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Polysome Fractionation to Analyze mRNA Distribution Profiles
多聚核糖体分馏分析mRNA分布特征   

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Abstract

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Keywords: mRNA translation(mRNA翻译), Protein synthesis(蛋白质合成), Ribosome(核糖体), Polysomes(多聚核糖体), Sucrose gradient(蔗糖梯度), Fractionation(分馏), RT-qPCR(RT-qPCR)

Background

Gene expression is regulated at many steps, including gene transcription, pre-mRNA splicing, and mRNA export to the cytoplasm, turnover and translation. Given the robust impact of post-transcriptional gene regulatory mechanisms on overall protein expression patterns in the cell, there is great interest in elucidating the processes that control these events. In particular, the steady-state mRNA levels of one-half of the transcriptome show poor correlation with the level of proteins translated from these mRNAs, indicating that protein levels in the cell are potently regulated at the level of mRNA translation and/or protein stability (Schwanhausser et al., 2011). A number of assays can be used to study how translation is regulated in response to different conditions – both at the transcriptome level and at the level of single mRNAs. Traditionally, Western blot analysis, puromycin labeling, and 35S-methionine/cysteine labeling assays have been used to measure the efficiency mRNA translation.

The method discussed here focuses on analyzing the sizes of polysomes that form on a given mRNA. The premise of this analysis is that mRNAs found in larger polysomes are expected to be translated robustly, while mRNAs present in smaller polysomes or devoid of ribosome components are expected to be translated poorly or remain untranslated. This protocol allows the capture of actively translating mRNAs by ‘freezing’ translating ribosomes and thus permitting the measurement of the relative size of polysomes forming on given mRNAs. This method has been successfully used in dozens of studies to analyze how RBPs and microRNAs affect the translation of target mRNAs and can be used to explore the role of polysome-associated proteins and noncoding RNAs on global translation and the translation of specific mRNAs.

Materials and Reagents

  1. Tube, thin-wall, polypropylene, 13.2-ml (Beckman Coulter, catalog number: 331372 )
  2. 9” Pasteur pipet (Kimble Chase Life Science and Research Products, catalog number: 883350-0009 )
  3. 15 ml tube
  4. Posi-Click 1.7-ml microcentrifuge tube (Denville Scientific, catalog number: C2171 )
  5. ThermoGridTM rigid strip 0.2-ml PCR tubes [(Denville Scientific, catalog number: C18064 (1000859) ]
  6. MicroAmp® optical 384-well reaction plate (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309849 )
  7. MicroAmp® optical adhesive film (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311971 )
  8. Piercing needle
  9. 100-mm dish
  10. Bromophenol blue (BPB) (Sigma-Aldrich, catalog number: B0126 )
  11. Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 )
  12. Dimethyl sulfoxide (DMSO)
  13. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 20012027 )
  14. RiboLock RNase inhibitor (40 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
  15. TRIzol® reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596018 )
  16. Chloroform
  17. Isopropanol
  18. GlycoBlueTM coprecipitant (15 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9515 )
  19. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  20. Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9930 )
  21. Random primers (150 ng/µl) (Sigma-Aldrich, catalog number: 11034731001 )
  22. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0193 )
  23. Maxima reverse transcriptase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0741 )
  24. KAPA SYBR® FAST ABI prism 2x qPCR master mix (Kapa Biosystems, catalog number: KK4605 ), or SYBR Green from other vendors
  25. EDTA
  26. Sucrose (Sigma-Aldrich, catalog number: S1888 )
  27. NaCl
  28. Tris-HCl
  29. MgCl2
  30. KCl
  31. Nonidet P-40
  32. DTT
  33. 5x RT buffer (250 mM Tris-HCl [pH 8.3 at 25 °C], 375 mM KCl, 15 mM MgCl2, 50 mM DTT, provided with Maxima Reverse Transcriptase)
  34. cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11873580001 )
  35. 2.2 M sucrose (MW 342.3) (see Recipes)
  36. 10x salts solution (see Recipes)
  37. Chase solution (60% sucrose) (see Recipes)
  38. Cycloheximide (CHX) (1,000x) (see Recipes)
  39. 25x protease inhibitors (see Recipes)
  40. Polysome extraction buffer (PEB) (see Recipes)

