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May 2021

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An Aptamer-based mRNA Affinity Purification Procedure (RaPID) for the Identification of Associated RNAs (RaPID-seq) and Proteins (RaPID-MS) in Yeast
基于适体的 mRNA 亲和纯化程序 (RaPID) 用于鉴定酵母中的相关 RNA (RaPID-seq) 和蛋白质 (RaPID-MS)   

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Abstract

RNA-RNA and RNA-protein interactions are involved in the regulation of gene expression. Here, we describe an updated and extended version of our RNA purification and protein identification (RaPID) protocol for the pulldown of aptamer-tagged mRNAs by affinity purification. The method takes advantage of the high affinity interaction between the MS2 RNA aptamer and the MS2 coat protein (MCP), as well as that between streptavidin-binding peptide (SBP) and streptavidin. Thus, it employs MCP-SBP fusions to affinity purify MS2-tagged target RNAs of interest over immobilized streptavidin. Purified aptamer-tagged mRNAs, along with any associated RNAs and proteins, are then sent for RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS), which allows for the identification of bound cohort RNAs and proteins, respectively.


Keywords: MS2 (MS2), RNA aptamer (RNA适体), RNA affinity purification (RNA亲和纯化), RaPID (RaPID), Streptavidin-binding peptide (链霉亲和素结合肽), Yeast (酵母)

Background

RNA-binding proteins (RBPs) and RNA-associated proteins regulate every step in the gene expression pathway. There are also frequent occasions of regulation via RNA-RNA interactions (Guil and Esteller, 2015). Thus, identifying protein-RNA and RNA-RNA interactions is key to understanding RNA biology.


A wide list of methods is available to study RNA-protein interactions. These are classified further as RNA-centric methods or protein-centric methods. RNA-centric approaches characterize proteins bound to a specific RNA of interest, while RNAs bound to a specific protein are studied in protein-centric methods (Ramanathan et al., 2019). RNA-centric approaches use both in vitro and in vivo methods. The simplest method of RNA tagging and pulldown is either by 5′- or 3′-end RNA biotinylation (Zheng et al., 2016), or S1 aptamer tagging used for the in vitro pull down of an RNA of interest with immobilized streptavidin. In vivo methods include RNA affinity purification (RAP) (Hacisuleyman et al., 2014; McHugh and Guttman, 2018), tandem RNA isolation procedure (TRIP) (Matia-González et al., 2017), MS2 in vivo biotin-tagged RAP (MS2-BioTRAP) (Tsai et al., 2011), and CHART (Capture Hybridization Analysis of RNA Targets) (Simon et al., 2011). RAP has been used to study non-coding RNA, such as Xist (McHugh et al., 2015), whereas TRIP is used to study polyadenylated RNA (Matia-González et al., 2017). MS-BioTRAP employs the use of the MS2 aptamer system, in which the MS2 stem loop-tagged RNA and the MS2 coat protein are ectopically expressed (Tsai et al., 2011); however, their over-expression may not reflect the normal physiological level of RNA. CHART capture oligonucleotides are designed to specifically hybridize to the RNA of interest (Simon et al., 2011). In contrast to the RNA-centric approach, cross-linking immunoprecipitation (CLIP) is the most common protein-centric method used to characterize RNAs bound to a protein of interest (Ule et al., 2003; Licatalosi et al., 2008). While all potentially effective, the described methods have not been combined to study RNA-RNA and RNA-protein interactions together.


In addition to these methods, there are several transcriptome-wide approaches to study RNA-RNA and RNA-protein interactions in vivo and in vitro. Approaches like PARIS (psoralen analysis of RNA interactions and structures) (Lu et al., 2018), SPLASH (sequencing of psoralen crosslinked, ligated, and selected hybrids) (Aw et al., 2017), LIGR-seq (ligation of interacting RNA followed by high-throughput sequencing) (Sharma et al., 2016), and MARIO (mapping RNA interactome in vivo) (Nguyen et al., 2016) are used to study transcriptome-wide RNA-RNA interactions, but cannot be used to study the interactions of a specific transcript.


We developed RaPID (RNA purification and identification), which is an MS2 aptamer-based mRNA affinity purification technique (Slobodin and Gerst, 2010 and 2011). The method allows for the specific isolation of MS2 aptamer-labeled mRNAs from yeast or mammalian cells (Slobodin and Gerst, 2010 and 2011), and subsequent analysis of associated RNAs and proteins (Figure 1) using RNA seq and mass-spectrometry, respectively. The advantage of RaPID is the use of the high affinity interaction between the MS2 aptamer and MS2 coat protein (MCP), as well as that of the streptavidin-binding peptide (SBP), which is fused to MCP, to streptavidin beads (Slobodin and Gerst, 2010 and 2011). Hence, the method allows for the specific purification of MS2-tagged mRNAs and their associated proteins and RNAs from cells.


The RaPID-mass spectrometry (RaPID-MS) and RaPID RNA sequencing (RaPID-seq) protocols presented here are updated and extended versions of the protocols previously described by Slobodin and Gerst (2010 and 2011), and Haimovich et al. (2016), which were used to analyze RNA-protein and RNA-RNA interactions in yeast cells, respectively. Using these methods (see Figure 2), we identified both known (Slobodin and Gerst, 2010 and 2011) and novel (Zabezhinsky et al., 2016) RNA-protein interactions, as well as the phenomenon of mRNA multiplexing (Nair et al., 2021). Briefly, the endogenous yeast gene of interest is tagged with the MS2 aptamer (Haim et al., 2007; Haim-Vilmovsky and Gerst, 2009), and the cells transformed with a plasmid expressing MCP fused to both GFP and SBP (MCP-GFP-SBP). The cells are cultured under the required experimental conditions, following which MCP-GFP-SBP expression is induced and the cells fixed to preserve the ribonucleoprotein (RNP) complexes. Next, the cells are lysed and the RNP complexes precipitated by affinity purification using streptavidin-conjugated beads. The associated RNAs and proteins are then analyzed using RNA-seq and mass spectrometry, respectively.



Figure 1. Schematic of the RaPID strategy.

Cells co-expressing the MS2 aptamer-tagged RNA and MCP-GFP-SBP binding protein are fixed and lysed, as described in the text. Following which, the lysates are incubated with immobilized streptavidin beads and the MS2 aptamer-tagged mRNA along with associated untagged RNAs and RNA-binding proteins (RBPs) is precipitated. Protein and RNA extraction of the precipitates is then performed to identify RBPs by mass spectrometry and RNAs by RNA-seq or qRT-PCR.



Figure 2. Schematic flowchart of the RaPID-seq/mass-spectrometry procedure.

Each major step is presented by graphical representation. A. Preparation of cells bearing the MS2 aptamer tagged mRNA and MCP-GFP-SBP. B. Cell culture. C. Induction of MCP-GFP-SBP protein expression. D. Crosslinking, harvesting, freezing of cells. E. Lysis, measurement of protein concentration. F. Add beads, incubation. G. Centrifugation, washes. H. Crosslink reversal, elution. I. Sample for RNA-seq. J. Library prep and sequencing. K. Analysis of gene expression. L. Sample for mass spectrometry. M. SDS-PAGE and silver staining. N. Mass spectrometry.

Materials and Reagents

  1. Yeast strains

    Wild-type strains used successfully with this procedure include BY4741 (EUROSCARF; MATa his3D1 leu2D0 met15D0 ura3D0), although any S. cerevisiae lab strain should suffice, provided it is mutated in the HIS3 and URA3 genes. Yeast can be stored at 4°C for up to 2 weeks on plates, or indefinitely when frozen at -80°C.


  2. Reagents and disposables

    1. 17-gauge needle or 23-gauge needle (any vendor)

    2. 1.7 mL plastic tubes (autoclaved) (any vendor)

    3. Sterile 15-mL polypropylene centrifuge tubes (any vendor)

    4. Sterile Nuclease-free Barrier tips (10 µL, 200 µL, 1,000 µL) (any vendor)

    5. Sterile 50 mL polypropylene centrifuge tubes (Greiner, catalog number: 227270)

    6. Glass beads, 0.5 mm in diameter (Biospec products, catalog number: 11079-105)

    7. Ultra-pure water (Biological Industries, catalog number: 01-866-1B)

    8. Sterile double distilled water (DDW)

    9. Lithium acetate dihydrate (LiOAc) (Sigma, catalog number: L6883)

    10. Salmon sperm DNA (ssDNA) (Sigma, catalog number: D1626)

    11. Ultrapure water (Molecular Biology Grade Water, nuclease free) (Sigma, catalog number: W4502)

    12. Ethanol absolute (Bio Lab, catalog number: 05250521)

    13. Formaldehyde 37% (Sigma, catalog number: F8775-25ML)

    14. Glycine free base (Sigma, catalog number: G7126)

    15. Liquid Nitrogen

    16. Recombinant Rnasin Ribonuclease Inhibitor (Promega, catalog number: 20006332)

    17. cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail ×25 (Roche, catalog number: 11836170001)

    18. BCA protein assay kit (Pierce, catalog number: 23225)

    19. Streptavidin-conjugated SepharoseTM beads (GE Healthcare, catalog number: 17-5113-01)

    20. Yeast tRNA (Sigma, catalog number: R8508)

    21. Avidin solution (Sigma, catalog number: A9275)

    22. Bovine Serum albumin (BSA), lyophilized powder, ≥96% (Sigma, catalog number: A7906)

    23. Biotin (Sigma, catalog number: B4501)

    24. Dimethylsulfoxide (DMSO) (Sigma, catalog number: D8418)

    25. MPC protein precipitation reagent (a component of the MasterPureTM Yeast RNA purification kit; Epicentre Biotechnologies, catalog number: MPY03100)

    26. Glycogen 20 mg/mL solution (Fermentas, catalog number: R0561)

    27. 3 M sodium acetate (NaOAc) solution, pH 5.2 (Fermentas, catalog number: R1181)

    28. qScript Flex cDNA Kit (Quantabio, catalog number: 95049-100-2)

    29. DNaseI (Quantabio, catalog number: 95150-100)

    30. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma, catalog number: T9281-25ML)

