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.
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 (eIF4FH), 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.
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 intiation1,4. Cap-dependent translation has been historically synonymous with eIF4F, however, stress-specific variations of eIF4F have become a trending topic5-8.
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 translation9,10. 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 stress6,13. 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 eIF4FH6,13. 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 assays16-19. As the cap-dependent translation machinery is emerging as flexible and adaptable with inter-changing parts6-8,13, 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 eIF4FH 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, eIF4FH 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 eIF4FH 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.
1. Preparations for Cell Culture
2. Initiation of Cell Culturing
3. Subculturing
4. Physioxic Exposure
5. Preparing Buffers for Cap-binding Assay
6. Cell Lysis
7. Cap-binding Assay
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 colorectal carcinoma (HCT116) in Figure 2. Cells are maintained at the indicated oxygen availability for 24 hr to ensure that the dissolved oxygen in the liquid media has equilibrated with the air within the hypoxia workstation. The input lane (IN) represents 10% of the whole cell lysate before the m7GTP-linked agarose beads are added to capture cap-binding proteins. This input lane is used as a baseline to measure enrichment of a target protein in the m7GTP pulldown. The GTP lane is the eluate from the m7GTP beads that were elute with 1 mM GTP. This step allows for the detection of proteins that also recognize non-methylated GTP. The cap-binding proteins eIF4E and eIF4E2 recognize m7GTP specifically, but their homolog eIF4E3 (not shown here) also binds to non-methylated GTP. The ability of eIF4E and eIF4E2 to bind the 5' mRNA cap structure (m7GTP) can be measured by comparing the intensity of their bands in the m7GTP column relative to the intensity in the input lane (total available pool).
This quantification can be performed by measuring the pixel intensity of the protein bands for three biological replicates with an image analysis software such as ImageJ. Results are expressed as relative means of density units (RDU) ± standard error of the mean. Raw values prior to normalization from both experimental and control samples were compared by unpaired two-tailed Student's t-test. P < 0.05 was considered statistically significant. This representative experiment shows that the two cell lines have different ranges of oxygen for their eIF4E and eIF4E2 usage. Figure 1 shows that in HRPTEC only eIF4E binds strongly to m7GTP at 8% O2 (Figure 1A), that both eIF4E and eIF4E2 are significantly associated with m7GTP from 3-5% O2 (Figure 1B–C), and that only eIF4E2 binds strongly to m7GTP at 1% O2 (Figure 1D). In Figure 2, in HCT116 cells, the oxygen-dependent usage of these cap-binding proteins is different: only eIF4E binds significantly to m7GTP at 12% O2 (Figure 2A), both cap-binding proteins bind significantly to m7GTP in the 5-8% O2 range (Figure 2B–C), and only eIF4E2 binds significantly to m7GTP at 3% O2 (Figure 2D). The absence of elution from the m7GTP column with GTP shows that eIF4E and eIF4E2 are specific to m7GTP and do not recognize non-methylated GTP. The bar graphs represent quantification of three biological replicates using ImageJ. Our results demonstrate that two major cap-binding proteins, eIF4E and eIF4E2, differ in their ability to bind the mRNA m7GTP cap structure in an oxygen-dependent manner.
Based on previous literature that these two proteins initiate the translation of unique classes of mRNAs, this shift in cap-binding activity could result in different proteomes generated to adapt to changes in oxygen availability. This technique can be used to characterize novel translation initiation factors that interact with the 5' mRNA cap (or with cap-binding proteins) through a targeted western blot approach or a broad approach such as mass spectrometry analysis of an m7GTP eluate.
Figure 1: eIF4E and eIF4E2 activities overlap during physioxia in HRPTEC from 3-5% O2. Capture assays using m7GTP beads in human renal proximal tubular epithelial cell (HRPTEC) lysates exposed to (A) 8%, (B) 5%, (C) 3% or (D) 1% O2 for 24 hr. GTP, GTP wash to measure specificity for m7GTP; m7GTP, proteins bound to m7GTP beads after GTP wash. Representative Western blots are shown and data from at least three independent experiments were quantified by ImageJ and expressed as relative density units (RDU) relative to 10% input (IN) of whole cell lysate. *P < 0.05 (unpaired two-tailed Student's t-test) was considered a significant change when comparing the enrichment of eIF4E or eIF4E2 in the cap-bound fraction (m7GTP) to the IN. Data, mean ± standard error of the mean (SEM) of three independent experiments. This research was originally published in the Journal of Biological Chemistry. Timpano, S. and Uniacke, J. Human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. 2016; 291(20): 10772-8224. Please click here to view a larger version of this figure.
