agilent, catalog number: 5182-0723)

4. Deep-well Plate 96/1,000 µl, Protein LoBind (Eppendorf, catalog number: 0030504208)

5. Ultrafree-MC Centrifugal Filter, 0.45-µm pore size, hydrophilic PVDF, 0.5-ml volume, non-sterile (Merck Millipore, catalog number: UFC30HVNB)

6. Stericups (Merck Millipore)

7. 1.5-ml tubes (e.g., Sarstedt, catalog number: 72.690.001)

8. 100-mm cell culture dishes (e.g., Corning, catalog number: 430791)

9. Cell scraper

10. Embryo collection cages (Flystuff.com, catalog number: 59-101)

11. w¹¹¹⁸ or any other flies of the required genotype

12. Ice bucket

13. DPBS, no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 14190144)

14. Tris-buffered saline (TBS)

15. HEPES

16. NaOH

17. Tris

18. Trisodium citrate dehydrate (Merck, Sigma-Aldrich, catalog number: C8532)

19. NaCl

20. MgCl₂·6H₂O

21. EDTA disodium salt

22. CHAPS, Sol-Grade (Anatrace, catalog number: C316S)

23. Glycerol

24. Dithiothreitol (DTT)

25. Heparin sodium salt from porcine intestinal mucosa (Merck, Sigma-Aldrich, catalog number: H3393)

26. Glass beads, acid-washed, 425-600 μm (Merck, Sigma-Aldrich, catalog number: G8772)

27. Cycloheximide (Merck, Sigma-Aldrich, catalog number: C7698)

28. Sodium dodecyl sulfate (SDS)

29. Pepsin (Merck, Sigma-Aldrich, catalog number: P7125)

30. RNase A (Merck, Sigma-Aldrich, catalog number: R5000)

31. 10 mg/ml BSA solution

32. Trichloroacetic acid solution (Merck, Sigma-Aldrich, catalog number: T0699)

33. TRI Reagent (Merck, Sigma-Aldrich, catalog number: T3934)

34. SUPERase•In^TM RNase Inhibitor (20 U/μl) (Thermo Fisher Scientific, catalog number: AM2694)

35. cOmplete^TM, EDTA-free Protease Inhibitor Cocktail (Roche, catalog number: 11873580001)

36. Pierce^TM 660 nm Protein Assay Kit (Thermo Fisher Scientific, catalog number: 22662)

37. HyperLadder 1 kb (BIOLIBE, catalog number: BIO-33053)

38. 2× SSC, 1% SDS solution

39. Filtered Milli-Q (MQ) water

40. Filtered 20% ethanol (EtOH)

41. Apple juice agar (CSH_Protocols 2011) dispensed in 100-mm plates

42. Polysome extraction buffer (see Recipes)

43. SEC running buffer (see Recipes)

44. 50× protease inhibitor solution (see Recipes)

45. Pepsin solution (see Recipes)

46. RNase A solution (see Recipes)

47. 20× SSC (see Recipes)

48. Standard HeLa cell lysates (see Recipes)

Note: If the vendor/manufacturer is not described in the standard laboratory chemicals above, these can be obtained from Merck (Sigma-Aldrich), VWR, or Formedium.

Equipment

1. Ultimate 3000 Bio-RS HPLC with Diode Array Detector (Thermo Fisher Scientific)

2. Vortex mixer (e.g., Scientific Industries, Vortex-Genie 2)

3. Refrigerated microcentrifuge (e.g., Thermo Fisher Scientific, Fresco^TM 17 Microcentrifuge)

4. Eppendorf BioPhotometer

5. Biospec Mini-Beadbeater-24

Software

1. Chromeleon software (Thermo Fisher Scientific)

Procedure

1. uHPLC setup

   1. Prepare and filter all solutions and SEC running buffer (see Recipes): SEC running buffer in line ‘A,’ MQ water in line ‘B’, and 20% EtOH in line ‘C’.

   2. Purge the pump with the default settings (3 ml/min, 5 min).

   3. In the column compartment, attach a 2,000 Å pore size SEC column for mammalian samples or a 1,000 Å pore size SEC column for drosophila or yeast samples.

   4. Flush the column with at least 2 column volumes (CV) of MQ water, using a flow rate of 0.2-0.8 ml/min at room temperature.

   5. Set the temperature of the column compartment at 5°C and that of the autosampler/fraction collection compartment at 4°C (Note 1).