    31. 30% acrylamide/Bis 29:1 (Bio-Rad, catalog number: 161-0156)

    32. Silver Stain Kit (Pierce, catalog number: 24612)

    33. TE buffer (see Recipes)

    34. 1 M LiOAc (see Recipes)

    35. 0.1 M LiOAc (see Recipes)

    36. PEG solution (see Recipes)

    37. ssDNA (see Recipes)

    38. Selective medium (see Recipes)

    39. Glycine solution (see Recipes)

    40. Yeast lysis buffer (see Recipes)

    41. Complete yeast lysis buffer (see Recipes)

    42. Yeast tRNA (see Recipes)

    43. Avidin solution (see Recipes)

    44. Yeast tRNA (see Recipes)

    45. BSA solution (see Recipes)

    46. Washing buffer (see Recipes)

    47. Biotin solution (see Recipes)

    48. 2× Cross-link reversal buffer (see Recipes)

    49. 5× Protein sample buffer (see Recipes)


  3. Plasmids

    1. pUG34-MS2-CP-GFP-SBP (aka pMCP-GFP-SBP) (Slobodin and Gerst, 2010)

    2. pMS2-SL plasmid (Haim-Vilmovsky and Gerst, 2009)

Equipment

  1. 1 L flask

  2. 3 L Erlenmeyer flask

  3. -80°C freezer

  4. Sterile surgical blades for the excision of protein bands from gel (Jai Surgical Blades, catalog number: 400-11)

  5. Orbital shaker incubator (MRC, TOU-120)

  6. Pipette aid (recommended: S1 pipet filler, Thermo Fisher Scientific, catalog number: 9501)

  7. (Optional) Vacuum trap

  8. Chemical (fume) hood

  9. Ultrospec 10 Cell Density Meter (Biochrom, catalog number: 634-0882)

  10. Rotator/Mixer Intelli-Mixer (Thomas Scientific, catalog number: RM-2M)

  11. Benchtop refrigerated centrifuge (Eppendorf, catalog number: 5810 R)

  12. Benchtop refrigerated microcentrifuge (Eppendorf, catalog number: 5417 R)

  13. Centrifuge (refrigerated) suitable for large volumes (Sorvall, RC5C plus with SLA3000 rotor) or equivalent centrifuge

  14. Bottles for Sorvall centrifuge, 500 mL (e.g., Thermo, Nalgene, catalog number 10430613)

  15. Digital Disruptor Genie (Scientific Industries, catalog number: SI-DD38 120V). This should be pre-cooled in a cold room prior to the experiment.

  16. Standard equipment for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments

  17. Sterile box capable of accommodating acrylamide gels

Software

  1. Proteome Discoverer (version 1.4)

Procedure

  1. MS2 tagging and growing the yeast cell culture

    The MS2 aptamer is a short RNA stem-loop (SL) structure originating from the MS2 bacteriophage. MS2 tagging, i.e., inserting 12-24 repeats of the MS2-SL, allows the visualization and pulldown of endogenously tagged mRNAs using MCP fused to a fluorescent protein, like GFP (Figure 2). Addition of the SBP moiety allows for the pulldown of MCP-GFP using immobilized streptavidin. However, to identify nonspecific signals during the pull-down experiments several controls are needed, such as untagged cells and cells tagged with a control mRNA.

    1. Tag the gene of interest with MS2 aptamer a multiple binding sites for the RNA-binding MS2 coat protein (MCP), using a PCR-based genomic-tagging strategy and homologous recombination as previously described (Haim et al., 2007; Haim-Vilmovsky and Gerst, 2009). See also Note 1.

    2. Transform the strain having the gene tagged with MS2 aptamer with plasmids expressing the MCP-GFP-SBP using standard yeast LiOAc transformation (Gietz and Schiestl, 2007), and plate on SC-His synthetic media agarose plate.

    3. Pick a single colony that has been transformed with MS2-CP-GFP-SBP containing the HIS3 selection marker, inoculate it into a 50-mL test tube with 7-8 mL of selective medium (SC-His), and grow in a shaking incubator at 30°C for 6-10 h.

    4. Dilute to 500 mL culture for an overnight culture. For cells at mid-logarithmic phase, the final OD600 the next morning should be between 0.4-0.8. Cultures at different OD600 values can be used if other cell states are required, but further steps may need modification according to the amount of cells obtained. To calculate the starter culture volume to take, follow these steps:

      1. Measure the cell number or OD600 of a culture and determine the doubling time using the formula below. For the wild-type strain (BY4741) used in this experiment, the doubling time was 2 h.


        [log10 (Nt/N0)]/log10(2) = g

        g – number of generations

        N0 – # of cells or OD600 at start

        Nt – # of cells or OD600 at the end

        t = time cultured

        d – doubling time (d=t/g)


      2. Determine the amount of starter culture to add to a larger, overnight culture using this formula:


        N0 – OD600 at which to start

        Nt – OD600 that you want at the end

        g – number of generations the culture will go through before harvesting

        N0 = Nt/2g

        g = t/d


    5. After overnight growth, measure absorbance of the culture and, if needed, keep growing the culture at least up to OD600  = 0.4. Collect the cells by centrifugation at 1,100 × g for 5 min at 4°C, using a SLA3000-type rotor in a Sorvall centrifuge, and discard the growth medium.

    6. Resuspend the cell pellet by gentle pipetting (do not vortex) in 200 mL of fresh growth medium lacking methionine, transfer to 1 L flask, and incubate at 30°C for 45 min with shaking to induce the expression of the MS2-CP-GFP-SBP. See also Note 2.

    7. Fix the cells with formaldehyde by directly adding 270 μL of formaldehyde to a final concentration of 0.05%, and incubate at room temperature (RT) with slow shaking for 10 min.

    8. Terminate the cross-linking reaction by adding 25 mL of glycine solution to a final concentration of 0.125 M and incubate for an additional 2 min.

    9. Centrifuge the cells, discard the supernatant according to chemical waste procedures at your institute, and resuspend the cells using a pipette (do not vortex) in 10 mL of PBS in a 50 mL tube.

    10. Centrifuge the cells at 2,500 × g for 5 min at 4°C.

    11. Discard the PBS completely and keep the tube inverted for 1-2 min on tissue paper to remove residual PBS.

    12. Quickly freeze the cell pellet using liquid nitrogen. For storage, transfer the cells to a -80°C freezer. The cells may be stored for prolonged periods under these conditions.


  2. Yeast Cell Lysis

    Note: All the steps mentioned below should be performed on ice.

    1. Place the frozen cell pellet on ice and thaw by adding 4 mL of complete yeast lysis buffer.

    2. Once the cells are thawed, transfer 0.7 mL into 1.5 mL microcentrifuge tubes prefilled with a volume of 0.5 mL of acid-prewashed and oven-baked glass beads.

    3. Vortex the cells using the disruptor for 20 min at 4°C at maximum speed. Turn off the shaker and let stand for 5 min to prevent excessive heating, then turn on for an additional 20 min.

    4. Centrifuge microfuge tubes in a bench-top centrifuge precooled to 4°C at 1,000 × g for 2 min. Transfer the supernatant into fresh microfuge tubes, and centrifuge again at 15,000 × g for 10 min.

    5. Collect the supernatants from each sample (i.e., each individual culture) into disposable 15 mL test tubes. Around 4 mL of supernatant is obtained after centrifugation. Measure the protein concentration with the BCA protein assay kit according to the manufacturer’s instructions.

    6. Transfer the total amount of protein extract desired for RaPID-seq/MS (typically 80-100 mg per pulldown are used for an RaPID-seq or RaPID-MS experiment, whereas 5-10 mg are used for simple pulldowns for qRT-PCR or Western analysis) to a fresh 15 mL tube, add the avidin solution (10 μg of avidin per 1 mg of protein extract), and incubate at 4°C for 1 h with constant rotation/shaking. This step is done to block any biotinylated protein from binding to the streptavidin beads. See Notes 3 and 4 regarding protein measurements and the required amounts for each type of experiment.

    7. Separate the amounts desired for the isolation of RNA and protein into different tubes. In addition, place aside two tubes, each with 1% of initial amount of total extract. One tube is used for “input RNA” (proceed to Step 14 for RNA isolation), and can be used to normalize for gene expression when performing either qRT-PCR or Nanostring experiments (see Note 5). The other tube will be used for “input protein” (proceed to Step 15). These samples are kept on ice and processed in parallel to the eluate, or can be kept frozen at -80°C until used.

    8. In parallel, aliquot 30 µL of streptavidin-conjugated beads. Wash the beads twice by adding 1 mL of ice-cold PBS; once in lysis buffer, swirl (do not vortex) 2-3 times, and centrifuge at 1,000 × g. Block the beads for 1 h at 4°C with 2% BSA and tRNA (0.1 mg per 100 μL of beads) prepared in lysis buffer. After 1 h, centrifuge and wash once with lysis buffer.

    9. Discard the buffer from the washed beads, add the avidin-blocked total cell extract to the beads along with 0.1 mg of yeast tRNA, and incubate at 4°C for 2 h to overnight with constant rotation.

    10. Centrifuge the tubes at 1,000 × g for 2 min and remove the supernatant. Transfer the beads to 1.7 mL tubes and wash them three times with lysis buffer and twice with washing buffer, with centrifugation (as above) between washes.

    11. Perform a last wash in ice-cold PBS and remove the excess buffer.

    12. Add 150 μL of the biotin elution solution to the beads, and incubate for 1 h at 4°C with rotation, for elution of the mRNA-protein/RNA complexes from the beads.

    13. Centrifuge at 1,000 × g for 2 min, transfer the eluate into a fresh microfuge tube, centrifuge once again, and transfer the eluate into a new tube to assure that no beads were carried over.

    14. To reverse crosslink the fraction destined for RNA isolation, add an equal volume of the cross-link reversal buffer to the eluate and incubate at 70°C for 45 min.

    15. To reverse crosslink the fraction destined for protein isolation, add an appropriate volume of the 5× protein sample buffer to reach the 1× concentration, and incubate at 70°C for 45 min.