Figure 2. eIF4E and eIF4E2 activities overlap during physioxia in HCT116 from 5-8% O2. Capture assays using m7GTP beads in HCT116 human colorectal carcinoma lysates exposed to (A) 12%, (B) 8%, (C) 5% or (D) 3% O2 for 24 hr. GTP, GTP wash to measure specificity for m7GTP; m7GTP, proteins bound to m7GTP beads after GTP wash. Representative Western blots are shown and data from at least three independent experiments were quantified by ImageJ and expressed as relative density units (RDU) relative to 10% input (IN) of whole cell lysate. *P < 0.05 (unpaired two-tailed Student's t-test) was considered a significant change when comparing the enrichment of eIF4E or eIF4E2 in the cap-bound fraction (m7GTP) to the IN. Data, mean ± standard error of the mean (SEM) of three independent experiments. This research was originally published in the Journal of Biological Chemistry. Timpano, S. and Uniacke, J. Human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. 2016; 291(20): 10772-8224. Please click here to view a larger version of this figure.
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 maintain protein-protein interactions and post-translational modifications (PTMs). Several protein-protein interactions mediate the binding of the eIF4E family to the 5' cap. The 4E-BP binds and represses eIF4E by blocking its interaction with eIF4G and preventing the recruitment of the 43S pre-initiation complex38. The eIF4E/4E-BP complex remains bound to the 5' mRNA cap, blocking any initiation from that transcript, and can be used as a marker for eIF4E inhibition in m7GTP affinity columns. Conversely, the interaction of eIF4E with eIF4G on m7GTP columns could be a marker for active translation initiation. PTMs are another method to regulate the activity of translation initiation factors. For example, phosphorylation of eIF4E by Mnk1 can increase its affinity for the 5' mRNA cap39. The protein modifier encoded by the Interferon Stimulated Gene 15 (ISG15) is a PTM that increases the cap-binding activity of eIF4E2 in response to interferon induction via pathogen infection40. The ISG15 gene contains a hypoxia response element in its promoter suggesting that this system could regulate eIF4E2 in low oxygen. Very little is known about how PTMs may alter the activity of cap-binding proteins during physiologically relevant oxygen conditions. This technique followed by mass spectrometry analysis could reveal novel oxygen-regulated and physiologically relevant PTMs that mediate the activity of translation initiation factors.
An alternative method to capturing with an m7GTP cap analog would be to immunoprecipitate a known cap-binding protein such as eIF4E or eIF4G and perform mass spectrometry to identify associated proteins. A limitation of this strategy is that cap-binding proteins often have alternative cellular roles such as within stress granules41 or in cap-independent translation1,4. Therefore, utilizing m7GTP cap analogs is the best, most reliable method to isolate cap-binding proteins, especially when investigating novel cap-associated proteins. A limitation of utilizing an m7GTP cap analog is that some cap-binding proteins such as the scavenger decapping enzyme DcpS not only interact with the m7GTP mRNA cap, but also require the second base for binding42. Furthermore, decapping enzymes in general, such as the Dcp1/Dcp2 complex, can hydrolyze m7GTP and effectively reduce resin capacity. Therefore, some cap-binding proteins may be lost in this analysis. To bypass these caveats, enzymatically resistant mRNA cap analogs can be utilized. Two non-hydrolyzable cap analogs, mononucleotide m7GpCH2pp or dinucleotide m7GpCH2ppA have been shown to capture DcpS, Dcp1, and Dcp243. These mRNA cap analogs are not available commercially, but must be chemically synthesized de novo. Another limitation of this protocol is that only cap-binding proteins or proteins interacting with cap-binding proteins via "piggybacking" will be captured. While cap-dependent translation initiation is the major form of initiation, cap-independent mechanisms are especially prevalent during conditions of cellular stress1. However, in the context of this technique and emerging trends in the field of translation initiation6-8,24, identifying novel stress-induced factors that participate in cap-dependent initiation is a promising area of research.
