Yeast three-hybrid system for detecting and analyzing RNA-protein interactions
Yeast three-hybrid system for detecting and analyzing RNA-protein interactions
The yeast three-hybrid system can be used to analyze protein-RNA interactions. In the yeast three-hybrid system, RNA-protein interactions lead to transcription of reporter genes, and protein-RNA interactions can be detected by cell growth, clone color, or specific enzyme activity. This experiment is derived from the "RNA Laboratory Guidebook" edited by Xiaofei Zheng.
Operation method
Screening of interacting proteins for known RNA sequences
Principle
The yeast three-hybrid system can be used to analyze protein-RNA interactions. In the yeast three-hybrid system, RNA-protein interactions lead to the transcription of reporter genes, and protein-RNA interactions can be detected by cell growth, clone color, or specific enzyme activity.
Materials and Instruments
YPD and SD medium Salmon Sperm DNA PEG 3350 TE buffer LiAc DMSO Z buffer Move I. Introduction of RNA plasmids and cDNA libraries For more product details, please visit Aladdin Scientific website.
Typically, the RNA plasmid is first transformed into the host strain, L40coat, to obtain cells containing the RNA plasmid, and then a cDNA library incorporating the transcription activation region is transformed (hybrid RNAs have little or no toxicity to the host cell, unlike some hybrid protein decoys used in two-hybrid screening, so it is not necessary to cotransform the two plasmids), and the transformed mixture is spread on medium deficient in leucine and histidine (e.g., ADE2 or URA3 selection). (e.g., ADE2 or URA3 selection), and the transformation mixture is spread on medium lacking leucine and histidine, without the need to maintain the RNA plasmid. This allows cells to activate HIS3 in the absence of RNA or loss of RNA plasmid, enabling the use of clonal color screening.
Below are easy methods for yeast receptor cell preparation and transformation of plasmids and libraries.
1. Yeast Sensation Cell Preparation (LiAc Method)
The reagents to be prepared include: YPD and SD medium; various defective amino acid additives: salmon DNA (10 mg/mL, denatured at 100℃ for 20 min before use, and then put on ice immediately); 50% PEG 3350; TE buffer; LiAc (1 mol/L, pH 7.5); DMSO.
(1) Transfer the yeast to the corresponding culture plate (YPD) once 2~3 days in advance and use it within one week.
(2) Pick a few clones of 2~3 mm in diameter and insert them into a large test tube containing 10 ml of YPD, shake vigorously and break them up.
(3) Incubate at 30℃, 230~270 r/min for 16~18 h, and incubate until OD600＞1.5.
(4) Transfer the overnight culture to a triangular flask containing 200 ml of YPD and incubate until the OD600 is 0.2~0.3.
(5) Cultivate at 30℃, 230~270 r/min for 3 h, and incubate until the OD600 is 0.4~0.6.
(6) Dispense the culture into 50 ml centrifuge tubes and centrifuge at 2200 r/min for 5 min at room temperature.
(7) Discard the supernatant and resuspend with 25~50 ml of sterile water or TE.
(8) Centrifuge at 2200 r/min for 5 min at room temperature.
(9) Discard the supernatant and add 1 ml of freshly prepared sterile 1XTE/LiAc. The preparation of sensory cells is completed.
2. Plasmid/library transformation
(1) Prepare 10 ml of PEG/LiAc solution.
(2) Mix the plasmid or library DNA with salmon sperm DNA, add 0.1 ml of receptor cells and shake well.
(3) Add 0.6 ml of PEG/LiAc and shake well.
(4) Cultivate at 30℃, 200 r/min for 30 min.
(5) Add 70 μl of DMSO, turn upside down and mix gently.
(6) Heat-excite at 42℃ for 15 min.
(7) Place on ice for 1~2 min.
(8) Centrifuge at 14000 r/min for 5 s at room temperature.
(9) Discard the supernatant and resuspend the yeast cells with 0.5 ml TE.
(10) Coat the plate and incubate at 30°C for about 3~4 days to grow colonies.