    16. De-crosslinked RNA and protein samples can be kept at -20°C.

Data analysis

  1. Analysis of precipitated RNA

    1. After reverse crosslinking, add 175 μL of MPC protein precipitation reagent to each 300 μL of eluate and vortex vigorously for 10 s.

    2. Centrifuge for 10 min at 4°C at ≥10,000 × g.

    3. Carefully transfer the supernatant into a new microfuge tube. Add glycogen (80 μg/mL final concentration) and NaOAc (0.3 M final concentration), and vortex thoroughly for 20 s. Add an equal volume of isopropanol, mix by inverting the tubes ~20 times, and incubate overnight at -20°C.

    4. Centrifuge the tubes (12,000 × g for 10 min at 4°C). The pellet containing the RNA should be visible. Carefully discard the supernatant.

    5. Wash the pellet with 70% ice-cold ethanol. Centrifuge for 5 min at 4°C at ≥10,000 × g. Carefully discard the supernatant.

    6. Heat the tube in a dry bath at 50°C for 10 min with an open cap, to evaporate residual ethanol. Do not over-dry, since this will make the pellet hard to dissolve.

    7. Dissolve the pellet in 30 μL of Ultra-pure water. Heat at 50°C for 5-10 min to achieve complete solubility. Store the dissolved RNA in -20°C or -80°C freezers for long term storage.

    8. Construct the library for RNA-seq as previously described (Levin et al., 2010). Oligo(dT) selection is optional, depending upon whether polyA+ RNAs alone are desired or not.

    9. Reverse transcribe RNA with SuperScript III (Invitrogen) at 55°C, and amplify the cDNA with Herculase (Stratagene) in the presence of 5% DMSO for 16 cycles of PCR, followed by a cleanup with 1.8× volumes of AMPure beads (Beckman-Coulter, IN).

    10. Sequence the library to a depth of ~107 reads using standard methodologies (e.g., we used an Illumina HiSeq2000 sequencer with paired-end 76 base reads; Nair et al., 2020).


  2. Data analysis of RaPID RNA seq

    1. Estimate the gene expression levels from the RNA-Seq data using Kallisto (Bray et al., 2016) and targeting the Saccharomyces cerevisiae (S. cerevisiae) reference transcriptome (derived from the Saccharomyces Genome Database and leveraging the Saccharomyces cerevisiae S288C genome version R64-2-1). An example of the read counts acquired for various RNAs for different MS2-tagged mRNAs is provided in Figure 3A.

      Note: Consider the 100 bases downstream of the annotated 3' UTR of each gene as part of the mRNA, in order to cover possible unannotated 3’UTR regions.

    2. Identify the differentially expressed genes. We used edgeR (Robinson et al., 2009), with the dispersion parameter manually set to 0.1. Those genes reported as at least four-fold differentially expressed (FDR < 0.001) were retained as significantly differentially expressed. After log transformation of the data [log2(TPM +1)], the gene expression values from the control sample are subtracted from the experimental samples. Expression quantitation, differential expression, and plotting of heat maps were facilitated through use of the transcriptome analysis modules integrated into the Trinity software suite (Haas et al., 2013). It is difficult to determine in advance how many RNAs are enriched with a specific MS2-tagged bait mRNA, since it depends on the MS2-tagged mRNA, the yeast strain used (e.g., MATa or MATα, or a specific deletion strain), and the growth conditions employed. From our experience, we have found up to ten interacting RNAs identified by RaPID-seq. It is important to validate these results by other methods (e.g., co-smFISH, RaPID pulldowns followed by qRT-PCR). We successfully identified and validated such RNA-RNA interactions for various mRNAs (e.g., encoding mating factors, heat-shock proteins, MAP kinases etc.).



      Figure 3. Examples of read counts from RaPID-seq with various MS2-tagged mRNAs and a silver stain gel for RaPID-MS.

      (A) Shown is a screenshot of the “Counts.matrix” excel sheet from Supplementary File 1 from Nair et al. (2021). It shows the number of reads assigned to each gene (gene names are in column A) for various MS2-tagged target mRNAs (column headers). Cntrl – control untagged strain. rRNA, tRNA and snRNAs were filtered out. The sheet (as shown) is sorted from largest to smallest for EXO70 mRNA (column L). Blue labeled cells show mating-related genes that are enriched in RaPID-seq pulldowns of EXO70, SRO7 and OM45. Note that the MS2-tagged EXO70 target mRNA is highly enriched for its own RNA. Many RNAs are common to all strains, including the untagged control cells, and are therefore considered as background. Only a few chosen genes are shown out of ~7,100 genes identified. (B) Silver stain of SDS-PAGE gel for RaPID-MS (reproduced from Nair et al., 2021). Yeast strains expressing MS2 aptamer-tagged MFA1, MFA2, and STE2 either with or without their 3’UTRs, along with ASH1 were grown and processed for RaPID-MS. The arrow indicates a band of ~150 kDa that exists in the STE2 lane (and all MFA1 and MFA2 lanes), but not in the ASH1 or STE2 ∆3’UTR lane. This band was cut from the gel and sent for MS. Blue square – indicates the band of MCP-GFP-SBP. kDa – kilodaltons.


  3. Analysis of precipitated protein

    1. To identify unknown proteins, there are two alternatives. The first is to separate the proteins on a medium-sized 1.5 mm thick gel (e.g., 20 cm × 15 cm, Bio-Rad) 9% SDS-PAGE gel. Use a comb that will allow loading an entire sample volume per lane (about 180 µL).

    2. Perform silver staining using a Silver Stain Kit (Pierce) according to manufacturer’s instructions. It is fully compatible with subsequent mass-spectrometry analysis.

    3. Analyze the staining results. If specific bands are detected (see Figure 3B), excise them with a sterile blade and send to mass-spectrometry for identification.

    4. The second possibility is to send the entire sample for mass spectrometry.

    5. We recommend discussing protein sample preparation and submission, peptide measurements, and data analysis with personnel of the selected proteomics facility prior to the experiment. In Section D we provide some details to allow a basis for discussion.


  4. Data analysis of RaPID-MS

    Reversed-phase nano-liquid chromatography-electrospray ionization tandem mass spectrometry and data analysis was done at the Israel National Center for Personalized Medicine (Weizmann Institute, Rehovot, Israel). The gel slice was processed and trypsinized at the core facility. A nanoUPLC (10 μm tip; New Objective; Woburn, MA, USA) coupled to a quadrupole orbitrap mass spectrometer (Q Exactive HF) using a FlexIon nanospray apparatus (Proxeon) was used. The parameters used are: MS1 resolution was set to 120,000 (at m/z 200), mass range of m/z 300-1650, AGC of 3e6 and maximum injection time was set to 60 ms. MS2 resolution was set to 30,000, quadrupole isolation m/z 1.7, AGC of 1e5, dynamic exclusion of 20 s, and maximum injection time of 60 ms. Data is processed at the core facility using Proteome Discoverer (version 1.4), with two search algorithms – SequestHT and Mascot, using the UniprotKB yeast protein database. A list of common lab contaminants is added and the following variable modifications as well: Carbamidomethyl on C, Oxidation on M, and N-terminal acetylation. Data is filtered for maximum 1% false discovery rate.


    By using RaPID-MS, we successfully identified and validated novel proteins that interact with the MS2-tagged mRNA, and its associated mRNAs (from RaPID-seq) (Slobodin and Gerst, 2010; Zabezhinsky et al., 2016; Nair et al., 2021). Although in each case we focused on a single excised band and on a single identified protein for further exploration, this protocol can potentially yield multiple RNA interacting proteins to study.


    Discussion

    To validate RaPID RNA-seq and RaPID-MS results it is recommended to perform subsequent RaPID experiments using qRT-PCR (RaPID-qPCR) and western blotting, respectively. It is also important to include in these follow-ups both aptamer-tagged and non-tagged strains expressing the MS2 coat protein. This allows for measuring the quantitative enrichment of mRNA and protein from the tagged strains versus non-tagged strains, and is indicative of specificity in the pulldown of RNA and protein, respectively. It is also highly advisable to use a proper negative control (e.g., an unrelated tagged strain), to normalize gene expression using the input sample, and to validate the results with additional experiments. For example, RaPID-seq results indicating RNA-RNA interactions can be validated using single-molecule fluorescence in situ hybridization (smFISH) experiments, using dual sets of labeled probes that can detect potential interaction partners. This protocol does not distinguish RNA-RNA interaction via base paring or through mutual protein binding. Protocols using psoralen crosslinking [e.g., PARIS (psoralen analysis of RNA interactions and structures) (Lu et al., 2018) or SPLASH (sequencing of psoralen crosslinked, ligated, and selected hybrids) (Aw et al., 2017)] could be complementary to RaPID for this purpose. To validate RaPID-MS results, we recommend performing the reciprocal experiment – i.e., to affinity purify the protein and test whether the mRNA is co-precipitated with the protein.


    The uniqueness of RaPID is its ability to precipitate a single RNA species from the thousands expressed, due to the high affinity between the MS2 aptamer and MCP (Slobodin and Gerst, 2010 and 2011). While this non-biased affinity purification approach is potentially applicable to any aptamer-binding component pair (e.g., PP7, S1m) (Larson et al., 2011; Leppek and Stoecklin, 2014), RaPID has already proven effective in precipitating known and novel protein-RNA, as well as RNA-RNA, interactions, using single target mRNAs as bait (Slobodin and Gerst, 2010; Zabezhinsky et al., 2016; Nair et al., 2021). Our protocol has been used on a variety of mRNAs in yeast, some of which were expressed at low levels (e.g., UGO1; <5 copies per cell) and some highly expressed (e.g., HSP104; >100 copies per cell after heat-shock), working well in all cases. Thus, the protocol should be suitable for most mRNAs. Nevertheless, we recommend the use of live imaging with the MS2 system or single-molecule FISH, to estimate general transcript abundance and to scale up the protocol for rare transcripts. Finally, we note that RaPID dovetails well with m-TAG, an MS2 aptamer genome-tagging approach originally developed to allow for the localization of endogenously expressed messages in the yeast (Haim-Vilmovsky and Gerst, 2009). Together, the two approaches can be useful towards developing transcriptome-wide mRNA interaction maps for yeast.

