- Email: [email protected]
Polysome Analysis of Mammalian Cells
CHAPTER TEN
Polysome Analysis of Mammalian Cells Shan L. He, Rachel Green1 Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Duration 4.2 Preparation 4.3 Caution 4.4 Tip 5. Step 1 Immobilize Ribosomes on mRNA in Cells 5.1 Overview 5.2 Duration 5.3 Tip 6. Step 2 Prepare Cell Lysate for Polysome Analysis 6.1 Overview 6.2 Duration 7. Step 3 Size Fractionation of Polysomes 7.1 Overview 7.2 Duration 7.3 Tip 7.4 Tip 7.5 Tip References
184 184 185 185 186 186 187 187 187 188 188 188 188 188 188 188 189 189 189 190 190 190 192
Abstract To assess the global translational level of mammalian cells (see similar protocols for bacteria and yeast on Analysis of polysomes from bacteria, Polysome Profile Analysis - Yeast and Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
Methods in Enzymology, Volume 530 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420037-1.00010-5
#
2013 Elsevier Inc. All rights reserved.
183
184
Shan L. He and Rachel Green
1. THEORY Translation of mRNA starts with the association of a small ribosome subunit and a large ribosome subunit to form a complete ribosome (monosome). As the ribosome moves along the mRNA molecule during translation elongation, additional ribosomes can initiate translation on the same RNA molecule, forming polysomes. Since ribosome loading on mRNA is determined by the rate of translational initiation and elongation, polysome analysis can be used to assess the global translational level. This analysis first uses a high concentration of cycloheximide to freeze ribosomes on mRNA, followed by size-fractionation by sucrose density gradient centrifugation to separate populations of mRNAs with various numbers of ribosomes loaded. When combined with techniques to detect specific genes, such as quantitative real time PCR and Northern blotting, this analysis can examine the translational level of a specific gene. When combined with genome-wide analysis techniques such as microarrays, the translational level of each gene in the whole genome can be addressed.
2. EQUIPMENT Refrigerated centrifuge Gradient maker [e.g., Gradient Master™ (Biocomp)] Ultracentrifuge SW41 Ti rotor Brandel gradient fractionators Brandel syringe pump UV spectrophotometer with chart recorder Aspirator Polyallomer ultracentrifuge tubes (14 89 mm) Syringe Cell scraper Needles Filter barrier pipettor tips 1.75-ml microcentrifuge tubes 1.75-ml tube rack
185
Polysome Analysis of Mammalian Cells
3. MATERIALS HEPES Potassium hydroxide (KOH) Potassium chloride (KCl) Magnesium chloride (MgCl2) Dithiothreitol (DTT) Cycloheximide Sodium chloride (NaCl) Sodium phosphate dibasic (Na2HPO4) Potassium phosphate monobasic (KH2PO4) Protease inhibitor tablet, EDTA-free RNase inhibitor Sucrose NP-40 Fluorinert
3.1. Solutions & buffers Step 1 PBS Component
Final concentration
Amount
NaCl
137 mM
8g
KCl
2.7 mM
0.2 g
Na2HPO4
10 mM
1.44 g
KH2PO4
1.76 mM
0.24 g
Adjust pH to 7.4. Add water to 1 l
Lysis buffer Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.4
10 mM
1M
100 ml
KCl
150 mM
3M
500 ml
MgCl2
10 mM
2M
50 ml
DTT
1 mM
1M
10 ml
Cycloheximide
100 mg ml
1
20 mg ml
1
50 ml Continued
186
Shan L. He and Rachel Green
NP-40
2%
20%
1 ml
Protease inhibitor tablet (EDTA-free)
–
–
1 tablet
RNase inhibitor
6 U ml
20 U ml
1
1
3 ml
Add water to 10 ml
Step 2 15% Sucrose buffer Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.4
10 mM
1M
2.5 ml
KCl
150 mM
3M
12.5 ml
MgCl2
10 mM
2M
1.25 ml
Cycloheximide
100 mg ml
DTT
1 mM
1M
0.25 ml
Sucrose
15% (w/w)
–
39.68 g
Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.4
10 mM
1M
2.5 ml
KCl
150 mM
3M
12.5 ml
MgCl2
10 mM
Cycloheximide
100 mg ml
DTT
1 mM
1M
0.25 ml
Sucrose
50% (w/w)
–
153.7 g
1
20 mg ml
1
1.25 ml
Add water to 250 ml
50% Sucrose buffer
2M 1
20 mg ml
1.25 ml 1
Add water to 250 ml
4. PROTOCOL 4.1. Duration Preparation
About 1 h
Protocol
About 5–6 h
1.25 ml
Polysome Analysis of Mammalian Cells
187
4.2. Preparation Make the lysis buffer and the sucrose gradient buffers. Keep all the buffers on ice or at 4 C. Prepare a linear sucrose gradient. First add 5.25 ml of 50% (w/w) sucrose gradient buffer to the bottom of an ultracentrifuge tube. Carefully add 5.25 ml of 15% (w/w) sucrose buffer on top of the 50% (w/w) sucrose buffer. Cap the tube and use a Gradient Master™ to mix the two layers to make a linear 15–50% gradient.