Equipment

  1. SW 41 Ti rotor package (Beckman Coulter, catalog number: 331336 )
  2. Manual pipettor (SP Scienceware - Bel-Art Products - H-B Instruments, catalog number: F37911-1010 )
  3. Cell scraper
  4. Vortexer
  5. Refrigerated centrifuge (Eppendorf, model: 5430 R )
  6. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ND-ONE-W )
  7. OptimaTM XE 90K - preparative ultracentrifuge (Beckman Coulter, catalog number: A94471 )
  8. Spectrophotometer
  9. PCR strip tube rotor, mini centrifuge C1201 [Denville Scientific, catalog number: C1201-S (1000806) ]
  10. Veriti® 96-Well thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4375786 )
  11. Eppendorf ThermoMixer® F1.5 (Eppendorf, catalog number: 5384000012 )
  12. MPS 1000 mini plate spinner (Next Day Science, catalog number: C1000 )
  13. Density gradient fractionation system (Brandel, catalog number: BR-188 )
  14. QuantStudio 5 Real-Time PCR System, 384-well (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: A28140 )

Procedure

  1. Preparation of sucrose gradients (Table 1) (Notes 3 and 4)

    Table 1. Preparation of sucrose stock solutions


    1. Prepare stock solutions for 10%, 20%, 30%, 40% or 50% sucrose as mentioned in the table and store it at 4 °C until use.
    2. Prepare the sucrose gradients a day before the polysome fractionation experiment and pre-cool the SW41Ti rotor and buckets at 4 °C overnight.
    3. Using prepared solutions containing 10%, 20%, 30%, 40% or 50% sucrose (see Table 1), begin by adding 2.2 ml of 10% sucrose gradient to the bottom of the thin-wall ultracentrifuge tube.
    4. Underlay each subsequent layer (2.2 ml each of 20%, 30%, 40%, then 50%) using a 9” Pasteur pipet and manual pipettor (Figure 1; Note 1).
    5. Leave gradients at 4 °C overnight to allow the gradient to become linear (Figure 2; Notes 6 and 7)
    6. Make chase solution (see Recipes), a high-density sucrose solution which will push the sample through the absorbance detector, with a speck of bromophenol blue (BPB) powder added for slight color (optional step to observe the transition between samples and the chase solution) and store at 4 °C.


    Figure 1. Schematic of sucrose gradient preparation

  2. Cytoplasmic lysate preparation
    1. Add 10 µl of 100 mg/ml cycloheximide (CHX) in DMSO to the cells containing 10 ml culture media resulting in 100 µg/ml CHX and incubate for 10 min at 37 °C (Note 2).
    2. Remove media and wash the cells three times with ice cold PBS supplemented with 100 µg/ml CHX.
    3. Trypsinize for 3 min or long enough for the cells to come off the plate or scrape the cells with cell scraper and transfer the cells to a 15-ml tube using cold PBS with CHX to rinse the plate (Note 8).
    4. Pellet the cells by centrifugation for 5 min at 500 x g.
    5. Cell pellets can be stored at -80 °C or immediately used for cytoplasmic lysate preparation.
    6. Disrupt the cell pellet by pipetting 0.5 to 1.0 ml polysome extraction buffer (PEB) containing 100 µg/ml CHX, 1x protease inhibitors and 1:1,000 dilution of RiboLock RNase inhibitor.
    7. Transfer the cell suspension to a 1.7-ml microfuge tube and incubate on ice for 10 min with occasional inverting every two minutes (do not vortex).
    8. Centrifuge at 12,000 x g for 10 min at 4 °C to pellet the nuclei and debris.
    9. Transfer approximately 9/10 of the total volume of the supernatant (cytoplasmic lysate) to a fresh tube.
    10. Measure the protein concentration by Bradford assay and/or the total RNA concentration by Nanodrop.
    11. This supernatant can also be stored at -80 °C and fractionated another time or proceed to polysome fractionation.