Notes

  1. In the current protocol, we have used the version 3 (V3) MS2 aptamer for mRNA pulldown experiments, as originally developed for m-TAG (Haim et al., 2007). However, there are other versions of the MS2 aptamer developed by Singer group. For example, the V6 MS2 aptamer (Tutucci et al., 2018) may be advantageous since it does not show the accumulation of 3’UTR decay products that V3 may sometimes yield, particularly upon gene overexpression using a non-native promoter and/or under conditions that induce P-body formation (Haimovich et al., 2016). We recommend using a 12 or 24 SL cassette, since it is also useful for single molecule mRNA imaging. Shorter cassettes will be very difficult to image, but may work for RaPID. However, having more repeats increases the chance of the mRNA binding to the streptavidin beads. We do not have experience using less than 12 SL repeats.

  2. Induction of the binding protein does not necessarily allow for visualization of endogenously-expressed RNP granules, since the MCP-GFP SBP fusion has only one GFP (unlike in m-TAG, where MCP is fused to three GFPs). We performed a 45 min induction step, which was sufficient. Prolonged induction (i.e., >90 min in our yeast strains) can lead to excessive expression of the MCP-GFP-SBP and, possibly, to a higher level of background of precipitated proteins using RaPID. To optimize RaPID for other strains/growth conditions, users can perform RaPID using different induction times and compare the affinity purified RNA and proteins from the mRNA of interest with the background obtained from pulldowns using control strains or from different conditions.

  3. The amount of the protein extract needed for performing RaPID-seq and/or RaPID-mass spectrometry in yeast is typically between 80-100 mg. Between 5-10 mg of total protein extract is sufficient for the identification of precipitated proteins using western analysis, or for the analysis of RNA by qRT-PCR. Thus, the culture volume of yeast can vary, depending upon the desired downstream application, as well as the efficiency of lysis steps.

  4. For each RaPID reaction, an equal amount of cell extract is recommended. Hence, the concentration of total cellular protein should be accurately measured. The lysates used for this procedure are usually concentrated; we dilute them 1:10 and 1:100 in order to accurately determine the protein concentration. However, starting with an equal amount of cells for each reaction is also advisable.

  5. For normalization of gene expression for qRT-PCR, equal amounts of RNA from input samples of tagged and untagged or control strains are taken for cDNA preparation using the cDNA kit, according to manufacturer’s instructions. qRT-PCR is performed using gene specific primers. Fold enrichment is calculated using the following formula:

    Fold enrichment = 2–[(Ct tagged RaPID – Ct tagged input) – (Ct untagged RaPID – Ct untagged input)]

    Ct refers to cycle threshold.

Recipes

  1. Tris-ethylenediaminetetraacetic acid (EDTA) buffer (TE)

    10 mM Tris-HCl and 1 mM EDTA in double distilled (or fully deionized) water (DDW)

    pH 7.5

  2. 1 M LiOAc

    Lithium acetate 10.2% (wt/vol) in TE

    Filter-sterilize and store at RT for up to 1 year.

  3. 0.1 M LiOAc

    Mix 0.1 volumes of 1 M LiOAc (pH 7.5) with 0.9 volumes of TE.

    Prepare the solution under sterile conditions and store at RT for up to 1 year.

  4. 50% (wt/vol) polyethylene glycol (PEG) 3350

    Dissolve 250 g of PEG 3350 in 300 mL of TE buffer, while stirring and warming to 50°C.

    Fill to 500 mL, and filter sterilize.

  5. Salmon sperm DNA (ssDNA)

    The DNA is dissolved in ultrapure water at a concentration of 10 mg/mL.

    The solution will need to be stirred for at least 2-4 h at RT to dissolve the DNA. Shearing the DNA will help to reduce the viscosity by passing the DNA solution rapidly 12 times through a 17-gauge needle or once through a 23-gauge needle.

  6. Selective medium

    Synthetic complete (SC) were mixed essentially according to the protocol of Rose et al. (1990).

    In brief, 1 L of synthetic medium is prepared by mixing 7 g of synthetic dry mix (mix composed of 294 g of yeast nitrogen base with ammonium sulfate and without amino acid; 0.3 g each of arginine, cysteine, and proline; 0.45 g each of isoleucine, lysine, and tyrosine; 0.75 g each of glutamic acid, phenylalanine, and serine; 1.0 g each of aspartate, threonine, and valine) with 850 mL DDW, adding 350 µL of 10 N NaOH, followed by stirring and autoclaving in a large (i.e., 3 liters) Erlenmeyer flask. After cooling the autoclaved mixture to 55°C, add 100 mL of either prewarmed 20% glucose (wt/vol) along with 10 mL of a sterile-filtered 100 amino acid stock solution (see below). The 100 amino acid stock is composed of up to six amino acids/bases (e.g., adenine, histidine, leucine, methionine, tryptophan, and uracil) for the preparation of specific selective media. For example, SC medium contains all six, whereas synthetic medium lacking histidine (SC-H) would contain all except histidine. The 100 amino-acid stock solution is prepared by first adding 0.4 g of each amino acid/base required to 150 mL of DDW, followed by the addition of 3 mL of concentrated HCl while stirring, developing the volume up to 200 mL, and then sterile filtering. Liquid media can be stored at RT for up to 2 months. The 100 amino-acid stock is stored up to 1 year at 4°C, whereas the synthetic dry mix can be stored up to 1 year at RT.

    ! CAUTION: NaOH and HCl are very corrosive and can cause severe burns. May cause serious permanent eye damage. Very harmful upon ingestion. Harmful by skin contact or by inhalation. Use safety glasses, adequate ventilation, and neoprene or PVC gloves!

  7. Glycine solution

    1 M glycine solution at pH 7.0

  8. Yeast lysis buffer

    20 mM Tris-HCl at pH 7.5

    150 mM NaCl

    0.5% (vol/vol) Triton X-100

    1.8 mM MgCl2

    Prepared using RNase-free water

  9. Complete yeast lysis buffer

    Supplement Yeast lysis buffer with the following reagents (to be freshly added before use):

    1× proteinase inhibitor cocktail

    1 mM dithiothreitol (DTT)

    80 U/mL RNAsin® ribonuclease inhibitor

  10. Avidin solution

    1 mg/mL prepared in PBS

    Store at -20°C

  11. Yeast tRNA

    10 mg/mL solution in nuclease free water

    Store at -20°C

  12. BSA solution

    4% solution in PBS

    Store at 4°C

    Prepared from BSA fraction V

  13. Washing buffer

    20 mM Tris-HCl at pH 7.5

    300 mM NaCl

    0.5% (vol/vol) of NP-40

  14. Biotin solution

    0.2 M stock solution in dimethylsulfoxide (DMSO)

    Store at 4°C for up to two weeks.

    For elution, prepare a 6 mM solution in PBS prewarmed to 37°C.

    This solution should be prepared directly before use and be well mixed.

  15. 2× Cross-link reversal buffer

    100 mM Tris-HCl at pH 7.0

    10 mM EDTA

    20 mM DTT

    2% (wt/v) of sodium dodecylsulfate (SDS)

  16. 5× Protein sample buffer

    400 mM Tris-HCl at pH 6.8

    50% (v/v) of glycerol

    10% (wt/v) SDS

    0.5% (wt/v) Bromophenol blue

    Store at RT.

    Before use, add fresh 0.5 M DTT or 5% (v/v) of β-mercaptoethanol.

    Warning! DTT and β-mercaptoethanol may be harmful upon inhalation or skin contact. Use hood while preparing buffers containing these agents.

Acknowledgments

This work was supported by grants to J.E.G. from the Takiff Family Foundation and Jeanne and Joseph Nissim Center for Life Sciences (Weizmann Institute of Science), and the Israel Science Foundation (#578/18). R.R.N. was supported by a VATAT Fellowship for Postdoctoral Fellows from China and India (Israel Council of Higher Education). J.E.G. holds the Besen-Brender Chair in Microbiology and Parasitology (Weizmann Institute of Science).

This protocol was described in: Nair, R. R., Zabezhinsky, D., Gelin-Licht, R., Haas, B. J., Dyhr, M. C., Sperber, H. S., Nusbaum, C. and Gerst, J. E. (2021). Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology. eLife 10: e66050.

Competing interests

The authors declare they have no competing financial or non-financial interests.

References


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简介

[摘要] RNA-RNA和RNA-蛋白质相互作用参与基因表达的调节。在这里,我们描述了我们的 RNA 纯化和蛋白质鉴定 (RaPID) 协议的更新和扩展版本,用于通过亲和纯化来下拉适体标记的 mRNA。该方法利用了 MS2 RNA 适配体和 MS2 外壳蛋白 (MCP) 之间以及链霉亲和素结合肽 (SBP) 和链霉亲和素之间的高亲和力相互作用。因此,它采用 MCP-SBP 融合来亲和纯化 MS2 标记的目标 RNA,而不是固定化的链霉亲和素。然后将纯化的适配体标记的 mRNA 以及任何相关的 RNA 和蛋白质送去进行 RNA 测序 (RaPID-seq) 或质谱 (RaPID-MS),从而分别识别结合的群组 RNA 和蛋白质。