4.3. Caution RNase-free conditions are important to prevent degradation of the RNA. Disposable gloves should be worn at all times and changed frequently. All reagents should be autoclaved or filter-sterilized.
4.4. Tip Take care when adding the 15% (w/w) sucrose buffer so there is a clean interface between the heavy and light layers of sucrose. See Fig. 10.1 for the flowchart of the complete protocol.
Figure 10.1 Flowchart of the complete protocol, including preparation.
188
Shan L. He and Rachel Green
5. STEP 1 IMMOBILIZE RIBOSOMES ON mRNA IN CELLS 5.1. Overview Treat the cells with cycloheximide to freeze ribosomes on the mRNA, preventing ribosomes from falling off or running off the mRNA during the subsequent experimental steps.
5.2. Duration 10 min 1.1 Add cycloheximide to the cells to a final concentration of 100 mg ml 1. 1.2 Return the cells to the incubator for 10 min.
5.3. Tip A confluent monolayer of mammalian cells from a 10-cm dish should provide sufficient material for the polysome analysis.
6. STEP 2 PREPARE CELL LYSATE FOR POLYSOME ANALYSIS 6.1. Overview Harvest and lyse the cycloheximide-treated cells (for more information on how to lyse mammalian cells, see Lysis of mammalian and Sf 9 cells).
6.2. Duration 30 min 2.1 Aspirate the media. 2.2 Wash the cells with 10 ml PBS containing 100 mg ml 1 cycloheximide. Aspirate completely. 2.3 Harvest the cells by scraping. 2.4 Add 1 ml cold lysis buffer to the cells and triturate 5 times to help lyse the cells. 2.5 Transfer the cells to a 1.75-ml microcentrifuge tube and leave on ice for 5 min to ensure complete lysis. 2.6 Centrifuge at 13 000 rpm, 4 C for 10 min. 2.7 Transfer the supernatant to a new tube and measure the absorbance at 260 nm.
Polysome Analysis of Mammalian Cells
189
Figure 10.2 Flowchart of Step 2.
2.8 Calculate the amount of the sample needed to give 40 A260 units and dilute it to 800 ml with lysis buffer. See Fig. 10.2 for the flowchart of Step 2.
7. STEP 3 SIZE FRACTIONATION OF POLYSOMES 7.1. Overview Separate different populations of polysomes by ultracentrifugation. Collect fractions of the separated polysomes across the gradient.