  3. Polysome fractionation
    1. Carefully layer equal amounts of cytoplasmic lysate (1 ml or less containing 1.0-1.5 mg protein or 100-300 µg RNA) to the top of a 10-50% sucrose gradient. Be sure all tubes are equally balanced.
    2. Using a Beckman ultracentrifuge, centrifuge the gradients for 90 min in a SW41Ti swinging bucket rotor at 190,000 x g (~39,000 rpm) at 4 °C with maximum acceleration and brake (Figure 2).


      Figure 2. Schematic of polysome fractionation

    3. Keep the gradients on ice after centrifugation. To begin, place the gradient onto the fractionating system - the piercing needle will puncture the bottom of the tube.
    4. Collect 1 ml/min fractions into 12 tubes per gradient and immediately transfer the fractions to an ice bucket.
    5. The 254 nm spectrophotometer within the optical unit of the fractionation system reads the RNA amount in each fraction and plots a chart which displays the polysome profile of the gradient (Figure 3A; Note 5).
    6. Analyze the fractions as desired or store the fractions at -80 °C until use.

  4. RNA and cDNA preparation from sucrose gradient
    1. Take 0.5 ml of each sucrose fraction in a fresh 1.7-ml tube, and add 0.5 ml TRIzol and 200 µl chloroform.
    2. Vortex or shake vigorously for 10 sec and centrifuge at 13,000 x g for 15 min at 4 °C.
    3. Transfer 500 µl of the upper layer to a fresh microfuge tube containing 1 ml isopropanol and 2 µl of 15 mg/ml Glycoblue.
    4. Precipitate the RNA by incubating overnight at -20 °C followed by centrifugation at 13,000 x g for 15 min at 4 °C.
    5. Discard the supernatant and wash pellet once with 1 ml ice-cold 70% ethanol.
    6. Centrifuge at 13,000 x g for 15 min at 4 °C and discard the supernatant.
    7. Centrifuge for few seconds at 4 °C to collect the remaining ethanol at the bottom of the tube.
    8. Discard the ethanol and air dry RNA pellet for 5-10 min at 25 °C.
    9. Dissolve RNA in 12 µl nuclease-free water (Note 9).
    10. Use the whole RNA solution for reverse transcription by preparing a 20-µl reaction containing 12 µl of prepared RNA, 1 µl random hexamers, 4 µl 5x RT buffer, 1 µl RiboLock, 1 µl dNTP mix and 1 µl Maxima reverse transcriptase.
    11. Mix and centrifuge for a few seconds to settle the reaction mixture at the bottom of the tube.
    12. The cDNA synthesis was performed using the thermal cycler set at 25 °C for 10 min, 50 °C for 30 min and 5 min at 85 °C.
    13. The cDNA product can be stored at -20 °C or -80 °C, or used immediately for RT-qPCR analysis.

  5. RT-qPCR analysis of mRNA distribution over the sucrose gradient
    1. Dilute the cDNAs (20 µl volume) with nuclease-free water to 1,000 µl final volume.
    2. To amplify specific transcripts, prepare the gene-specific forward and reverse primer mix at a final concentration of 1 µM in nuclease-free water. Use primer sets for mRNAs of interest as well as for 1 or 2 mRNAs encoding housekeeping proteins (e.g., ACTB mRNA or GAPDH mRNA).
    3. Prepare 20 µl qPCR reactions in a 384-well plate, add 10 µl 2x SYBR Green PCR mix, 5 µl cDNA, and 5 µl primer mix.
    4. Cover the plate with optical adhesive film and vortex at maximum speed for 10 sec to mix the reaction.
    5. Centrifuge the plate for 30 sec using Plate Spinner to settle the reactions at the bottom of the wells.
    6. Use a QuantStudio 5 Real-Time PCR System for qPCR with a cycle set up of 3 min at 95 °C and 40 cycles of 5 sec at 95 °C plus 20 sec at 60 °C.
    7. It is recommended to analyze dissociation curves to verify that the primer set is optimal.
    8. Using the cycle threshold (CT) values, the percent (%) distribution for the mRNAs across the gradients can be calculated using the ΔCT method (Tables 2 and 3).