[背景] RNA 结合蛋白 (RBP) 和 RNA 相关蛋白调节基因表达途径的每一步。还经常有通过 RNA-RNA 相互作用进行调节的情况(Guil 和 Esteller,2015) 。因此,识别蛋白质-RNA 和 RNA-RNA 相互作用是理解 RNA 生物学的关键。
有很多方法可用于研究 RNA-蛋白质相互作用。这些被进一步分类为以 RNA 为中心的方法或以蛋白质为中心的方法。以 RNA 为中心的方法表征与特定目标 RNA 结合的蛋白质,而以蛋白质为中心的方法研究与特定蛋白质结合的 RNA(Ramanathan等人,2019 年)。以 RNA 为中心的方法同时使用体外和体内方法。最简单的 RNA 标记和下拉方法是通过 5'-或 3'-末端 RNA 生物素化 (Zheng et al ., 2016),或 S1 适体标记,用于在体外用固定的链霉亲和素下拉目标 RNA。体内方法包括 RNA 亲和纯化 (RAP) (Hacisuleyman et al ., 2014; McHugh and Guttman, 2018) 、串联 RNA 分离程序 (TRIP) (Matia-González et al ., 2017)、MS2体内生物素标记 RAP (MS2-BioTRAP) (Tsai et al ., 2011) 和 CHART (Capture Hybridization Analysis of RNA Targets) (Simon et al ., 2011)。 RAP 已用于研究非编码 RNA,例如 Xist (McHugh et al ., 2015),而 TRIP 用于研究多腺苷酸化 RNA (Matia-González et al ., 2017)。 MS-BioTRAP 使用 MS2 适配子系统,其中 MS2 茎环标记的 RNA 和 MS2 外壳蛋白异位表达(Tsai等,2011);然而,它们的过度表达可能并不能反映 RNA 的正常生理水平。 CHART 捕获寡核苷酸旨在与感兴趣的 RNA 特异性杂交(Simon等人,2011)。与以 RNA 为中心的方法相比,交联免疫沉淀 (CLIP) 是最常见的以蛋白质为中心的方法,用于表征与感兴趣的蛋白质结合的 RNA ( Ule等人,2003;Licatalosi等人,2008)。虽然所有这些方法都可能有效,但尚未将所描述的方法结合起来研究 RNA-RNA 和 RNA-蛋白质相互作用。
除了这些方法之外,还有几种转录组范围的方法来研究体内和体外的 RNA-RNA 和 RNA-蛋白质相互作用。诸如 PARIS(RNA 相互作用和结构的补骨脂素分析)(Lu等人,2018 年)、SPLASH(补骨脂素交联、连接和选择的杂种的测序)(Aw等人,2017 年)、LIGR-seq(相互作用的连接RNA 随后进行高通量测序)(Sharma等人,2016 年)和 MARIO(体内映射 RNA 相互作用组)(Nguyen等人,2016 年)用于研究转录组范围的 RNA-RNA 相互作用,但不能用于研究特定转录本的相互作用。
我们开发了 RaPID(RNA 纯化和鉴定),这是一种基于 MS2 适体的 mRNA 亲和纯化技术(Slobodin 和 Gerst,2010 和 2011) 。该方法允许从酵母或哺乳动物细胞中特异性分离 MS2 适体标记的 mRNA (Slobodin 和 Gerst,2010 和 2011),并随后分别使用 RNA seq 和质谱分析相关的 RNA 和蛋白质(图 1)。 RaPID 的优点是利用 MS2 适体和 MS2 外壳蛋白 (MCP) 之间的高亲和力相互作用,以及与 MCP 融合的链霉亲和素结合肽 (SBP) 与链霉亲和素珠(Slobodin 和格斯特,2010 年和 2011 年) 。因此,该方法允许从细胞中特异性纯化 MS2 标记的 mRNA 及其相关蛋白和 RNA。
此处介绍的 RaPID 质谱 (RaPID-MS) 和 RaPID RNA 测序 (RaPID-seq) 协议是 Slobodin 和 Gerst(2010和2011)以及 Haimovich等人先前描述的协议的更新和扩展版本。 (2016),分别用于分析酵母细胞中的 RNA-蛋白质和 RNA-RNA 相互作用。使用这些方法(参见图 2),我们确定了已知的(Slobodin 和 Gerst,2010 和 2011)和新的(Zabezhinsky等人,2016)RNA-蛋白质相互作用,以及 mRNA 多路复用现象(Nair等人。 , 2021)。简而言之,用 MS2 适体标记内源性酵母基因(Haim等人,2007 年;Haim-Vilmovsky 和 Gerst,2009 年),并用表达 MCP 的质粒转化细胞,融合 GFP 和 SBP(MCP-GFP -收缩压)。在所需的实验条件下培养细胞,然后诱导 MCP-GFP-SBP 表达并固定细胞以保存核糖核蛋白 (RNP) 复合物。接下来,裂解细胞并使用链霉亲和素结合珠通过亲和纯化沉淀 RNP 复合物。然后分别使用 RNA-seq 和质谱分析相关的 RNA 和蛋白质。


图 1. RaPID 策略示意图。
如文中所述,将共表达 MS2 适体标记的 RNA 和 MCP-GFP-SBP 结合蛋白的细胞固定并裂解。随后,裂解物与固定的链霉亲和素珠一起孵育,MS2 适体标记的 mRNA 以及相关的未标记 RNA 和 RNA 结合蛋白 (RBP) 被沉淀。然后对沉淀物进行蛋白质和 RNA 提取,以通过质谱法鉴定 RBP,通过 RNA-seq 或 qRT-PCR 鉴定 RNA。


图 2. RaPID-seq/质谱程序的示意图。
每个主要步骤都通过图形表示。 A. 制备带有 MS2 适体标记的 mRNA 和 MCP-GFP-SBP 的细胞。 B. 细胞培养。 C. MCP-GFP-SBP 蛋白表达的诱导。 D. 细胞的交联、收获、冷冻。 E. 裂解,蛋白质浓度的测量。 F. 添加珠子,孵育。 G. 离心、洗涤。 H. 交联逆转,洗脱。 I. RNA-seq 样本。 J. 图书馆准备和测序。 K. 基因表达分析。 L. 质谱样品。 M. SDS-PAGE 和银染。 N. 质谱法。

关键字:MS2, RNA适体, RNA亲和纯化, RaPID, 链霉亲和素结合肽, 酵母

材料和试剂

 

A. 酵母菌株

该程序成功使用的野生型菌株包括 BY4741EUROSCARFMAT a his3D1 leu2D0 met15D0 ura3D0 ),尽管任何S. cerevisiae实验室菌株都应该足够,只要它在HIS3URA3基因中发生突变。酵母可以在 4 °C的盘子上储存长达 2 周,或在 -80 °C冷冻时无限期储存

 

B. 试剂和一次性用品

1. 17 号针或 23 号针(任何供应商)

2. 1.7 mL 塑料管(高压灭菌)(任何供应商)

3. 无菌 15-mL 聚丙烯离心管(任何供应商)

4. 无菌无核酸酶屏障吸头(10 µL200 µL1,000 µL)(任何供应商)

5. 无菌 50 mL 聚丙烯离心管(Greiner,目录号:227270

6. 玻璃珠,直径 0.5 mmBiospec 产品,目录号:11079-105

7. 超纯水(Biological Industries,目录号:01-866-1B

8. 无菌双蒸水 (DDW)

9. 乙酸锂二水合物(LiOAc)(Sigma,目录号:L6883

10. 鲑鱼精子DNAssDNA)(Sigma,目录号:D1626

11. 超纯水(分子生物学级水,无核酸酶)(Sigma目录号: W4502

12. 无水乙醇(Bio Lab,目录号:05250521

13. 甲醛37%(Sigma,目录号:F8775-25ML

14. 甘氨酸游离碱(Sigma,目录号:G7126

15. 液氮

16. 重组Rnasin核糖核酸酶抑制剂(Promega,目录号:20006332

17. cOmplete TM Mini,不含 EDTA 的蛋白酶抑制剂混合物× 25Roche,目录号:11836170001

18. BCA 蛋白质测定试剂盒(Pierce,目录号:23225

19. 链霉亲和素缀合的 Sepharose TM珠(GE Healthcare,目录号:17-5113-01

20. 酵母tRNASigma,目录号:R8508

21. 抗生物素蛋白溶液(Sigma,目录号:A9275

22. 牛血清白蛋白(BSA),冻干粉, ≥96 %(Sigma,目录号:A7906

23. 生物素(Sigma,目录号:B4501

24. 二甲基亚砜(DMSO)(Sigma,目录号:D8418

25. MPC 蛋白沉淀试剂(MasterPure TM Yeast RNA 纯化试剂盒的一个组成部分;Epicenter Biotechnologies,目录号:MPY03100

26. 糖原20 mg / mL溶液(Fermentas,目录号:R0561

27. 3 M乙酸钠(NaOAc)溶液,pH 5.2Fermentas,目录号:R1181

28. qScript Flex cDNA 试剂盒(Quantabio,目录号:95049-100-2

29. DNaseIQuantabio,目录号:95150-100

30. NNN'N'-四甲基乙二胺(TE MED)(Sigma,目录号:T9281-25ML

31. 30%丙烯酰胺/ Bis 291Bio-Rad,目录号:161-0156

32. Silver Stain KitPierce,目录号:24612

33. TE 缓冲液(见配方)

34. 1 M LiOAc(见配方)

35. 0.1 M LiOAc(见配方)

36. PEG溶液(见配方)

37. ssDNA(见食谱)

38. 选择性培养基(见配方)

39. 甘氨酸溶液(见配方)

40. 酵母裂解缓冲液(见配方)

41. 完整的酵母裂解缓冲液(参见食谱)

42. 酵母 tRNA(见食谱)

43. 抗生物素蛋白溶液(见配方)

44. 酵母 tRNA(见食谱)

45. BSA 溶液(见配方)

46. 洗涤缓冲液(见配方)

47. 生物素溶液(见食谱)

48. 交联反向缓冲液(见配方)

49. 蛋白质样品缓冲液(见配方)

 

C. 质粒

1. pUG34-MS2-CP-GFP-SBP(又名 pMCP-GFP-SBP Slobodin Gerst2010

2. pMS2-SL 质粒Haim-Vilmovsky Gerst2009

 

设备

 

1. 1升烧瓶

2. 3升锥形瓶

3. -80°C 冰箱

4. 用于从凝胶中切除蛋白质条带的无菌手术刀片(Jai Surgical Blades,目录号:400-11

5. 轨道摇床培养箱 (MRC, TOU-120)

6. 移液器辅助装置(推荐:S1 移液器填充器,Thermo Fisher Scientific,目录号:9501

7. (可选)真空阱

8. 化学(通风)罩

9. Ultrospec 10 细胞密度计(Biochrom,目录号: 634-0882

10. 旋转器/混合器 Intelli-MixerThomas Scientific,目录号:RM-2M

11. 台式冷冻离心机(Eppendorf,目录号:5810 R

12. 台式冷冻微量离心机(Eppendorf,目录号:5417 R

13. 适用于大容量的离心机(冷藏)(SorvallRC5C plus SLA3000 转子)或同等离心机

14. 用于 Sorvall 离心机的瓶子,500 mL例如ThermoNalgene,目录号 10430613

15. Digital Disruptor GenieScientific Industries,目录号:SI-DD38 120V)。这应该在实验前在冷室中进行预冷。

16. 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 实验的标准设备

17. 可容纳丙烯酰胺凝胶的无菌盒

软件

 