   6. Equilibrate the column by flushing with at least 2 CV SEC buffer at a flow rate of 0.8 ml/min.

   7. Start a blank run to normalize the baseline at 260 and 280 nm.

   8. Check an operating pressure of ~160 bar at 5°C using a flow rate of 0.8 ml/min.

   9. Inject 100 µl 1 mg/ml BSA solution to block the sites of non-specific interactions.

   10. Inject 10 µl 1 mg/ml BSA solution to run as part of the standard and check the separation (Figure 2).

   11. Inject 10 µl HyperLadder solution to run as part of the standard and check the separation (Figure 2).

   12. Inject standard HeLa cell lysates containing 10 µg RNA (Figure 2) (Note 2).

   13. Start another blank run.




2. Sample Preparation (all centrifugations at 4°C)

   1. Lysates of human cell lines

      1. Wash cells in one 100-mm dish (~80% confluence; approximately ~5 × 10⁶ cells) twice with ice-cold PBS.

      2. Harvest cells by scraping in 1 ml ice-cold PBS and collect them in a 1.5-ml tube on ice.

      3. Centrifuge at 500 × g for 5 min and remove the supernatant carefully using an aspirator.

      4. Rinse cells with 1 ml ice-cold PBS if needed.

      5. Cell pellets can either be snap-frozen in liquid nitrogen and stored at -80°C or immediately used for lysate preparation (Note 4).

      6. Lyse cells by pipetting, vortexing for 10 s in 250 μl polysome extraction buffer, and incubating on ice for 15 min.

      7. Centrifuge at 17,000 × g for 20 min to pellet the debris.

      8. Collect and filter the supernatants through 0.45-μm Ultrafree-MC HV centrifugal filter units, spinning at 12,000 × g for 2-5 min (Note 3).

      9. Transfer the filtrates into new 1.5-ml tubes and leave on ice.

      10. Lysates can be snap-frozen in liquid nitrogen and stored at -80°C (Note 4).

      11. Measure RNA and protein concentrations in the filtrates using a BioPhotometer and a Pierce^TM 660 nm Protein Assay Kit, respectively.

      12. Equalize the concentrations of the samples across the conditions by diluting in polysome extraction buffer (Note 5).

      13. Transfer lysates into a vial (12 × 32 mm), close the cap, and keep on ice.

      14. (Optional) Place 5 µl protease inhibitors (50× stock solution) and 2 U RNase inhibitors in each well of a 96 deep-well plate.

   2. Lysates of Drosophila embryos

      1. Set up egg collection cups with w¹¹¹⁸ or any other required genotype of flies. Change the apple juice agar plates 2-4 times every day for 2 days.

      2. On the day of embryo collection, change the apple juice agar plate twice after 30-min collections. Discard the plates.

      3. Subsequently, collect embryos in a fresh plate for 30 min at 25°C.

      4. Age the collected embryos for an additional 40 min. At this point, the embryos will be 40-70-min-old and therefore will still be in the syncytial stage.

      5. Dechorionate the collected and aged embryos with 5% bleach until most of the embryos rise to the surface of the solution.

      6. Wash the embryos thoroughly with tap water and dry on a tissue.

      7. Collect the dechorionated embryos using a paintbrush and transfer them to a 1.5-ml tube.

      8. At this point, embryos can either be snap-frozen and stored at -80°C or processed further.

      9. Add 400 μl polysome extraction buffer, lyse the embryos with a pestle that fits into the 1.5-ml tube, and incubate on ice for 15 min.

      10. Follow the steps ‘g to n’ described in the ‘Lysates of human cell lines.

   3. Lysates of budding yeasts

      1. Grow yeast cultures on YPD or selective media. Take a sweep of the yeast strain of interest grown on a YPD plate with clean toothpick and inoculate a 5-ml culture.

      2. Grow overnight to the early stationary phase. In the morning, inoculate a 500-ml culture with 5 ml overnight culture and grow in a 2-L Erlenmeyer glass flask at 30°C until it reaches OD₆₀₀ ~1.0.

      3. To stabilize the polysomes before harvesting, add cycloheximide (CHX) at a final concentration of 0.1 mg/ml and incubate for 10 min on ice.