      Table 2. % of mRNA A distribution over sucrose gradient in control cells


      Table 3. % of mRNA A distribution over sucrose gradient in treated cells

Data analysis

The density gradient fractionation system generates a polysome profile for each sample, which includes, from light to heavy fractions, fractions without ribosomal material, and fractions with 40S, 60S, 80S (monosomes), and low- and high-molecular-weight (LMW and HMW) polysomes. As these peaks did not appear to be different in the treated cells compared to control of the hypothetical example in Figure 3A, the treatment does not seem to have a major effect on the global mRNA translation. However, the % distribution of mRNA A across the gradient shows that this mRNA was most abundant in the actively translating fractions (fractions 8 to 10, spanning high-molecular-weight polysomes) of the gradient containing control sample (Table 2 and Figure 3B, solid line). In the treated cells, the distribution of mRNA A displayed a leftward shift on the gradient and was most abundant in fractions 5 to 7 (spanning low-molecular-weight polysomes) (Table 3 and Figure 3B, dotted line), indicating that mRNA A associated with smaller polysomes after treatment of cells. These data are consistent with the notion that translation of mRNA A is suppressed in treated cells without changes in global mRNA translation (Figures 3A and 3B). The distribution of mRNAs encoding housekeeping proteins (e.g., GAPDH mRNA or ACTB mRNA) should be measured and plotted similarly (Figure 3C; Note 10). The fact that GAPDH mRNA did not show significant change in distribution pattern supports the notion that the reduced sizes of mRNA A polysomes following treatment is specific (Figures 3B and 3C).


Figure 3. Effect of treatment on distribution of mRNA A across the sucrose gradient. A. Cytoplasmic lysates from control and treated cells were fractionated through sucrose gradients. Global RNA polysome profiles generated by the density gradient fractionation system are shown. B. The relative distribution of the % mRNA A (left) and GAPDH mRNA (right), encoding a housekeeping protein, over the sucrose gradient was studied by RT-qPCR analysis of the RNA in each of the 12 gradient fractions.

Notes

  1. Add the sucrose gradient layers slowly without introducing any air bubbles.
  2. For good polysome peaks, use fresh lysates from one 100-mm dish not more than 80% confluent. Too few cells will result in smaller peaks, perhaps below the detector sensitivity settings. Cells which are overly confluent may not be in a proliferative state with active translation, so the polysome peaks will not appear as robust.
  3. Use either nuclease-free water or DEPC-treated water for all buffers and solutions.
  4. Do not autoclave sucrose.
  5. Fractionation of a 10-50% sucrose gradient without cell lysate should yield an absorbance profile that is a straight, flat line. If the profile generates a sloped line, then the sucrose itself is contributing to the absorbance readings and may interfere with the resolution of polysome peaks in the experimental lysates.
  6. The gradients are OK to use within 16 h to 3 days (at 4 °C).
  7. The linear gradient can be frozen at -20 °C or -80 °C for 2 weeks (or more) and used immediately after thawing.
  8. For differentiated cells (neurons, muscle) or senescent cells, it is better to add PEB directly to the plate and scrape the cells.
  9. Do NOT measure RNA concentrations in the polysome fractions as this will vary between fractions, with some fractions having very little RNA.
  10. There is no normalization of the RT-qPCR values for mRNA A to the control mRNA. The mRNAs encoding housekeeping proteins (e.g., GAPDH mRNA or ACTB mRNA) are processed side by side simply to test if generic mRNAs are distributed similarly across the gradients for different treatment conditions.
  11. To confirm that mRNAs fractionated along the gradients are indeed associated with polysomes, the cell lysates can be incubated with 20 mM EDTA for 10 min on ice before loading on the sucrose gradient. Unlike the control sample, the EDTA-treated sample should show no polysome peaks and increased peaks for monosomes and ribosomal subunits. In the EDTA-treated sample, the distribution of mRNAs encoding housekeeping proteins and the mRNA of interest should not peak in the polysome fractions and instead should accumulate in the unbound and monosome fractions, showing an overall shift toward lighter fractions of the gradient.