1. 蛋白质组发现者(1.4 版)

 

程序

 

A. MS2 标记和培养酵母细胞培养物

MS2 适体是源自 MS2 噬菌体的短 RNA 茎环 (SL) 结构。 MS2 标记,. 插入 12-24 个重复的 MS2-SL,允许使用与荧光蛋白(如 GFP)融合的 MCP 对内源性标记的 mRNA 进行可视化和下拉(图 2)。 SBP 部分的添加允许使用固定的链霉亲和素下拉 MCP-GFP。然而,为了在下拉实验中识别非特异性信号,需要几个对照,例如未标记的细胞和用对照 mRNA 标记的细胞。

1. 使用基于 PCR 的基因组标记策略和如前所述的同源重组Haim等人2007 年;Haim-Vilmovsky格斯特,2009。另见注 1

2. 使用标准酵母 LiOAc 转化(GietzSchiestl, 2007) 将带有 MS2 适体标记的基因的菌株转化为表达 MCP-GFP-SBP 的质粒,并在 SC-His 合成培养基琼脂糖板上进行平板。

3. HIS3选择标记 MS2-CP-GFP-SBP 转化的单个菌落,将其接种到具有 7-8 mL 选择性培养基 (SC-His) 50 mL 试管中,并在摇动中生长30°C 培养箱 6-10 小时。

4. 稀释至 500 mL培养过夜培养。对于处于对数中期的细胞,第二天早上最终的 OD 600应该在 0.4-0.8 之间。如果需要其他细胞状态,则可以使用不同 OD 600值的培养物,但可能需要根据获得的细胞数量来修改进一步的步骤。要计算要采取的起始培养体积,请按照下列步骤操作:

a. 测量培养物的细胞数或 OD 600并使用以下公式确定倍增时间。对于本实验中使用的野生型菌株(BY4741),倍增时间为2小时。

 

[log 10 (N t /N 0 )]/log 10 (2) = g

g - 代数

N 0 –开始时的细胞数或 OD 600

N t –最后的细胞数或 OD 600

t = 培养时间

d——倍增时间(d=t/g

 

b. 使用以下公式确定添加到更大的过夜培养物中的起始培养物的量:

 

N 0 – OD 600开始

N t – OD 600最后你想要的

g – 文化在收获前将经历的世代数

N 0 = N/2

g = /

 

5. 过夜生长后,测量培养物的吸光度,如果需要,继续培养培养物,至少达到 OD 600 = 0.41,100 ×离心收集细胞 g 4 °C 5 分钟, Sorvall 离心机中使用 SLA3000 型转子,弃去生长培养基。

6.  200 mL 不含蛋氨酸的新鲜生长培养基中轻轻吹打(不要涡旋)重悬细胞 pell et,转移到 1 L 烧瓶中,并在 30°C 下摇动孵育 45 分钟以诱导 MS2-CP- 的表达GFP-收缩压。另见注 2

7. 直接加入 270 μL 甲醛至终浓度为 0.05%,用甲醛固定细胞,并在室温 (RT) 下缓慢摇动孵育 10 分钟。

8. 溶液添加到最终浓度为 0.125 M 并再孵育 2 分钟来终止交联反应。

9. 离心细胞,根据您研究所的化学废物程序丢弃上清液,然后使用移液器(不要涡旋)在 50 mL 管中的 10 mL PBS 中重悬细胞。

10. ×离心细胞 g 4°C 5 分钟。

11. 完全丢弃 PBS,并在纸巾上将管倒置 1-2 分钟,以去除残留的 PBS

12. 使用液氮快速冷冻细胞沉淀。储存时,将细胞转移到 -80°C 冰箱中。在这些条件下,细胞可以长期储存。

 

B. 酵母细胞裂解

下面提到的所有步骤都应在冰上进行。

1. 将冷冻细胞颗粒置于冰上,加入 4 mL 的完全酵母裂解缓冲液解冻。

2. 细胞解冻后,将 0.7 mL 转移到 1.5 mL 微量离心管中,其中预填充 0.5 mL 的酸预洗和烤箱烘烤的玻璃珠。

3. 使用破坏器在 4°C 下以最大速度涡旋细胞 20 分钟。关闭摇床,静置 5 分钟以防止过热,然后再打开 20 分钟。

4. ×预冷至 4°C 的台式离心机中离心微量离心管 g 2 分钟。将上清液转移到新鲜的微量离心管中,再次以 15,000 ×离心 g 10 分钟。

5. 即每个单独的培养物)的上清液收集到一次性 15 mL 试管中。离心后获得约 4 mL 的上清液。根据制造商的说明,用检测试剂盒中 BCA 蛋白测量蛋白质浓度。

6. 转移 RaPID-seq/MS 所需的蛋白质提取物总量(通常每次下拉 80-100 mg 用于 RaPID-seq RaPID-MS 实验,而 5-10 mg 用于 qRT-PCR Western 分析)到新的 15 mL 管中,加入抗生物素蛋白溶液(每 1 mg 蛋白质提取物加入 10 μg 抗生物素蛋白),并在 4°C 下孵育 1 小时,同时不断旋转/摇动。完成此步骤是为了阻止任何生物素化蛋白质与链霉亲和素珠结合。关于蛋白质测量和每种实验所需的量,请参见注释 3 4

7. 将分离 RNA 和蛋白质所需的量分离到不同的管中。此外,将两个试管放在一边,每个试管都含有初始总提取量的 1% 一根管子用于输入 RNA”(进行第 14 步进行 RNA 分离), 并且可用于在进行 qRT-PCR Nanostring 实验时对基因表达进行标准化(见注 5)。另一个试管将用于输入蛋白(进行第 15 步)。这些样品保存在冰上并与洗脱液平行处理,或者可以在 -80°C 下冷冻直至使用。

8. 同时,等分 30 μL 的链霉亲和素结合珠。通过加入 1 mL 的冰冷 PBS 清洗珠子两次;加入裂解缓冲液一次,涡旋(不要涡旋)2-3 次,然后以 1,000 ×离心 。用裂解缓冲液中制备的 2% BSA tRNA(每 100 μL 珠子 0.1 mg)在 4°C 下封闭珠子 1 小时。 1 小时后,离心并用裂解缓冲液洗涤一次。

9. 丢弃洗涤过的珠子中的缓冲液,将亲和素封闭的总细胞提取物与 0.1 mg 酵母 tRNA 一起添加到珠子中,并在 4°C 下孵育 2 小时至过夜,并持续旋转。

10. ×离心管 g持续2 分钟并去除上清液。将珠子转移到 1.7 mL 管中,用裂解缓冲液洗涤 3 次,用洗涤缓冲液洗涤两次,洗涤之间进行离心(如上)。

11. 在冰冷的 PBS 中进行最后一次洗涤并去除多余的缓冲液。

12. 向磁珠中加入 150 μL 生物素洗脱溶液,并在 4°C 下旋转孵育 1 小时,以从磁珠中洗脱 mRNA-蛋白质/RNA 复合物。

13. 1,000 ×离心机 g 2 min,将洗脱液转移到新的微量离心管中,再次离心,将洗脱液转移到新管中以确保没有珠子带入。

14. 要反向交联用于 RNA 分离的部分,向洗脱液中加入等体积的交联反向缓冲液,并在 70°C 下孵育 45 分钟。

15. 为了使用于蛋白质分离的级分反向交联,加入适当体积的 蛋白质样品缓冲液以达到 浓度,并在 70°C 下孵育 45 分钟。

16. 去交联的 RNA 和蛋白质样品可以保存在 -20°C

 

数据分析

 

A. 沉淀 RNA 分析

1. 反向交联后,每 300 μl 洗脱液中加入 175 μL MPC 蛋白沉淀试剂,剧烈涡旋 10 s

2. × 4°C 离心 10 分钟

3. 小心地将上清液转移到新的离心管中。添加糖原(80 μg/mL 最终浓度)和 NaOAc0.3 M 最终浓度),并彻底涡旋 20 秒。加入等体积的异丙醇,颠倒离心管 20 次混匀, -20 °C 孵育过夜。

4. 离心管(12,000 × g 4 °C 10 分钟)。含有 RNA 的颗粒应该是可见的。小心丢弃上清液。

5.  70% 冰冷的乙醇清洗颗粒。 ≥10,000 × 4°C 离心 5 分钟 。小心丢弃上清液。

6. 将试管在 50°C 的干浴中加热 10 分钟,盖上盖子,以蒸发残留的乙醇。不要过度干燥,因为这会使颗粒难以溶解。

7. 将颗粒溶解在 30 μL 的超纯水中。在 50°C 加热 5-10 分钟以达到完全溶解。将溶解的 RNA 储存在 -20°C -80°C 的冰箱中,以便长期储存。

8. 如前所述构建 RNA-seq (Levin et al ., 2010)Oligo(dT) 选择是可选的,取决于是否需要单独的 polyA+ RNA

9.  55°C 下用 SuperScript III (Invitrogen) 反转录 RNA,并在 5% DMSO 存在下用 Herculase (Stratagene) 扩增 cDNA 16 个循环,然后用 1.8体积的 AMPure 珠子 (Beckman-印第安纳州库尔特)。

10. 使用标准方法对文库进行测序,深度约为 10 7 个读数(例如,我们使用了 Illumina HiSeq2000 测序仪,具有配对末端 76 个碱基读数;Nair等人2020)。

 

B. RaPID RNA seq的数据分析

1. 使用 Kallisto (Bray et al ., 2016) 并针对酿酒酵母( S. cerevisiae ) 参考转录组(源自酿酒酵母基因组数据库并利用酿酒酵母S288C 基因组版本 R64- )从 RNA-Seq 数据中估计基因表达水平2-1)。图 3A 中提供了为不同 MS2 标记的 mRNA 获取的各种 RNA 的读取计数示例。