      4. Spin the cells for 10 min at 4,000 × g and discard the supernatant.

      5. Wash the cell pellet with ice-cold TBS containing 0.1 mg/ml CHX.

      6. Resuspend the pellet in 10 ml polysome extraction buffer containing 0.1 mg/ml CHX.

      7. Separate the resuspended cells into 1-ml aliquots in 2-ml tubes.

      8. Cells can be snap-frozen in liquid nitrogen and stored at -80°C (Note 4).

      9. Add an equal volume of glass beads to the tube and mix.

      10. Disrupt the cells in a glass bead beater at 4°C; 2 min bead beating with 2 min interval on ice.

      11. Repeat this process 6 times.

      12. Take 1 μl disrupted cells and dilute to 10 μl; check under the microscope for ghosting of cells.

      13. Centrifuge the disrupted cells at 6,000 × g, 4°C for 5 min.

      14. Transfer the supernatant to a pre-cooled 1.5-ml microcentrifuge tube.

      15. Clarify the lysates by two successive centrifugations at 11,300 × g, 4°C for 5 min, each time transferring the supernatant carefully to a new pre-cooled 1.5-ml tube.

      16. Lysates can be snap-frozen in liquid nitrogen and stored at -80°C (Note 4).

      17. Follow steps ‘k to n’ described in the ‘Lysates of human cell lines.’




3. Ribo Mega-SEC run

   1. Place the vial and the plate in the autosampler/fraction collection compartment.

   2. Inject 1/5 volume to an equal volume of the lysates and decide the time at which to collect the fractions based on the chromatogram.

   3. Set the time for fraction collection in the sequence (Note 6).

   4. Analyze the samples using the sequence set up.




4. Sample preparation from Ribo Mega-SEC fractions

   1. For western blotting, precipitate the proteins by trichloroacetic acid (TCA) precipitation followed by an ethanol wash or methanol/chloroform precipitation and resuspend the protein pellets in SDS sample buffer. Where necessary, add Tris to the samples to adjust the pH.

   2. To prepare samples for proteomics analysis, digest RNA in the samples with benzonase, reduce and alkylate the proteins, and clean up the proteins using FASP (Wisniewski et al., 2009; Potriquet et al., 2017), which efficiently removes salts and detergents such as heparin and CHAPS. SP3-based protein clean up (Hughes et al., 2014) is an alternative approach that can be used if samples do not need to contain heparin for separating molecules or protein complexes other than ribosomes.

   3. To prepare RNA from the fractions, extract RNA using TRIzol LS (Thermo) or an equivalent product. Please note that RNA needs to be re-purified by LiCl precipitation for RNA-Seq or RT-qPCR to remove heparin (Jung et al., 1997).




5. Column maintenance after analyzing samples

   1. Flush with MQ water using at least 2 CV, with a flow rate of 0.2-0.8 ml/min at room temperature (Note 7, Note 8).

   2. Flush with 20% EtOH using at least 2 CV, with a flow rate of 0.2-0.4 ml/min at room temperature.

   3. Take the column from the uHPLC and store it according to the manufacturer’s instructions.




6. Column cleanup

   1. Flush with MQ water using at least 5 CV, with a flow rate of 0.2-0.8 ml/min at room temperature.

   2. Flush with pepsin solution using at least 5 CV, with a flow rate of 0.2-0.8 ml/min at 37°C; incubate the column for 1 h at 37°C without flow (Note 9).

   3. Flush with RNase A solution using at least 5 CV, with a flow rate of 0.2-0.8 ml/min at 37°C; incubate the column for 1 h at 37°C without flow (Note 9).

   4. Flush with 2× SSC, 1% SDS solution using at least 10 CV, with a flow rate of 0.2-0.8 ml/min at 50°C.

   5. Rinse the column with at least 10 CV MQ water, using a flow rate of 0.2-0.8 ml/min at room temperature.

Data analysis

We use the Chromeleon software to run a uHPLC and to analyze data. To compare the data, we draw a mean profile together with standard deviation across biological replicates [see our original paper (Yoshikawa et al., 2018)]. We also use the area under the curve calculated by the Chromeleon software (Figure 3).

 As an example, we compared polysomes by Ribo Mega-SEC using lysates from HeLa cells treated with or without CHX or Emetine. Better recovery was seen from Emetine-treated cells as compared with CHX-treated cells (Figure 3). We also detected sufficient yield of polysomes for most experiments from untreated cells, despite the yield being lower than from either CHX- or Emetine-treated cells (Figure 3).