It should be noted that a competitive inhibitor of a product of the HIS3 gene, 3-aminotriazole (3-AT), can be added to the plate to select for stronger interactions. Some RNAs can weakly activate reporter genes on their own, and many proteins can weakly activate reporter genes independently of hybridizing RNA, both of which produce "false positives." To eliminate activation produced by RNA decoys, 3-AT should be titrated with a strain carrying only the RNA plasmid prior to initiating transformation.To reduce the number of protein false positives ("RNA-independent" positives), 3-ATs in the range of 2 to 5 mmol/L are a good starting point.They provide Data on the affinity of RNA-protein interactions in vitro are informative in determining the concentration of 3AT that should be used. In a screen for an SLBP that interacts strongly with its RNA target in vitro, the inclusion of 25 mmol/L 3-AT greatly reduces background.
II. Removal of RNA-independent false positives by clonal coloration
Two classes of positives can be obtained from the starting transformation. One class of transformations requires hybridizing RNA to activate the HIS3 reporter gene; these are called "RNA-dependent." The second class of positives can activate HIS3 in the presence or absence of hybridizing RNA, and these are called "RNA-independent." RNA-independent positives may carry proteins that can bind to the promoter region of the reporter gene or may interact directly with the Lex A-MS2 packaged fusion protein, and such RNA-independent transformations may be very numerous, even accounting for all clones. RNA-independent transformations may be very numerous, even accounting for more than 95% of all clones.
To remove RNA-independent false positives, we used the method of attaching the ADE2 gene to an RNA plasmid. The host strain was a mutant of ade2. When adenylate levels in the culture plate become low, the cells begin to synthesize adenylate and accumulate a reddish purine metabolite due to the absence of the enzyme encoded by ADE2. This accumulation causes the cells to appear pink or red, whereas cells carrying the bison ADE2 gene are white.
In the initiating transformation, only the activating HIS3 is selected, not the maintaining RNA plasmid, and for RNA-dependent positivity, HIS3 selection is not a direct selection of the RNA plasmid that carries the ADE2 gene; these transformants are therefore white, and will remain so if they continue to be grown in the absence of an exogenous His. However, non-dependent positives do not require the RNA plasmid to activate the HIS3 gene, so that plasmids working at a frequency of a few percent per generation may be lost, and so these false positives generate pink colonies or white colonies that are partially pink.
The starting transformation plates are usually incubated at 30°C for one week. This time allows the positive clones to accumulate the HIS3 gene product and grow, and provides enough time to produce color. If the pink color is not strong after one week, an overnight stay at 4°C may enhance the effect. Discard pink or partially pink clones and select completely white colonies for further analysis. We usually select all white colonies (typically a few large, many small clones) and again select clones containing both cDNA and RNA plasmids on selective media. Most of the small white clones are RNA-independent and cannot grow. White clones that can grow on selective medium are used for further analysis.
Identifying RNA-dependent positives by clone color is not perfect; many RNA-independent positives can be identified and discarded. Most white clones are probably RNA-independent. It is important to rigorously remove the remaining RNA-independent activators from the white clones before recovering the plasmid in E.coli.
Analysis of β-Gal Activity
In order to confirm that the white clone containing the cDNA is activated by yeast three-hybridization, the expression level of the lacZ gene needs to be examined. In the L40coat strain, the lacZ gene is integrated into the chromosome under the control of the Lex-A binding site.
Analysis of β-Gal activity can be analyzed by detecting the conversion of lactose to a luminescent product, using clones immersed in filter paper or cell lysates. The filter paper assay produces qualitative results, while the liquid assay is more quantitative.
Reagents to be prepared include, Z buffer: 60 mmoI/L Na2HPO4-7H2O, 40 mmol/L NaH2PO4-H2O, 10 mmol/L KCl, 1 mmol/L MgSO4-7H2O, 50 mmol/L β-mercaptoethanol, pH 7.0, autoclaved; Z buffer/X-Gal solution: 100 Z buffer/X-Gal solution: 100 ml Z buffer, 0.27 ml β-mercaptoethanol, 1.67 ml X-Gal reservoir.
1. Qualitative (filter paper) assay
(1) Scratch clones from Step 2 "Removal of RNA-independent false positives by clone color" onto suitable selective medium (SD-Leu-Ura) and incubate overnight.
(2) Duplicate clones onto plate-sized nitrocellulose membrane or filter paper (Whatman, 3 MM ).
(3) Treat for 20 s in liquid nitrogen.
(4) Dissolve the membrane, colony side up, at room temperature (about 2 min ).
