Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Summary

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m⁷GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Abstract

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m⁷GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4F^H), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m⁷GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Introduction

Translational control is emerging as an equally important step to transcriptional regulation in gene expression, especially during periods of cellular stress¹. A focal point of translation control is at the rate-limiting step of initiation where the first steps of protein synthesis involve the binding of the eukaryotic initiation factor 4E (eIF4E) to the 7-methylguanosine (m⁷GTP) 5' cap of mRNAs². eIF4E is part of a trimeric complex named eIF4F that includes eIF4A, an RNA helicase, and eIF4G, a scaffolding protein required for the recruitment of other translation factors and the 40S ribosome³. Under normal physiological conditions, the vast majority of mRNAs are translated via a cap-dependent mechanism, but under periods of cellular stress approximately 10% of human mRNAs contain 5' UTRs that could allow cap-independent translation intiation^1,4. Cap-dependent translation has been historically synonymous with eIF4F, however, stress-specific variations of eIF4F have become a trending topic^5-8.

Various cellular stresses cause eIF4E activity to be repressed via the mammalian target of rapamycin complex 1 (mTORC1). This kinase becomes impaired under stress, which results in the increased activity of one of its targets, the 4E-binding protein (4E-BP). Non-phosphorylated 4E-BP binds to eIF4E and blocks its ability to interact with eIF4G causing the repression of cap-dependent translation^9,10. Interestingly, a homolog of eIF4E named eIF4E2 (or 4EHP) has a much lower affinity for 4E-BP¹¹, perhaps allowing it to evade stress-mediated repression. Indeed, initially characterized as a repressor of translation due to its lack of interaction with eIF4G¹², eIF4E2 initiates the translation of hundreds of mRNAs that contain RNA hypoxia response elements in their 3' UTR during hypoxic stress^6,13. This activation is achieved through interactions with eIF4G3, RNA binding protein motif 4, and the hypoxia inducible factor (HIF) 2α to constitute a hypoxic eIF4F complex, or eIF4F^H6,13. As a repressor under normal conditions, eIF4E2 binds with GIGYF2 and ZNF598¹⁴. These complexes were, in part, identified through Agarose-linked m⁷GTP affinity resins. This classic method¹⁵ is standard in the field of translation and is the best and most commonly used technique to isolate cap-binding complexes in pull down and in vitro binding assays^16-19. As the cap-dependent translation machinery is emerging as flexible and adaptable with inter-changing parts^6-8,13, this method is a powerful tool to rapidly identify novel cap-binding proteins involved in the stress response. Furthermore, variations in eIF4F could have broad implications as several eukaryotic model systems appear to use an eIF4E2 homolog for stress responses such as A. thaliana²⁰, S. Pombe²¹, D. melanogaster²², and C. elegans²³.

Evidence suggests that variations in eIF4F may not be strictly limited to stress conditions, but be involved in normal physiology²⁴. The oxygen supply to tissues (at capillary ends) or within tissues (measured via microelectrodes) varies from 2-6% in the brain²⁵, 3-12% in the lungs²⁶, 3.5-6% in the intestine²⁷, 4% in the liver²⁸, 7-12% in the kidney²⁹, 4% in muscle³⁰, and 6-7% in bone marrow³¹. Cells and mitochondria contain less than 1.3% oxygen³². These values are much closer to hypoxia than the ambient air where cells are routinely cultured. This suggests that what were previously thought of as hypoxia-specific cellular processes may be relevant in a physiological setting. Interestingly, eIF4F and eIF4F^H actively participate in the translation initiation of distinct pools or classes of mRNAs in several different human cell lines exposed to physiological oxygen or "physioxia"²⁴. Low oxygen also drives proper fetal development³³ and cells generally have higher proliferation rates, longer lifespans, less DNA damage and less general stress responses in physioxia³⁴. Therefore, eIF4F^H is likely a key factor in the expression of select genes under physiological conditions.

Here, we provide a protocol to culture cells in fixed physiological oxygen conditions or in a dynamic fluctuating range that is likely more representative of tissue microenvironments. One advantage of this method is that cells are lysed within the hypoxia workstation. It is not often clear how the transition from hypoxic cell culture to cell lysis is performed in other protocols. Cells are often first removed from a small hypoxia incubator before lysis, but this exposure to oxygen could affect biochemical pathways as the cellular response to oxygen is rapid (one or two min)³⁵. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m⁷GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. Exploring the activity and composition of eIF4F^H and other variations of eIF4F through the method described here will shed light on the intricate gene expression machineries that cells utilize during physiological conditions or stress responses.