Another factor to consider in this protocol is oxygen in the media. Culturing cells in a liquid medium provides a barrier between the cells and the atmosphere. It has been shown that liquid media can take up to 24 hr to equilibrate with the atmospheric gas composition36. Therefore, cells must be cultured for 24 hr in the desired oxygen availability prior to lysis, or the media can be pre-conditioned if shorter incubations are required. Pre-conditioning involves maintaining the media in the hypoxia workstation for at least 24 hr in a sterile culture flask that allows gas exchange. Any oxygenated solution introduced to the cells within the hypoxia workstation could rapidly alter cellular signaling pathways such as the cap-dependent translation initiation that is being examined in this protocol. Due to the sensitivity of the oxygen-sensing pathways in cells, any solution that will interact with cells prior to lysis such as PBS and trypsin-EDTA must also be pre-conditioned for at least 24 hr. Lysis and post-lysis wash buffers must be used cold and are therefore not pre-conditioned in the 37 °C hypoxia workstation. While some oxygen-dependent modifications to proteins could be active post-lysis, the cold buffers are required to minimize enzymatic activities and "shuffling" of protein-protein or protein-RNA complexes that could lead to artefacts. A modification to this protocol to increase stringency may be to pre-condition lysis and post-lysis wash buffers for 24 hr and then incubate them on ice within the workstation before use. Media and buffers should not be kept in the hypoxia workstation for more than three days because these solutions will concentrate due to water evaporation. For longer term studies (>3 days), the initial amount of cells seeded must be carefully considered. As cells divide and the surface area of the dish becomes crowded, other stresses such as media acidification could affect the activity of cap-binding proteins. On the final day of the experiment, cells should have a confluency of 80-90%.
There are four critical steps within this protocol: maintaining the cells in the hypoxia workstation until lysis, the amount of cells used, washing the beads, and the non-methylated GTP control. 1) There are several methods to maintain cells in low oxygen. Most of these include small chambers that can hold only cell culture dishes, but any manipulation or cell lysis must be performed outside the chambers in ambient air. Components of the oxygen-sensing machinery such as the hypoxia inducible factors are activated or inactivated in a matter of one or two minutes35. Therefore, as mentioned previously, lysing the cells in a hypoxia workstation is critical to ensure the activity of the cap-binding proteins is associated with the respective oxygen availability. 2) The number of cells suggested in this protocol (two 150 mm dishes) is adequate for strong m7GTP interactors such as the eIF4E family or members of the eIF4F complex (eIF4A and eIF4G). More cells, at least five 150 mm dishes, could be required to detect proteins with transient interactions or that only associate with the m7GTP mRNA cap through eIF4F. 3) Washing the beads after incubating them with the cell lysate can be performed in a stringent and less stringent manner. The protocol presented here offers a stringent option where lysis buffer is used to wash the beads five times. If proteins with weaker interactions to cap-binding proteins are of interest, one can perform fewer washes and utilize Tris-buffered saline instead of lysis buffer. 4) While it is important to pre-clear the cell lysate with unconjugated agarose to remove proteins that non-specifically interact with the bead, some cap-binding proteins cannot differentiate between the true mRNA cap structure, methylated GTP (m7GTP), and non-methylated GTP. One such protein is the eIF4E homologue eIF4E344. Eluting the beads with GTP will dislodge any protein that also recognizes non-methylated GTP, which could be of interest. The biological relevance of proteins that recognize both m7GTP and GTP has yet to be fully elucidated. This is emphasized by the few publications involving eIF4E3 (which is a bona fide cap-binding protein with a conserved cap-binding domain) relative to the eIF4E and eIF4E2 proteins, which do recognize m7GTP from non-methylated GTP.
This technique, as presented, can be performed as a targeted approach to identify m7GTP interactors of interest or as a broad approach by analyzing the m7GTP eluate via mass spectrometry. Several other techniques can follow physioxic exposure (protocol 4) to analyze cap-binding activity such as subcellular fractionation, immunofluorescence, co-immunoprecipitation, and polysome analysis. Translational control is emerging as an equal contributor to gene expression as transcription. Utilizing this technique to investigate the activity and composition of cap-binding complexes will shed light on how cap-dependent translation initiation is regulated in response to low oxygen.
The authors have nothing to disclose.
This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.
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 |
Dithiothreital | 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 (100 x) | 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 |