Figure 3. SEC separation profiles of lysates prepared from untreated, Cycloheximide (CHX)-treated or Emetine-treated HeLa cells. A) Lysates prepared from untreated HeLa cells (blue line) or cells treated with CHX (red line) or Emetine (green) were analyzed by Ribo Mega-SEC using a 2,000 Å SEC column with a flow rate of 0.8 ml/min. B) Quantitation of the area under the curve in the polysome region shown in Figure 1A. Dots indicate biological replicates. *P < 0.05, **P < 0.01.

Notes

1. We usually use the ‘Set Delay’ option to conduct steps 6-12 during the night, such that we can start analyzing samples the next morning.

2. To check reproducibility, injecting HeLa cell lysates at least twice is advisable when enough sample is available.

3. Filters tend to become clogged when lysates contain lipids. Where necessary, lysates remaining on the top of the filter unit need to be transferred to a new filter.

4. Snap-frozen lysates or cells can be kept for at least 6 months at -80°C.

5. Injection volume affects peak resolution; therefore, an equal volume should be injected across conditions.

6. We typically collect either 12, 24, or 48 fractions, ranging from polysomes to ‘small RNPs.’

7. Running two blanks, followed by injecting BSA and HyperLadder solution, is advisable to check the column condition. We typically see a good separation for up to ~50 injections of the lysates containing 20 µg RNA.

8. When new samples are to be analyzed the next day, the column is flushed with at least 2 CV MQ water and re-equilibrated with SEC buffer.

9. We found that rRNAs tend to interact with the fused silica in an SEC column. As rRNAs are protected by ribosomal proteins, we digest proteins using pepsin prior to RNase digestion to remove RNAs.

Recipes

1. Polysome extraction buffer

   20 mM HEPES-NaOH (pH 7.4)

   130 mM NaCl

   10 mM MgCl₂

   5% glycerol

   1% CHAPS

   0.2 mg/ml heparin

   Filter either through a high PVDF filter or using a Stericup to sterilize and remove any contaminating RNase traces. Store for up to 6 months at 4°C.

   Add before use:

   2.5 mM DTT (from a 1 M stock solution stored at -20°C)

   20 U SUPERase in RNase inhibitor/1 ml polysome extraction buffer

   cOmplete EDTA-free protease inhibitor (from 50× stock solution stored at -20°C; see Recipes for ‘50× protease inhibitor solution’ below)

2. SEC running buffer

   20 mM HEPES-NaOH (pH 7.4)

   60 mM NaCl

   10 mM MgCl₂

   5% glycerol

   0.3% CHAPS

   0.2 mg/ml heparin

   2.5 mM DTT

   Filter either through a high PVDF filter or using a Stericup to sterilize and remove any contaminating RNase traces. Ideally, always prepare fresh, but it can be stored at room temperature for 2 days.

3. 50× protease inhibitor solution

   1 tablet cOmplete^TM, EDTA-free protease inhibitor cocktail

   0.5 ml MQ water

   Resuspend well in a 1.5-ml tube and store at -20°C.

4. Pepsin solution

   20 μg/ml pepsin

   0.1 M acetic acid

   0.5 M NaCl

   Filter either through a high PVDF filter or using a Stericup to sterilize and remove any contaminating RNase traces. Always prepare fresh.

5. RNase A solution

   20 μg/ml RNase A

   10 mM Tris-HCl (pH 7.4)

   0.5 M NaCl

   Filter either through a high PVDF filter or using a Stericup to sterilize and remove any contaminating RNase traces. Always prepare fresh.

6. 20× SSC

   3 M NaCl

   0.3 M trisodium citrate dehydrate

7. 2× SSC, 1% SDS solution

   Filter either through a high PVDF filter or using a Stericup to sterilize and remove any contaminating RNase traces. Store at room temperature.

Acknowledgments

This protocol was adapted with minor modification from previous study published by Yoshikawa et al. (2018). We thank our colleagues in the Lamond laboratory for valuable suggestions. This research was supported by the following grants: Naito Foundation Grant for Studying Overseas (2013-413 to HY); Uehara Memorial Foundation Postdoctoral Fellowship (201430061 to HY); European Union's Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie Individual Fellowship (657087 to HY); and Tenovus Scotland Small Pilot grants (round 2017 to HY).

Competing interests

The authors declare no competing interests.

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