Protocol

1. Preparations for Cell Culture

1. Purchase commercially available stocks of human cells.
   NOTE: This protocol utilizes HCT116 colorectal carcinoma and primary human renal proximal tubular epithelial cells (HRPTEC).
2. Make 500 ml medium for culture of HCT116: Dulbecco's Modified Eagle Medium (DMEM)/High glucose medium supplemented with 7.5% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (P/S).
3. Make 500 ml medium for culture of HRPTEC: Epithelial cell medium supplemented with 5% FBS, 1% epithelial cell growth supplement, and 1% P/S.
4. Prepare 500 ml of 1x Phosphate-Buffered Saline (PBS): 140 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 15 mM KH₂PO₄. Adjust pH to 7.4 and sterilize by autoclaving for 40 min at 121 °C.

2. Initiation of Cell Culturing

1. Carefully aspirate the medium from a 80-90% confluent dish without disturbing the cells.
2. Aliquot 3-5 ml of 1x PBS per culture dish, gently rock each flask to coat the surface area of the dish, and carefully aspirate the 1x PBS. Repeat for a second 1x PBS wash.
3. Aliquot 3 ml of 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) into each culture dish and gently rock to evenly coat the cells with trypsin-EDTA. Incubate at 37 °C for 2-3 min.
4. Transfer the detached cells into a 15 ml centrifuge tube and centrifuge at 4,000 x g for 90 sec.
5. Resuspend cell pellet in 1 ml of complete DMEM.
6. Count cells using a haemocytometer. Calculate how many non-pyrogenic and polystyrene 100 mm cell culture dishes are required to achieve an initial seeding density of approximately 2,500-5,000 cells per cm².
7. Pre-warm complete cell culture medium made in steps 1.2 or 1.3 in a 37 °C water bath for 30-45 min.
8. Decontaminate the medium bottle and cell vial with 70% ethanol. From this point forward all operations should be performed aseptically.
9. Aliquot 6 ml of complete DMEM into as many 100 mm cell culture dishes required to use the entire volume of frozen cells within the seeding density mentioned in step 2.6.
10. Transfer the volume of cell suspension calculated in step 2.6 to each of the 100 mm culture dishes. Rock each plate manually to evenly distribute the cells over the surface of the plate.
11. Place the seeded 100 mm cell culture dish in the incubator at 37 °C in a 5% CO₂ atmosphere. Allow cells to incubate at least 24 hr before the next steps.

3. Subculturing

1. View each cell culture dish under a microscope to determine the percentage of cell confluency (% cells covering the area of the dish). Return cells to the incubator and pre-warm complete medium and 1x PBS. Proceed to the next step if the cells are 80-100% confluent.
2. Carefully aspirate the spent medium without disturbing the cells in each culture dish.
3. Aliquot 3-5 ml of 1x PBS per culture dish, gently rock each flask to coat the surface area of the dish, and carefully aspirate the 1x PBS. Repeat for a second 1x PBS wash.
4. Aliquot 3 ml of 0.05% trypsin-EDTA into each culture dish and gently rock to evenly coat the cells with trypsin-EDTA. Incubate at 37 °C for 2-3 min.
5. During this incubation, aliquot 10 ml of complete medium into as many culture dishes are required to obtain cell density of 2,500-5,000 cells per cm².
6. When the cells have detached from the plate, quickly add 3 ml of 1x defined trypsin inhibitor and gently swirl the dish for 1 min to ensure that all of the trypsin-EDTA has been neutralized.
7. Transfer the detached cells into a sterile 15 ml centrifuge tube and set aside. Add 3 ml 1x PBS to the dish to gather any remaining cells, and transfer that to the same 15 ml centrifuge tube.
8. Pellet the cells by centrifuging 150 x g for 5 min.
9. Aspirate the supernatant and resuspend the cell pellet in 3 ml of complete medium.
10. Count cells using a haemocytometer and seed 150 mm culture dishes containing 15 ml of complete medium to obtain a cell density of 2,500-5,000 cells per cm². Use two 150 mm for each cap-binding assay.
    NOTE: Oxygen diffusion to cells through liquid media is dependent on the volume³⁶. It is recommended to keep the volume of the media consistent between experiments whether adherent cells or cells in suspension are used. Media can be pre-conditioned (incubated in the workstation for 24 hr as discussed in Discussion) to avoid these variabilities in diffusion.
11. Place the seeded-culture dishes in the incubator at 37 °C in a 5% CO₂ atmosphere. Allow cells to incubate at least 24 hr before proceeding to the next steps.