Recipes

  1. 2.2 M sucrose (MW 342.3)
    753.06 g of sucrose in 1,000 ml of nuclease-free water (Notes 3 and 4)
    Store at 4 °C
  2. 10x salts solution
    100 mM NaCl
    20 mM Tris-HCl (pH 7.5 )
    5 mM MgCl2
    Store at 4 °C
  3. Chase solution (60% sucrose)
    40 ml 2.2 M sucrose
    5 ml H2O
    5 ml 10x salts solution
    Speck of bromophenol blue powder (BPB) - optional
    Store at 4 °C
  4. Cycloheximide (CHX) (1,000x)
    Stock solution is 100 mg/ml in dimethyl sulfoxide (DMSO )
    Aliquot and store at -20 °C
  5. 25x protease inhibitors
    Dissolve 1 tablet of cOmplete protease inhibitors in 2 ml RNase-free water
    Aliquot and store at -20 °C
  6. Polysome extraction buffer (PEB)
    20 mM Tris-HCl (pH 7.5)
    100 mM KCl
    5 mM MgCl2
    0.5% Nonidet P-40
    Store at 4 °C
    Add protease inhibitors, RNase inhibitor, and 100 µg/ml CHX to an aliquot of PEB before use

Acknowledgments

This work was supported by the National Institute on Aging Intramural Research Program, National Institutes of Health.

References

  1. Abdelmohsen, K., Panda, A. C., Kang, M. J., Guo, R., Kim, J., Grammatikakis, I., Yoon, J. H., Dudekula, D. B., Noh, J. H., Yang, X., Martindale, J. L. and Gorospe, M. (2014). 7SL RNA represses p53 translation by competing with HuR. Nucleic Acids Res 42(15): 10099-10111.
  2. Panda, A. C., Abdelmohsen, K., Martindale, J. L., Di Germanio, C., Yang, X., Grammatikakis, I., Noh, J. H., Zhang, Y., Lehrmann, E., Dudekula, D. B., De, S., Becker, K. G., White, E. J., Wilson, G. M., de Cabo, R. and Gorospe, M. (2016). Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis. Nucleic Acids Res 44(5): 2393-2408.
  3. Panda, A. C., Abdelmohsen, K., Yoon, J. H., Martindale, J. L., Yang, X., Curtis, J., Mercken, E. M., Chenette, D. M., Zhang, Y., Schneider, R. J., Becker, K. G., de Cabo, R. and Gorospe, M. (2014a). RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels. Mol Cell Biol 34(16): 3106-3119.
  4. Panda, A. C., Sahu, I., Kulkarni, S. D., Martindale, J. L., Abdelmohsen, K., Vindu, A., Joseph, J., Gorospe, M. and Seshadri, V. (2014b). miR-196b-mediated translation regulation of mouse insulin2 via the 5'UTR. PLoS One 9(7): e101084.
  5. Schwanhausser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W., Selbach, M. (2011). Global quantification of mammalian gene expression control. Nature 473; 337-342.