7.2. Duration 3h 3.1 Carefully load 800 ml of cell lysate on top of the linear sucrose gradient.
190
Shan L. He and Rachel Green
3.2 Put ultracentrifuge tubes into the rotor buckets and hang the buckets onto the swinging bucket rotor, making sure that they hang straight. 3.3 Spin at 40 000 rpm, 4 C for 2 h. 3.4 Insert the plastic tubing connected to the syringe of the syringe pump into a needle. Fill the syringe with fluorinert and force out all of the air. 3.5 Put the syringe in the syringe pump and set the pump speed to 1.5 ml min 1. 3.6 Assemble the gradient fraction collector together with the UV spectrophotometer. Connect the UV spectrophotometer to the chart recorder. 3.7 Turn on the lamp for the UV spectrophotometer 30 min before the end of the ultracentrifuge run to allow it to warm up. 3.8 Label twelve 1.75-ml microcentrifuge tubes and put them in a rack. 3.9 Remove the sample after the ultracentrifuge run and put it on the gradient fraction collector. 3.10 Put vacuum grease on the tip of the needle and puncture the ultracentrifuge tube. 3.11 Turn the chart speed of the chart recorder to 60 cm min 1 and turn on the syringe pump to start collecting fractions. 3.12 Collect 1–1.5 ml fractions in the 1.75-ml microcentrifuge tubes. 3.13 Analyze the polysome profiles from the chart recorder.
7.3. Tip Handle the sucrose gradient carefully to avoid disturbing the gradient.
7.4. Tip Before puncturing the ultracentrifuge tube, make sure that there is no air in the needle or the tubing connecting it to the syringe pump.
7.5. Tip Take care when puncturing the ultracentrifuge tube to avoid puncturing all the way through the tube. If this does happen, use vacuum grease to seal the leak. See Fig. 10.3 for the flowchart of Step 3.
Figure 10.3 Flowchart of Step 3.
192
Shan L. He and Rachel Green
REFERENCES Related Literature Stefani, G., Fraser, C. E., Darnell, J. C., & Darnell, R. B. (2004). Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells. The Journal of Neuroscience, 24, 7272–7276. Arava, Y., Wang, Y., Storey, J. D., Liu, C. L., Brown, P. O., & Herschlag, D. (2003). Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 100, 3889–3894.
Referenced Protocols in Methods Navigator Analysis of polysomes from bacteria. Polysome Profile Analysis - Yeast. Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae. Lysis of mammalian and Sf9 cells.
Polysome Analysis of Mammalian Cells Shan L. He, Rachel Green1 Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Duration 4.2 Preparation 4.3 Caution 4.4 Tip 5. Step 1 Immobilize Ribosomes on mRNA in Cells 5.1 Overview 5.2 Duration 5.3 Tip 6. Step 2 Prepare Cell Lysate for Polysome Analysis 6.1 Overview 6.2 Duration 7. Step 3 Size Fractionation of Polysomes 7.1 Overview 7.2 Duration 7.3 Tip 7.4 Tip 7.5 Tip References
184 184 185 185 186 186 187 187 187 188 188 188 188 188 188 188 189 189 189 190 190 190 192
Abstract To assess the global translational level of mammalian cells (see similar protocols for bacteria and yeast on Analysis of polysomes from bacteria, Polysome Profile Analysis - Yeast and Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
Methods in Enzymology, Volume 530 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420037-1.00010-5
#
2013 Elsevier Inc. All rights reserved.
183
184
Shan L. He and Rachel Green
1. THEORY Translation of mRNA starts with the association of a small ribosome subunit and a large ribosome subunit to form a complete ribosome (monosome). As the ribosome moves along the mRNA molecule during translation elongation, additional ribosomes can initiate translation on the same RNA molecule, forming polysomes. Since ribosome loading on mRNA is determined by the rate of translational initiation and elongation, polysome analysis can be used to assess the global translational level. This analysis first uses a high concentration of cycloheximide to freeze ribosomes on mRNA, followed by size-fractionation by sucrose density gradient centrifugation to separate populations of mRNAs with various numbers of ribosomes loaded. When combined with techniques to detect specific genes, such as quantitative real time PCR and Northern blotting, this analysis can examine the translational level of a specific gene. When combined with genome-wide analysis techniques such as microarrays, the translational level of each gene in the whole genome can be addressed.