4. Physioxic Exposure

1. View the cells under the microscope. For best results, ensure that cells are 70-80% confluent for a 24 hr exposure, 50-60% confluent for a 48 hr exposure, and 40-50% confluent for a 72 hr exposure.
2. Place the cells into a hypoxia workstation for the desired time (24, 48, or 72 hr depending on the experiment).
3. Set the workstation to the appropriate physioxia.
   NOTE: For example, a 3% O₂ setting would be accompanied by 5% CO₂ and 92% N₂.
   NOTE: If oxygen fluctuations within a dynamic range are desired over a specific timeline, program the schedule into the instrument or manually adjust the oxygen settings at the desired intervals.
4. Set the humidity to 60%. The workstation atmosphere is very dry nonetheless, and media will noticeably evaporate during long experiments (>48 hr). Keep a media reserve in a cell culture flask within the workstation (so that the media is equilibrated with the workstation atmosphere) to replenish the media in the cell culture dishes.
   NOTE: Any solution that will interact with the cells prior to lysis (such as PBS and trypsin-EDTA) should be pre-conditioned for 24 hr before use within the hypoxia workstation so that the dissolved oxygen equilibrates with the atmospheric oxygen³⁶.
5. Keep cells within the workstation until lysis.

5. Preparing Buffers for Cap-binding Assay

1. Prepare 1x Tris-buffered Saline (TBS).
   1. In 800 ml of dH₂O, dissolve 8.76 g NaCl and 6.05 g Tris Base.
   2. Bring the solution to a final pH of 7.4 using 1 M HCl.
   3. Bring the final volume to 1 L using dH₂O.
2. Prepare non-denaturing lysis buffer. Prepare lysis buffer the day of the cap-binding assay. Make extra lysis buffer to wash the blank agarose beads and the γ-aminophenyl-m⁷GTP agarose C10-linked beads (steps 7.9 and 7.13).
   1. In 7 ml of dH₂O, add 160 µl 5 M NaCl, 160 µl 1 M Tris-HCl pH 7.4, 40 µl 200 mM NaF, 40 µl 1 M MgCl₂, and 40 µl 1 mM sodium orthovanadate.
   2. Add 40 µl of Igepal to the 7 ml solution. Cut the tip of the pipette to help retrieve Igepal as it is extremely viscous.
   3. Mix by pipetting up and down, or vortex, the 7 ml solution until all of the Igepal has been dissolved.
   4. Raise the final volume of the solution to 8 ml and keep on ice.
3. Prepare 4x Sodium Dodecyl Sulfate (SDS)-PAGE Sample Buffer.
   1. In a beaker, combine 16 ml 1 M Tris-HCl pH 6.8, 12.64 ml glycerol, and 8 ml dithiothreitol (DTT).
   2. Add 3.2 g of SDS and 0.16 g of bromophenol blue. Allow powder to fully dissolve by mixing with a magnetic stir bar.
   3. Aliquot into 1.5 ml microcentrifuge tubes and store at -20 °C.