注意:将每个基因注释的 3'UTR 下游的 100 个碱基视为 mRNA 的一部分,以覆盖可能的未注释 3'UTR 区域。

2. 识别差异表达的基因。我们使用了 edgeR (Robinson et al ., 2009),将色散参数手动设置为 0.1。那些报告为至少四倍差异表达(FDR < 0.001)的基因被保留为显着差异表达。在对数据 [log 2 (TPM +1)] 进行对数转换后,从实验样本中减去来自对照样本的基因表达值。通过使用集成到 Trinity 软件套件中的转录组分析模块,促进了表达定量、差异表达和热图的绘制(Haas等人2013 年)。很难预先确定有多少 RNA 富含特定的 MS2 标记的诱饵 mRNA,因为它取决于 MS2 标记的 mRNA、使用的酵母菌株(例如 MATaMATα ,或特定的缺失菌株),以及所采用的生长条件。根据我们的经验,我们发现了多达 10 个由 RaPID-seq 鉴定的相互作用的 RNA。通过其他方法验证这些结果很重要(例如co-smFISHRaPID pulldowns,然后是 qRT-PCR)。我们成功地鉴定并验证了各种 mRNA 的这种 RNA-RNA 相互作用(例如,编码交配因子、热休克蛋白、MAP 激酶)。

 


3 来自 RaPID-seq 的读取计数示例,带有各种 MS2 标记的 mRNA 和用于 RaPID-MS 的银染凝胶。 

(A)显示的是来自 Nair等人的补充文件 1 “Counts.matrix”excel 表的屏幕截图。 2021 年)。它显示了分配给每个基因的读取数(基因名称在 A 列中),用于各种 MS2 标记的目标 mRNA(列标题)。 Cntrl - 控制未标记的应变。过滤掉 rRNAtRNA snRNA。对于EXO70 mRNAL 列) ,表(如图所示)从大到小排序。蓝色标记的细胞显示在EXO70 SRO7OM45 RaPID-seq pulldowns 中富集的交配相关基因。请注意,MS2 标记的EXO70目标 mRNA 高度富集其自身的 RNA。许多 RNA 对所有菌株都是通用的,包括未标记的对照细胞,因此被视为背景。在已鉴定的约 7,100 个基因中,仅显示了少数选定的基因。 (B)用于 RaPID-MS SDS-PAGE 凝胶的银染(转载自 Nair等人2021)。培养表达带有或不带有 3'UTR MS2 适体标记的MFA1 MFA2STE2的酵母菌株以及ASH1 ,并针对 RaPID-MS 进行处理。箭头表示存在于STE2车道(以及所有MFA1MFA2车道)中的 ~150 kDa 条带,但不在ASH1STE2 Δ3'UTR车道中。将该条带从凝胶上切下并送去进行 MS。蓝色方块——表示 MCP-GFP-SBP 的波段。 kDa——千道尔顿。

 

C. 沉淀蛋白分析

1. 要识别未知蛋白质,有两种选择。首先是在中等大小的 1.5 mm 厚凝胶(例如20 cm × 15 cmBio-Rad9% SDS-PAGE 凝胶上分离蛋白质。使用允许在每个泳道加载整个样品体积(约 180 μL)的梳子。

2. 根据制造商的说明,使用银染试剂盒 (Pierce) 进行银染。它与随后的质谱分析完全兼容。

3. 分析染色结果。如果检测到特定条带(参见图 3B),请用无菌刀片将其切除并发送到质谱仪进行识别。

4. 第二种可能性是将整个样品送去进行质谱分析。

5. 我们建议在实验前与所选蛋白质组学机构的人员讨论蛋白质样品制备和提交、肽测量和数据分析。在 D 节中,我们提供了一些细节以作为讨论的基础。

 

D. RaPID-MS的数据分析

反相纳米液相色谱-电喷雾电离串联质谱和数据分析在以色列国家个性化医学中心(Weizmann InstituteRehovotIsrael)进行。凝胶切片在核心设施进行处理和胰蛋白酶消化。使用了使用 FlexIon 纳米喷雾装置 (Proxeon) 耦合到四极轨道阱质谱仪 (Q Exactive HF)nanoUPLC10 μm 尖端;新物镜;WoburnMAUSA)。使用的参数是:MS1 分辨率设置为 120,000m/z 200),质量范围为 m/z 300-1650AGC 3e6,最大进样时间设置为 60 msMS2 分辨率设置为 30,000,四极杆隔离 m/z 1.7AGC 1e5,动态排除时间为 20 秒,最大进样时间为 60 毫秒。 核心设施使用 Proteome Discoverer1.4 版)处理数据,使用 UniprotKB 酵母蛋白数据库,使用两种搜索算法 - SequestHT Mascot 添加了常见实验室污染物列表以及以下变量修改:C 上的氨基甲酰甲基、M 上的氧化和 N 端乙酰化。过滤数据以获得最大 1% 的错误发现率。

通过使用 RaPID-MS,我们成功识别并验证了与 MS2 标记的 mRNA 及其相关 mRNA 相互作用的新蛋白质(来自 RaPID-seq)(Slobodin Gerst2010Zabezhinsky2016Nair , 2021)。尽管在每种情况下,我们都专注于单个切除带和单个已识别蛋白质以进行进一步探索,但该协议可能会产生多种 RNA 相互作用蛋白质进行研究。

 

讨论

为了验证 RaPID RNA-seq RaPID-MS 结果,建议分别使用 qRT-PCR (RaPID-qPCR) 和西方印迹进行后续 RaPID 实验。在这些后续行动中包括适体标记和非标记表达 MS2 外壳蛋白的菌株也很重要。这允许测量来自标记菌株与非标记菌株的 mRNA 和蛋白质的定量富集,并分别指示 RNA 和蛋白质的下拉特异性。还强烈建议使用适当的阴性对照(例如,不相关的标记菌株) 使用输入样本标准化基因表达,并通过额外的实验验证结果。例如,可以使用单分子荧光原位杂交 (smFISH) 实验验证表明 RNA-RNA 相互作用的 RaPID-seq 结果,使用可以检测潜在相互作用伙伴的双组标记探针。该协议不区分通过碱基配对或相互蛋白质结合的 RNA-RNA 相互作用。使用补骨脂素交联的方案 [例如PARISRNA 相互作用和结构的补骨脂素分析)(Lu等人2018)或 SPLASH(补骨脂素交联、连接和选择的杂交体的测序)(Aw等人2017 年)]为此目的对 RaPID 进行补充。为了验证 RaPID-MS 结果,我们建议进行交互实验——亲和纯化蛋白质并测试 mRNA 是否与蛋白质共沉淀。

由于 MS2 适体和 MCP 之间的高亲和力,RaPID 的独特之处在于它能够从数千个表达的 RNA 中沉淀出单个 RNA Slobodin Gerst2010 2011。虽然这种无偏的亲和纯化方法可能适用于任何适体结合组分对(例如PP7S1mLarson等人2011Leppek Stoecklin2014,但 RaPID 已经证明在沉淀已知和使用单靶 mRNA 作为诱饵的新型蛋白质-RNA 以及 RNA-RNA 相互作用Slobodin Gerst2010Zabezhinsky2016Nair2021。我们的方案已用于酵母中的多种 mRNA,其中一些以低水平表达(例如 UGO1 ;每个细胞<5 个拷贝)和一些高表达(例如 HSP104 ;加热后每个细胞>100 个拷贝) -shock),在所有情况下都能正常工作。因此,该协议应适用于大多数 mRNA。尽管如此,我们还是建议使用 MS2 系统或单分子 FISH 进行实时成像,以估计一般转录本丰度并扩大稀有转录本的协议。最后,我们注意到 RaPID m-TAG 非常吻合,m-TAG 是一种 MS2 适体基因组标记方法,最初开发用于在酵母中定位内源表达的信息 Haim-Vilmovsky Gerst2009。总之,这两种方法可用于开发酵母转录组范围的 mRNA 相互作用图。

 

 

 

 

笔记

 

1. 在当前协议中,我们使用版本 3 (V3) MS2 适配体进行 mRNA 下拉实验,如最初为 m-TAG 开发的 (Haim et al ., 2007)。不过,胜家集团还开发了其他版本的 MS2 适配体。例如,V6 MS2 适体(Tutucci等人2018)可能是有利的,因为它不会显示 V3 有时可能产生的 3'UTR 衰变产物的积累,特别是在使用非天然启动子和/或在诱导 P 体形成的条件下(Haimovich等人2016 年)。我们建议使用 12 24 SL 盒,因为它也可用于单分子 mRNA 成像。较短的磁带很难成像,但可能适用于 RaPID。然而,重复次数越多,mRNA 与链霉亲和素珠结合的机会就会增加。我们没有使用少于 12 SL 重复的经验。

2. 结合蛋白的诱导不一定允许内源性表达的 RNP 颗粒的可视化,因为 MCP-GFP SBP 融合只有一个 GFP(与 m-TAG 不同,其中 MCP 与三个 GFP 融合)。我们进行了 45 分钟的诱导步骤,这就足够了。长时间的诱导(,在我们的酵母菌株中 > 90 分钟)会导致MCP-GFP-SBP 的过度表达,并且可能导致使用 RaPID 的沉淀蛋白质的背景水平更高。为了针对其他菌株/生长条件优化 RaPID,用户可以使用不同的诱导时间执行 RaPID,并将来自感兴趣的 mRNA 的亲和纯化的 RNA 和蛋白质与使用对照菌株或来自不同条件的下拉获得的背景进行比较。

3. 在酵母中进行 RaPID-seq /RaPID-质谱分析所需的蛋白质提取物的量通常在 80-100 毫克之间。 5-10 mg 的总蛋白提取物足以使用西方分析鉴定沉淀的蛋白质,或通过 qRT-PCR 分析 RNA。因此,酵母的培养体积可能会有所不同,这取决于所需的下游应用以及裂解步骤的效率。