2. EQUIPMENT Refrigerated centrifuge Gradient maker [e.g., Gradient Master™ (Biocomp)] Ultracentrifuge SW41 Ti rotor Brandel gradient fractionators Brandel syringe pump UV spectrophotometer with chart recorder Aspirator Polyallomer ultracentrifuge tubes (14 89 mm) Syringe Cell scraper Needles Filter barrier pipettor tips 1.75-ml microcentrifuge tubes 1.75-ml tube rack
185
Polysome Analysis of Mammalian Cells
3. MATERIALS HEPES Potassium hydroxide (KOH) Potassium chloride (KCl) Magnesium chloride (MgCl2) Dithiothreitol (DTT) Cycloheximide Sodium chloride (NaCl) Sodium phosphate dibasic (Na2HPO4) Potassium phosphate monobasic (KH2PO4) Protease inhibitor tablet, EDTA-free RNase inhibitor Sucrose NP-40 Fluorinert
3.1. Solutions & buffers Step 1 PBS Component
Final concentration
Amount
NaCl
137 mM
8g
KCl
2.7 mM
0.2 g
Na2HPO4
10 mM
1.44 g
KH2PO4
1.76 mM
0.24 g
Adjust pH to 7.4. Add water to 1 l
Lysis buffer Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.4
10 mM
1M
100 ml
KCl
150 mM
3M
500 ml
MgCl2
10 mM
2M
50 ml
DTT
1 mM
1M
10 ml
Cycloheximide
100 mg ml
1
20 mg ml
1
50 ml Continued
186
Shan L. He and Rachel Green
NP-40
2%
20%
1 ml
Protease inhibitor tablet (EDTA-free)
–
–
1 tablet
RNase inhibitor
6 U ml
20 U ml
1
1
3 ml
Add water to 10 ml
Step 2 15% Sucrose buffer Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.4
10 mM
1M
2.5 ml
KCl
150 mM
3M
12.5 ml
MgCl2
10 mM
2M
1.25 ml
Cycloheximide
100 mg ml
DTT
1 mM
1M
0.25 ml
Sucrose
15% (w/w)
–
39.68 g
Component
Final concentration
Stock
Amount
HEPES–KOH, pH 7.4
10 mM
1M
2.5 ml
KCl
150 mM
3M
12.5 ml
MgCl2
10 mM
Cycloheximide
100 mg ml
DTT
1 mM
1M
0.25 ml
Sucrose
50% (w/w)
–
153.7 g
1
20 mg ml
1
1.25 ml
Add water to 250 ml
50% Sucrose buffer
2M 1
20 mg ml
1.25 ml 1
Add water to 250 ml
4. PROTOCOL 4.1. Duration Preparation
About 1 h
Protocol
About 5–6 h
1.25 ml
Polysome Analysis of Mammalian Cells
187
4.2. Preparation Make the lysis buffer and the sucrose gradient buffers. Keep all the buffers on ice or at 4 C. Prepare a linear sucrose gradient. First add 5.25 ml of 50% (w/w) sucrose gradient buffer to the bottom of an ultracentrifuge tube. Carefully add 5.25 ml of 15% (w/w) sucrose buffer on top of the 50% (w/w) sucrose buffer. Cap the tube and use a Gradient Master™ to mix the two layers to make a linear 15–50% gradient.
4.3. Caution RNase-free conditions are important to prevent degradation of the RNA. Disposable gloves should be worn at all times and changed frequently. All reagents should be autoclaved or filter-sterilized.
4.4. Tip Take care when adding the 15% (w/w) sucrose buffer so there is a clean interface between the heavy and light layers of sucrose. See Fig. 10.1 for the flowchart of the complete protocol.
Figure 10.1 Flowchart of the complete protocol, including preparation.
188
Shan L. He and Rachel Green
5. STEP 1 IMMOBILIZE RIBOSOMES ON mRNA IN CELLS 5.1. Overview Treat the cells with cycloheximide to freeze ribosomes on the mRNA, preventing ribosomes from falling off or running off the mRNA during the subsequent experimental steps.
5.2. Duration 10 min 1.1 Add cycloheximide to the cells to a final concentration of 100 mg ml 1. 1.2 Return the cells to the incubator for 10 min.