6. Cell Lysis

1. Prepare non-denaturing lysis buffer and keep on ice the day of the cap-binding assay.
2. Pre-warm 1x PBS and trypsin in a 37 °C water bath for 30 min.
3. Aliquot 980 µl of lysis buffer into a 1.5 ml microcentrifuge tube for each cap-binding assay being performed.
4. Add 10 µl of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 10 µl of 100x protease inhibitor cocktail to the 980 µl of lysis buffer. Keep on ice.
5. Discard the media from each 150 mm dish in a waste container within the hypoxia workstation.
6. Wash the cells with 3-4 ml of warm 1x PBS and discard all of the liquid.
7. Add 1 ml of warm 0.05% trypsin-EDTA to each dish and let sit for 2 min or until cells are no longer adhered to the plate.
8. Using a pipette, transfer the cells of one plate to a 1.5 ml microcentrifuge tube. Repeat as needed so that each plate of cells is transferred to a separate 1.5 ml microcentrifuge tube.
9. Centrifuge the 1.5 ml microcentrifuge tubes at 6,000 x g for 90 sec to pellet the cells.
10. Aspirate the trypsin using a pipette without disturbing the pellet. If the pellet is disturbed, re-centrifuge the 1.5 ml microcentrifuge tube at 6,000 x g for 90 sec.
11. Wash the cells with warm 1x PBS by gently pipetting 200 µl of PBS into the tube just above the pellet. Re-centrifuge if the pellet is disturbed.
12. Aspirate the PBS without disturbing the pellet.
13. Pipette 500 µl from the 1 ml prepared lysis buffer solution onto the first cellular pellet. Resuspend the pellet entirely by pipetting up and down.
14. Combine the resuspended cells with the second cell pellet and resuspend the second pellet by pipetting up and down. Repeat this process until all pellets have been combined and resuspended for each sample.
15. Combine the lysate with the remaining 500 µl of lysis buffer in a 1.5 ml microcentrifuge tube.
16. Remove samples from the hypoxia workstation.
    NOTE: After adding lysis buffer, all subsequent steps are to be performed in ambient air as the hypoxia workstation is set to 37 °C and the following steps are to be performed cold (4 °C or on ice).
17. Lyse the cells using gentle agitation by rotating the samples at 4 °C for 1.5-2 hr.
18. Centrifuge the lysed cells at 12,000 x g for 15 min at 4 °C to remove cellular debris.
19. Transfer the lysate to a new 1.5 ml microcentrifuge tube and discard the pellet.
20. Reserve a portion (5-10%) of the supernatant to be used as a whole cell lysate input control for future western blot analysis.

7. Cap-binding Assay

1. For each sample, transfer 50 µl of the blank agarose bead control slurry and 50 µl of the γ-aminophenyl-m⁷GTP agarose C10-linked bead slurry to separate 1.5 ml microcentrifuge tubes. Use scissors to remove the tip of the pipette to facilitate collection of the bead slurry.
2. Pellet the beads by centrifuging the slurry at 500 x g for 30 sec.
3. Remove the supernatant carefully and resuspend the beads in 500 µl of TBS.
4. Repeat steps 7.2 and 7.3. Pellet the beads at 500 x g for 30 sec and remove the supernatant.
5. Transfer the supernatant containing the lysate from step 6.19 to the 1.5 ml microcentrifuge tube containing the blank agarose beads.
   NOTE: The blank agarose beads act as a pre-clearing step to remove proteins from the lysate that non-specifically interact with the bead.
6. Incubate for 10 min at 4 °C with gentle agitation.
7. Pellet the blank agarose beads by centrifuging at 500 x g for 30 sec.
8. Transfer the lysate to the 1.5 ml microcentrifuge tube containing the γ-aminophenyl-m⁷GTP agarose C10-linked beads.
9. Wash the blank agarose beads by resuspending the beads in 500 µl of lysis buffer, pelleting the beads by centrifuging at 500 x g for 30 sec, and discarding the supernatant. Repeat this four times for a total of five washes.
10. Resuspend the blank agarose beads in 1x SDS-PAGE sample buffer, and boil the beads for 90 sec at 95 °C. Store at -20 °C for future western blot analysis to observe whether the protein of interest binds non-specifically to the bead.
11. Incubate the lysate with the γ-aminophenyl-m⁷GTP agarose C10-linked beads with gentle agitation for 1 hr at 4 °C to capture cap-binding proteins.
12. Pellet the γ-aminophenyl-m⁷GTP agarose C10-linked beads by centrifuging at 500 x g for 30 sec. Discard the supernatant.
13. Wash the γ-aminophenyl-m⁷GTP agarose C10-linked beads by repeating step 7.9.
14. Resuspend the beads in 600 µl of lysis buffer.
15. Add GTP to a final concentration of 1 mM.
16. Incubate the γ-aminophenyl-m⁷GTP agarose C10-linked beads + 1 mM GTP with gentle agitation for 1 hr at 4 °C. Note: This will disassociate proteins that non-specifically interact with m⁷GTP (i.e., also interact with non-methylated GTP).
17. Pellet the γ-aminophenyl-m⁷GTP agarose C10-linked beads by centrifuging at 500 x g for 30 sec.
18. Transfer the supernatant to a new 1.5 ml microcentrifuge tube and add 200 µl of 4x SDS-PAGE sample buffer (50 mM Tris-HCl, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). Store the GTP control sample at -20 °C for future western blot analysis³⁷. Wash the beads by repeating step 7.9.
19. Resuspend the beads in 1x SDS-PAGE sample buffer (50 mM Tris-HCl, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol), and boil at 95 °C for 90 sec. Store the m⁷GTP-bound fraction at -20 °C for future western blot analysis³⁷.