4. 对于每个 RaPID 反应,建议使用等量的细胞提取物。因此,应准确测量总细胞蛋白的浓度。用于此过程的裂解物通常是浓缩的;我们以 1:10 1:100 稀释它们,以准确确定蛋白质浓度。但是,也建议从每个反应的等量细胞开始。

5. 为了标准化 qRT-PCR 的基因表达,根据制造商的说明,使用 cDNA 试剂盒从标记和未标记或对照菌株的输入样本中提取等量的 RNA,用于 cDNA 制备。 qRT-PCR 使用基因特异性引物进行。使用以下公式计算倍数富集:

 

富集倍数 = 2 –[Ct标记的 RaPID – Ct标记的输入) Ct未标记的 RaPID – Ct未标记的输入)]

 

Ct 指周期阈值。

食谱

1. 三乙二胺四乙酸 (EDTA) 缓冲液 (TE)

10 mM Tris-HCl 1 mM EDTA 在双蒸(或完全去离子)水 (DDW)

酸碱度 7.5

2. 1 M LiOAc

TE 10.2% (wt/vol) 的醋酸锂

过滤灭菌并在室温下储存长达 1 年。

3. 0.1 M LiOAc

0.1 体积的 1 M LiOAc (pH 7.5) 0.9 体积的 TE 混合。

在无菌条件下制备溶液并在 RT 中储存长达 1 年。

4. 50% (wt/vol) 聚乙二醇 (PEG) 3350

250 g PEG 3350 溶解在 300 mL TE 缓冲液中,同时搅拌并升温至 50°C

加注至 500 mL,过滤除菌。

5. 鲑鱼精子 DNA (ssDNA)

DNA 10 mg/mL 的浓度溶解在超纯水中。

该溶液需要在室温下搅拌至少 2-4 小时以溶解 DNA 通过将 DNA 溶液快速通过 17 号针头 12 次或通过 23 号针头一次,剪切 DNA 有助于降低粘度。

6. 选择性培养基

合成完全 (SC) 基本上根据 Rose等人的方案混合1990)。

简而言之,1 L 合成培养基是通过混合 7 g 合成干混合物(由 294 g 酵母氮碱、硫酸铵和不含氨基酸的混合物组成;精氨酸、半胱氨酸和脯氨酸各 0.3 g;各 0.45 g异亮氨酸、赖氨酸和酪氨酸;谷氨酸、苯丙氨酸和丝氨酸各 0.75 g;天冬氨酸、苏氨酸和缬氨酸各 1.0 g)和 850 mL DDW,加入 350 µL 10 N NaOH,然后搅拌并高压灭菌一个大的(3 升)锥形瓶。将高压灭菌混合物冷却至 55 °C 后,加入 100 mL 的预热 20% 葡萄糖(重量/体积)以及 10 mL 的无菌过滤的 100 氨基酸库存溶液(见下文)。 100 种氨基酸原液由多达 6 种氨基酸/碱基(例如腺嘌呤、组氨酸、亮氨酸、蛋氨酸、色氨酸和尿嘧啶)组成,用于制备特定的选择性培养基。例如,SC 培养基包含所有六种,而缺乏组氨酸 (SC-H) 的合成培养基将包含除组氨酸之外的所有培养基。 100 个氨基酸储备液的制备方法如下:首先将 0.4 g 所需的每种氨基酸/碱加入 150 mL DDW,然后在搅拌下加入 3 mL HCl,使体积达到 200 mL,然后无菌过滤。液体培养基可在室温下储存长达 2 个月。 100 种氨基酸的原液在 4 °C 下可储存长达 1 ,而合成干混合物可在室温下储存长达 1 年。

!注意:NaOH HCl 具有很强的腐蚀性,会导致严重烧伤。可能造成严重的永久性眼损伤。摄入后非常有害。皮肤接触或吸入有害。使用安全眼镜、足够的通风和氯丁橡胶或 PVC 手套!

7. 甘氨酸溶液

1 M 甘氨酸溶液,pH 7.0

8. 酵母裂解缓冲液

20 mM Tris-HClpH 7.5

150 毫米氯化钠

0.5%(体积/体积)Triton X-100

1.8 毫米氯化镁2

使用无 RNase 水制备

9. 完全酵母裂解缓冲液

用以下试剂补充酵母裂解缓冲液(使用前新鲜添加):

1 ×蛋白酶抑制剂混合物

1 mM 二硫苏糖醇 (DTT)

80 U/mL RNAsin ®核糖核酸酶抑制剂

10. 抗生物素蛋白溶液

PBS 中制备 1 mg/mL

储存于 -20°C

11. 酵母tRNA

10 mg/mL 无核酸酶水溶液

储存于 -20°C

12. BSA 溶液

4% PBS溶液

储存于 4°C

BSA 馏分 V 制备

13. 洗涤缓冲液

20 mM Tris-HClpH 7.5

300 毫米氯化钠

NP-40 0.5% (vol/vol)

14. 生物素溶液

二甲亚砜 (DMSO)原液

4°C 下储存长达两周。

对于洗脱,在预热至 37°C PBS 中制备 6 mM 溶液。

该溶液应在使用前直接制备并充分混合。

15. 交联反向缓冲液

100 mM Tris-HClpH 7.0

10 毫米乙二胺四乙酸

20 毫米数字地面电视

2% (wt/v) 十二烷基硫酸钠 (SDS)

16. 蛋白质样品缓冲液

400 mM Tris-HClpH 6.8

50% (v/v) 的甘油 

10%(重量/体积)SDS

0.5% (wt/v) 溴酚蓝

存储在 RT

使用前,加入新鲜的 0.5 M DTT 5% (v/v) β-巯基乙醇。

警告! DTT β-巯基乙醇在吸入或皮肤接触时可能有害。在准备含有这些试剂的缓冲液时使用罩。

 

致谢

 

这项工作得到了塔基夫家族基金会、珍妮和约瑟夫尼西姆生命科学中心 (魏茨曼科学研究所) 和以色列科学基金会 (#578/18) JEG 的资助。 RRN 得到了中国和印度博士后研究员 VATAT 奖学金(以色列高等教育委员会)的支持。 JEG 担任微生物学和寄生虫学 Besen-Brender 主席(魏茨曼科学研究所)。

该协议描述于: Nair, RR, Zabezhinsky, D., Gelin-Licht, R., Haas, BJ, Dyhr, MC, Sperber, HS, Nusbaum, C. Gerst, JE (2021)多路 mRNA 组装成核糖核蛋白颗粒在酵母细胞生理控制中起类似操纵子的作用 埃莱夫10 e66050

 

利益争夺

 

作者声明他们没有相互竞争的财务或非财务利益。 

 

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Copyright Nair et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nair, R. R., Haimovich, G. and Gerst, J. E. (2022). An Aptamer-based mRNA Affinity Purification Procedure (RaPID) for the Identification of Associated RNAs (RaPID-seq) and Proteins (RaPID-MS) in Yeast. Bio-protocol 12(1): e4274. DOI: 10.21769/BioProtoc.4274.
  2. Nair, R. R., Zabezhinsky, D., Gelin-Licht, R., Haas, B. J., Dyhr, M. C., Sperber, H. S., Nusbaum, C. and Gerst, J. E. (2021). Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology. Elife 10 : e66050.
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ARGHYA SETT
IOCB, Prague
Further queries as follows:
-In procedure. step no 6. the shaking speed(how much RPM) to induce the expression of the MS2-CP-GFP-SBP is not mentioned. I think the shaking speed is a critical parameter for protein expression.
-For western blot, which primary/secondary antibody should we use to determine MS2-CP-GFP-SBP on the SDS gel. or fluorophore tagged MS2 aptamer can be used to visualize the pull-down fractions?
2022/5/15 12:27:18 回复
Rohini Nair
Weizmann Institute of Science

-In procedure. step no 6. the shaking speed(how much RPM) to induce the expression of the MS2-CP-GFP-SBP is not mentioned. I think the shaking speed is a critical parameter for protein expression.
170rpm
-For western blot, which primary/secondary antibody should we use to determine MS2-CP-GFP-SBP on the SDS gel. or fluorophore tagged MS2 aptamer can be used to visualize the pull-down fractions?
anti-GFP

2022/5/19 18:02:59 回复


Rohini Nair
Weizmann Institute of Science

-In procedure. step no 6. the shaking speed(how much RPM) to induce the expression of the MS2-CP-GFP-SBP is not mentioned. I think the shaking speed is a critical parameter for protein expression.
170rpm
-For western blot, which primary/secondary antibody should we use to determine MS2-CP-GFP-SBP on the SDS gel. or fluorophore tagged MS2 aptamer can be used to visualize the pull-down fractions?
anti-GFP

2022/5/19 18:03:00 回复


ARGHYA SETT
IOCB, Prague
The protocol is really well-written and elaborate aptamer based mRNA purification. Although I have few queries :
1. In the protocol, SC-his media recipe is missing. The exact composition of the selective media is not mentioned in the protocol.
2022/5/15 12:09:40 回复
Rohini Nair
Weizmann Institute of Science

In brief, synthetic medium is prepared by mixing 7 g of synthetic dry mix (Dry mix is prepared by mixing 394 g Yeast nitrogen base with ammonium sulphate with 75 g Amino acid (0.3 g each of arginine, cysteine and proline; 0.45 g each of isoleucine, lysine and tyrosine; 0.75 g each of glutamic acid, phenylalanine and serine; 1.0 g each of aspartate, threonine and valine;) with 850 ml DDW, adding 350 ul of 10 N NaOH, followed by stirring and autoclaving in a large (i.e., 3 liters). Cool to room temperature before use. 100 ml of of 20% glucose (wt/vol) is added, as required, along with 10 ml of a sterile-filtered 100 aminoacid stock solution (see below).

The 100 amino-acid stock is composed of up to six amino acids/bases (e.g., adenine, histidine, leucine, methionine, tryptophan and uracil) for the preparation of specific selective media. For example, SC medium contains all six, whereas synthetic medium lacking uracil (SC-U) would contain all but uracil. The 100 amino-acid stock solution is prepared by first adding 0.4 g of each amino acid/base required to 150 ml DDW, followed by the addition of 3 ml concentrated HCl while stirring, developing the volume up to 200 ml, and then sterile filtering.

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