5.3. Tip A confluent monolayer of mammalian cells from a 10-cm dish should provide sufficient material for the polysome analysis.
6. STEP 2 PREPARE CELL LYSATE FOR POLYSOME ANALYSIS 6.1. Overview Harvest and lyse the cycloheximide-treated cells (for more information on how to lyse mammalian cells, see Lysis of mammalian and Sf 9 cells).
6.2. Duration 30 min 2.1 Aspirate the media. 2.2 Wash the cells with 10 ml PBS containing 100 mg ml 1 cycloheximide. Aspirate completely. 2.3 Harvest the cells by scraping. 2.4 Add 1 ml cold lysis buffer to the cells and triturate 5 times to help lyse the cells. 2.5 Transfer the cells to a 1.75-ml microcentrifuge tube and leave on ice for 5 min to ensure complete lysis. 2.6 Centrifuge at 13 000 rpm, 4 C for 10 min. 2.7 Transfer the supernatant to a new tube and measure the absorbance at 260 nm.
Polysome Analysis of Mammalian Cells
189
Figure 10.2 Flowchart of Step 2.
2.8 Calculate the amount of the sample needed to give 40 A260 units and dilute it to 800 ml with lysis buffer. See Fig. 10.2 for the flowchart of Step 2.
7. STEP 3 SIZE FRACTIONATION OF POLYSOMES 7.1. Overview Separate different populations of polysomes by ultracentrifugation. Collect fractions of the separated polysomes across the gradient.
7.2. Duration 3h 3.1 Carefully load 800 ml of cell lysate on top of the linear sucrose gradient.
190
Shan L. He and Rachel Green
3.2 Put ultracentrifuge tubes into the rotor buckets and hang the buckets onto the swinging bucket rotor, making sure that they hang straight. 3.3 Spin at 40 000 rpm, 4 C for 2 h. 3.4 Insert the plastic tubing connected to the syringe of the syringe pump into a needle. Fill the syringe with fluorinert and force out all of the air. 3.5 Put the syringe in the syringe pump and set the pump speed to 1.5 ml min 1. 3.6 Assemble the gradient fraction collector together with the UV spectrophotometer. Connect the UV spectrophotometer to the chart recorder. 3.7 Turn on the lamp for the UV spectrophotometer 30 min before the end of the ultracentrifuge run to allow it to warm up. 3.8 Label twelve 1.75-ml microcentrifuge tubes and put them in a rack. 3.9 Remove the sample after the ultracentrifuge run and put it on the gradient fraction collector. 3.10 Put vacuum grease on the tip of the needle and puncture the ultracentrifuge tube. 3.11 Turn the chart speed of the chart recorder to 60 cm min 1 and turn on the syringe pump to start collecting fractions. 3.12 Collect 1–1.5 ml fractions in the 1.75-ml microcentrifuge tubes. 3.13 Analyze the polysome profiles from the chart recorder.
7.3. Tip Handle the sucrose gradient carefully to avoid disturbing the gradient.
7.4. Tip Before puncturing the ultracentrifuge tube, make sure that there is no air in the needle or the tubing connecting it to the syringe pump.
7.5. Tip Take care when puncturing the ultracentrifuge tube to avoid puncturing all the way through the tube. If this does happen, use vacuum grease to seal the leak. See Fig. 10.3 for the flowchart of Step 3.
Figure 10.3 Flowchart of Step 3.
192
Shan L. He and Rachel Green
REFERENCES Related Literature Stefani, G., Fraser, C. E., Darnell, J. C., & Darnell, R. B. (2004). Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells. The Journal of Neuroscience, 24, 7272–7276. Arava, Y., Wang, Y., Storey, J. D., Liu, C. L., Brown, P. O., & Herschlag, D. (2003). Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 100, 3889–3894.
Referenced Protocols in Methods Navigator Analysis of polysomes from bacteria. Polysome Profile Analysis - Yeast. Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae. Lysis of mammalian and Sf9 cells.