Results

Analysis of Cap-binding Ability in Response to Oxygen of eIF4E and eIF4E2 in an m⁷GTP Affinity Column

Figures 1 and 2 represent western blots of typical m⁷GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in two human cell lines: primary human renal proximal tubular epithelial cells (HRPTEC) in Figure 1 and colorect...

Discussion

The analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions can allow for the identification of novel oxygen-regulated translation initiation factors. The affinity of these factors for the 5' cap of mRNA or other cap-associated proteins can be measured by the strength of their association to m⁷GTP-linked Agarose beads. One caveat of this technique is that it measures the cap-binding potential of proteins post-lysis, but it is performed under non-denaturing conditions that...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
γ-aminophenyl-m7GTP agarose C10-linked beads	Jena Bioscience	AC-1555	Agarose-linked m7GTP
100 mm culture dish	Corning	877222	10-cm culture dish
150 mm culture dish	Thermofisher	130183	15 cm culture dish
AEBSF Hydrochloride	ACROS Organics	A0356829	AEBSF
Agarose Beads	Jena Bioscience	AC-0015	Agarose bead control
Bromophenol Blue	Fisher	BP112-25	Component of SDS-PAGE loading buffer
1.5 ml Centrifuge Tubes	FroggaBio	1210-00S	Used to centrifuge small volumes
15 ml Conical Centrifuge Tubes	Fisher	1495970C	Used in culturing primary cells
Defined trypsin inhibitor	Fisher	R007100	DTI
Dithiothreitol	Fisher	BP172-25	DTT
Epithelial cell medium (complete kit)	ScienCell	4101	Includes serum and growth factor supplements)
Glycerol	Fisher	BP229-1	Component of SDS-PAGE loading buffer
100 mM Guanosine 5'-triphosphate, 1 ml	Jena Bioscience	272076-0251M	GTP
HCT116 colorectal carcinoma	ATCC	CCL-247	Human cancer cell line
Human renal proximal tubular epithelial cells	ATCC	PCS-400-010	HRPTEC
Hyclone DMEM/High Glucose	GE Life Sciences	SH30022.01	Standard media for human cell culture
Hyclone Penicillin-Streptomycin solution	GE Life Sciences	SV30010	Antibiotic component of DMEM
H35 HypOxystation	Hypoxygen	N/A	Hypoxia workstation
Igepal CA-630	MP Biomedicals	2198596	Detergent component of lysis buffer
Monopotassium phosphate	Fisher	P288-500	KH2PO4
Potassium chloride	Fisher	P217-500	KCl
Magnesium chloride	Fisher	M33-500	MgCl2
Sodium chloride	Fisher	BP358-10	NaCl
Sodium fluoride	Fisher	5299-100	NaF (phosphatase inhibitor component of lysis buffer)
Disodium phosphate	Fisher	5369-500	Na2HPO4
Premium Grade Fetal Bovine Serum	Seradigm	1500-500	FBS
Protease Inhibitor Cocktail (100x)	Cell Signalling	58715	Component of lysis buffer
Sodium Dodecyl Sulfate	Fisher	BP166-100	SDS
Sodium Orthovanadate	Sigma	56508	Na3VO4
Tris Base	Fisher	BP152-5	Component of buffers
0.05% Trypsin-EDTA (1x)	Life Technologies	2500-067	Trypsin used to detach adherent cells