简介

真核细胞通过精确调节特定基因产物的表达来适应外部或内部信号的变化。蛋白质编码基因的表达受到转录和转录后水平的控制。在后面的步骤中,翻译的调节在需要蛋白质表达模式快速变化的细胞过程中特别重要。 mRNA的翻译效率由RNA结合蛋白(RBP)和非编码(nc)RNA如微RNA(Panda等人,2014a和2014b; Abdelmohsen等人)改变2014)。调节选择性mRNA翻译的因素的影响是RNA生物学中的一个关键问题。多核糖体(多核糖体)分馏分析是评估核糖体与给定mRNA的关联的有效方法。它提供了关于该mRNA的翻译状态的有价值的信息,这取决于与它们相关联的核糖体的数目,并且鉴定未翻译的mRNA(Panda等人,2016)。与许多核糖体相关的mRNA形成大量的多核糖体,预计将被主动翻译,而与少数或没有核糖体相关的mRNA有可能翻译不佳。总之,多聚糖分馏分析允许直接测定整个转录组水平的翻译效率以及个体mRNA。

背景 基因表达受到许多步骤的调控,包括基因转录,mRNA前剪接和mRNA输出到细胞质,转换和翻译。鉴于转录后基因调控机制对细胞中整体蛋白表达模式的强烈影响,对阐明控制这些事件的过程有很大的兴趣。特别地,转录组的一半的稳态mRNA水平与从这些mRNA翻译的蛋白质水平差异相关,表明细胞中的蛋白质水平以mRNA翻译水平和/或蛋白质稳定性被有效调节(Schwanhausser等人,2011)。可以使用许多测定来研究如何在转录组水平和单个mRNA水平上响应不同条件调节翻译。传统上,Western印迹分析,嘌呤霉素标记和 S-甲硫氨酸/半胱氨酸标记测定已被用于测量效率mRNA翻译。
 这里讨论的方法主要是分析在给定的mRNA上形成的多核糖体的大小。这个分析的前提是,预期在更大的多核糖体中发现的mRNA可以稳健地翻译,而存在于较小的多核糖体中或缺乏核糖体组分的mRNA预期被翻译得很差或保持未翻译。该协议允许通过“冻结”翻译核糖体捕获活跃的mRNA,从而允许测量在给定mRNA上形成的多核糖体的相对大小。该方法已被成功应用于数十项研究,以分析RBP和微小RNA如何影响靶mRNA的翻译,并可用于探索多核糖体相关蛋白和非编码RNA对全球翻译和特异性mRNA翻译的作用。

关键字:mRNA翻译, 蛋白质合成, 核糖体, 多聚核糖体, 蔗糖梯度, 分馏, RT-qPCR

材料和试剂

  1. 管,薄壁,聚丙烯,13.2毫升(Beckman Coulter,目录号:331372)
  2. 9"巴斯德吸管(Kimble Chase Life Science and Research Products,目录号:883350-0009)
  3. 15毫升管子
  4. Posi-Click 1.7-ml微量离心管(Denville Scientific,目录号:C2171)
  5. ThermoGrid TM 刚性条带0.2-ml PCR管[(Denville Scientific,目录号:C18064(1000859)]
  6. MicroAmp ®光学384孔反应板(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4309849)
  7. MicroAmp ®光学粘合膜(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4311971)
  8. 穿刺针
  9. 100毫米盘
  10. 溴酚蓝(BPB)(Sigma-Aldrich,目录号:B0126)
  11. 环己酰亚胺(CHX)(Sigma-Aldrich,目录号:C7698)
  12. 二甲基亚砜(DMSO)
  13. Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:20012027)
  14. RiboLock RNase抑制剂(40U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0381)
  15. TRIzol ®试剂(Thermo Fisher Scientific,Ambion TM ,目录号:15596018)
  16. 氯仿
  17. 异丙醇
  18. GlycoBlue TM共沉淀物(15mg/ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9515)
  19. 乙醇(Sigma-Aldrich,目录号:E7023)
  20. 无核酸酶水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9930)
  21. 随机引物(150ng /μl)(Sigma-Aldrich,目录号:11034731001)
  22. dNTP混合物(每种10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0193)
  23. Maxima逆转录酶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:EP0741)
  24. KAPA SYBR ® FAST ABI棱镜2x qPCR主混合物(Kapa Biosystems,目录号:KK4605)或其他供应商的SYBR Green
  25. EDTA
  26. 蔗糖(Sigma-Aldrich,目录号:S1888)
  27. NaCl
  28. Tris-HCl
  29. MgCl 2
  30. KCl
  31. Nonidet P-40
  32. DTT
  33. 5×RT缓冲液(250mM Tris-HCl [25℃下的pH8.3],375mM KCl,15mM MgCl 2,50mM DTT,Maxima Reverse Transcriptase提供)
  34. 完全不含EDTA的蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:11873580001)
  35. 2.2 M蔗糖(MW 342.3)(参见食谱)
  36. 10x盐溶液(参见食谱)
  37. 追逐溶液(60%蔗糖)(参见食谱)
  38. 环己酰亚胺(CHX)(1,000x)(参见食谱)
  39. 25x蛋白酶抑制剂(参见食谱)
  40. 多聚赖氨酸提取缓冲液(PEB)(见食谱)