(5) Cut several pieces of filter paper of the same size as the Petri dish and saturate the filter paper with Z buffer/300 μg/ml X-Gal. X-Gal should be used fresh and excess buffer should be removed.
(6) Place the filter membrane on the saturated filter paper and seal the plate with a sealing film.
(7) Incubate at 30°C overnight and inspect the membrane periodically to observe color change.
Strong interactions (e.g., TRE and IRP) will turn blue within 30 min, and weak interactions will eventually produce a blue color if the incubation time is prolonged. For this reason, it is important to check the membrane regularly to determine how long it takes for the color to appear.
2. Quantitative (liquid) assay (ONPG method)
The specific activity of β-Gal in yeast cell lysate can be measured using different substrates. It is usually analyzed by ONPG or CPRG (Chlorophenol Red-D-Galactopyranoside) colorimetric methods, CPRG being more sensitive but more expensive. Alternatively, luminescent substrates provide high sensitivity and are not expensive. The following method applies the luminescent substrate Galacton-Plus ( Tropix, Bedford, MA), which requires a device to detect luminescence. The following procedure was applied to a Monolight 2010 luminometer (Analytic Luminescent Laboratories, CA). The details of this analysis, such as sample volume, vary with the instrument used.
(1) For each set of interactions to be tested, culture 3 single colonies of yeast into 5 ml of selective medium (SD/Leu Trp His) and shake overnight at 30°C, 230~250 r/min.
(2) Incubate until the OD600 value is about 0.8.
(3) For each set of precipitate equivalent to 1.0 OD cells, resuspend with 100 μl lysis buffer (100 mmol/L potassium phosphate, pH 7.8, 0.2% Triton X-100 ).
(4) Freeze-thaw lysed cells. After immersion in liquid nitrogen for 30 s, the cells were placed at 37°C for 90 s for 3 consecutive cycles.
(5) Take 100 μl of the bacterial solution into a 1.5 ml centrifuge tube and set up a blank control tube (100 μl of Z buffer).
(6) Briefly mix the tubes.
(7) Centrifuge at 12000 g.
(8) Collect the supernatant for luminescence analysis and store the sample at -70°C if necessary.
(9) Add 10 μl of lysate to the tube of the luminometer. For strong interactions, it is necessary to dilute the lysate to ensure that it is analyzed within the linear range.
(10) Dilute Galacton substrate 1:100 with reaction buffer (100 mmol/L sodium phosphate, 1 mmol/L magnesium chloride, pH 8.0). Add 100 μl to each luminometer tube.
(11) Incubate at 25°C for 60 min.
(12) Determine the luminescence directly by luminometer.
(13) Determine the protein concentration in the lysate by Bradford or equivalent method.
(14) Protein concentration is normalized to chemiluminescence to obtain specific activity.
Purified RNA plasmid re-testing
In step 2, "Removing RNA-independent false positives by clone color", clone color screening can eliminate most, but not all, RNA-independent false positives. To determine that a positive is indeed RNA-dependent, the RNA plasmid needs to be screened for URA3 resistance. The expression of the reporter gene is then tested and candidate molecules that fail to activate the reporter gene are further analyzed.
To screen out cells that have lost the RNA plasmid, cells can be spread on plates containing 5-FOA (5-fluorouracil acid), which can be converted to the toxic 5-fluorouracil by the product of the URA3 gene. Cells lacking the URA3 gene product can be grown on plates containing 5-FOA, while those containing the URA3 gene product cannot. Cells lacking the URA3 gene product can grow on plates containing 5-FOA, while those containing the URA3 gene product cannot.
1. Pick positive clones from Step 2, "Remove RNA-independent false positives by clone color", onto SD-Leu plates, and grow for one day so that the cells lose the plasmid.
2. Replicate onto SD-Leu plates containing 0.1% 5-FOA and incubate at 30°C for several days.
3. Growing cells are streaked onto SD-Ura plates to determine loss of RNA plasmid. Screening for resistance to 5-FOA by simple 5-FOA is usually effective.
4. β-Gal activity is analyzed.
The ADE2 marker on the RNA plasmid is useful in detecting RNA plasmid loss. Cells that have lost the RNA plasmid may turn pink after a few days. If the number of positive clones is small, the more expensive 5-FOA can be dispensed with and the cells can be cultured directly in nutrient-rich medium overnight and then spread on SD-Leu plates. After a few days some clones will turn pink or partially pink. Clones that are uniformly pink are missing RNA plasmid and can be analyzed for β-Gal activity.