设备

  1. SW 41 Ti转子封装(Beckman Coulter,目录号:331336)
  2. 手动移液器(SP Scienceware - Bel-Art Products - H-B Instruments,目录号:F37911-1010)
  3. 细胞刮刀
  4. Vortexer
  5. 冷冻离心机(Eppendorf,型号:5430 R)
  6. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,目录号:ND-ONE-W)
  7. Optima TM XE 90K - 制备型超速离心机(Beckman Coulter,目录号:A94471)
  8. 分光光度计
  9. PCR带管转子,迷你离心机C1201 [Denville Scientific,目录号:C1201-S(1000806)]
  10. Veriti ® 96孔热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4375786)
  11. Eppendorf ThermoMixer ® F1.5(Eppendorf,目录号:5384000012)
  12. MPS 1000迷你板旋转器(翌日科学,目录号:C1000)
  13. 密度梯度分馏系统(Brandel,目录号:BR-188)
  14. QuantStudio 5实时PCR系统,384孔(Thermo Fisher Scientific,Applied Biosystems TM,目录号:A28140)

程序

  1. 蔗糖梯度的制备(表1)(注3和4)

    表1.蔗糖储备溶液的制备


    1. 准备表中提到的10%,20%,30%,40%或50%蔗糖的储备溶液,并将其储存在4°C直到使用。
    2. 在多核糖分馏实验前一天准备蔗糖梯度,并在4℃预冷SW41Ti转子和桶过夜。
    3. 使用含有10%,20%,30%,40%或50%蔗糖的制备溶液(见表1),首先向薄壁超速离心管的底部加入2.2ml 10%蔗糖梯度。
    4. 使用9"巴斯德移液器和手动移液器(图1;注1),将每个后续层(每个2.2ml,分别为20%,30%,40%,然后50%)。
    5. 将梯度保持在4°C过夜,使梯度变成线性(图2;注6和7)
    6. 做一个高浓度的蔗糖溶液(请参阅食谱),这个高浓度的蔗糖溶液会将样品推入吸光度检测器,加入少许颜色的溴酚蓝(BPB)粉末斑点(观察样品与追踪之间的转换溶液)并在4℃下储存。