V. Determining Binding Specificity with Mutant and Control RNAs
In order to determine the specificity of RNA binding, re-introduce plasmids encoding different hybridization RNAs into the strain. If there are fewer positive clones, different RNA plasmids can be transformed into the strain, while the rest of the plasmids are generally introduced by the mating method, which can be done with the R40 coat strain. The following is a simple procedure for the mating method.
1. Culture R40coat transformant colonies carrying specifically hybridized RNA plasmids (e.g. mutant to wild type) on SD-Ura plates.
2. Copy the Ura clone from the 5-FOA plate to the YPD plate by the grid method.
3. Replicate each R4coat colony to the same YPD plate.
4. incubate at 30°C overnight for mating.
5. Replicate plates onto SD-Leu-Ura plates for diploid selection.
6. β-Gal live pieces were analyzed.
RNA is used for specificity analysis. Step 4 "Purified RNA Plasmid Reassay" Surviving positive clones carry proteins that preferentially bind hybridizing RNA relative to cellular RNA. Although these positives recognize some features of the hybridized RNA, they are not necessarily factors associated with biological activity. Thus, the ideal control is a fine mutation affecting biological function or affecting interactions in a sequence-specific manner. In some cases, antisense decoys can be used as secondary structure or base composition controls.
True RNA-dependent grading of positive clones. Initially, the physiological relevance of positive clone grading is unpredictable because of the many functions of the proteins (when randomly screening libraries), the strength of the interactions, the concentration of 3-AT used in the starting transformation, and other parameters.
What if there are no tiny RNA mutations available? Another method must be devised to identify these positive clones. Obviously, functional testing is ideal, but in many tissues and systems it is time-consuming and labor-intensive. cDNA sequences can be used to obtain information by directly comparing them to known RNA-binding proteins, or to an expected molecular mass based on e.g. UV cross-linking. Since each case is specific, we will not provide a generalized discussion here. However, it must be recalled that secondary screening is critical.
Identification of positive cDNAs
1. Raise a cDNA plasmid from yeast cells that shows the expected RNA binding specificity and transform it into E.coli. Yeast cells may contain multiple cDNA plasmids, only one of which encodes a protein that binds RNA. therefore, plasmids should be extracted from multiple E.coli transformants and retransformed into yeast to ensure that the correct plasmid is obtained. Below is a simple how-to, with all steps performed at room temperature.
(1) Toothpick yeast monoclones into 50 μl of lysis solution [ 2% Triton X-100, 1% SDS, 100 mmol/L NaCl, 10 mmol/L Tris-Cl (pH 8.0), 1 mmol/L EDTA].
(2) Add 50 μl of phenol: chloroform and 0.1 g of acid-washed glass beads.
(3) Mix and spin at high speed for 2 min.
(4) Centrifuge at high speed for 5 min.
(5) Transfer the supernatant to a clean tube and ethanol precipitate the DNA. 70% ethanol wash and dry the precipitate.
(6) Resuspend the precipitate with 10 μl of water. Electrotransform E.coli with 1 μl of water.
2. In order to easily determine how much plasmid is in a yeast transformant, we usually use a lysed yeast clone for PCR amplification, using the flanking sequences of the inserted fragments as primers. The following is the procedure for PCR of yeast clones.
(1) Touch the yeast clone with a sterile tip.
(2) Soak the tip in 10 μl of reaction buffer [ 1.2 mol/L sorbitol, 100 mmol/L sodium phosphate (pH 7.4), 2.5 mg/ml zymolysis enzyme ], blow up and down several times, and incubate at 37℃ for 5 min.
(3) Apply 1 μl to 20 μl of the system for the PCR reaction. If desired, the PCR product can be purified by chromatography or in-gel and sequenced directly.
VII. Functional or other screening
Additional steps are often required to characterize the biological significance of those positive clones. As noted earlier (Step 5, "Determine Binding Specificity with Mutant and Control RNAs"), these screens are unique and dependent on the interaction and the organization being studied. It is not surprising that an unrelated RNA-binding protein would be identified in a screen with RNA decoys, but rather a plausible interacting molecule.