    图1.蔗糖梯度准备示意图

  2. 细胞质裂解物制备
    1. 在含有10ml培养基的细胞中加入10μl100mg/ml放线菌酮(CHX)的DMSO溶液,得到100μg/ml CHX,37℃孵育10分钟(注2)。
    2. 取出培养基并用补充有100μg/ml CHX的冰冷PBS洗涤细胞三次。
    3. 胰蛋白酶消化3分钟或足够长以使细胞从板上脱落或用细胞刮刀刮擦细胞,并用CHX的冷PBS将细胞转移到15-ml管中以冲洗板(注8)。
    4. 通过在500×g下离心5分钟来造粒细胞。
    5. 细胞沉淀物可以储存在-80°C或立即用于细胞质裂解液制备。
    6. 通过吸取含有100μg/ml CHX,1×蛋白酶抑制剂和1:1,000稀释的RiboLock RNA酶抑制剂的0.5至1.0ml多聚糖提取缓冲液(PEB)来破坏细胞沉淀物。
    7. 将细胞悬浮液转移到1.7微升的微量离心管中,并在冰上孵育10分钟,每两分钟偶尔反转(不要旋涡)。
    8. 在4℃下以12,000xg离心10分钟以沉淀核和碎屑。
    9. 将上清液(胞质裂解物)的总体积的约9/10转移到新鲜管中
    10. 通过Bradford测定法测定蛋白质浓度和/或Nanodrop的总RNA浓度
    11. 该上清液也可以储存在-80℃,再次分馏,或进行多分支分馏
  3. 多聚糖分馏
    1. 小心地将等量的细胞质裂解物(含有1.0-1.5mg蛋白质或100-300μgRNA的1ml或更少)分层至10-50%蔗糖梯度的顶部。确保所有的管均衡。
    2. 使用Beckman超速离心机,在最大加速度和制动下,在4°C下,以最大加速度和制动力,以190,000 x g(〜39,000 rpm)的SW41Ti摆动叶轮转子将梯度离心90分钟(图2)。


      图2.多聚体分馏示意图

    3. 离心后将梯度保持在冰上。首先,将梯度放置在分馏系统上 - 穿刺针将穿刺管的底部。
    4. 每个梯度收集1ml/min级分至12管,并立即将馏分转移到冰桶中
    5. 分离系统光学单元内的254 nm分光光度计读取每个级分中的RNA量,并绘制显示梯度多聚体谱的图表(图3A;注5)。
    6. 根据需要分析馏分,或将馏分储存在-80°C直至使用。

  4. RNA和cDNA制备蔗糖梯度
    1. 在新鲜的1.7-ml管中取0.5ml各蔗糖级分,加入0.5ml TRIzol和200μl氯仿。
    2. 涡旋或剧烈振荡10秒,并在4℃下以13,000 x g离心15分钟。
    3. 将500μl上层转移到含有1ml异丙醇和2μl15mg/ml Glycoblue的新鲜微量离心管中。
    4. 通过在-20℃下孵育过夜沉淀RNA,然后在4℃下以13,000×g离心15分钟。
    5. 弃去上清液,用1ml冰冷的70%乙醇洗涤沉淀一次
    6. 在4℃下以13,000×g离心15分钟,弃去上清液。
    7. 在4℃离心几秒钟,收集管底部剩余的乙醇
    8. 在25℃下弃去乙醇和空气干燥的RNA颗粒5-10分钟
    9. 将RNA溶解在12μl无核酸酶的水中(注9)
    10. 通过制备含有12μl制备的RNA,1μl随机六聚体,4μl5x RT缓冲液,1μlRiboLock,1μldNTP混合物和1μlMaxima逆转录酶的20μl反应,使用整个RNA溶液进行逆转录。 />
    11. 混合并离心几秒钟以使反应混合物在管底沉降。
    12. 使用在25℃下设定10分钟,50℃30分钟和85℃5分钟的热循环仪进行cDNA合成。
    13. cDNA产物可以在-20°C或-80°C保存,或立即用于RT-qPCR分析。

  5. 蔗糖梯度上mRNA分布的RT-qPCR分析
    1. 用无核酸酶的水稀释cDNA(20μl体积)至最终体积为1,000μl
    2. 为了扩增特异性转录物,在无核酸酶的水中制备终浓度为1μM的基因特异性正向和反向引物混合物。使用感兴趣的mRNA的引物组以及编码内切蛋白(例如,/或> ACTB mRNA或GAPDH mRNA)的1或2个mRNA。 br />
    3. 在384孔板中准备20μlqPCR反应,加入10μl2x SYBR Green PCR混合液,5μlcDNA和5μl引物混合物。
    4. 用光学粘合剂膜覆盖板,并以最大速度涡旋10秒以混合反应。
    5. 使用Plate Spinner将板离心板30秒,以平衡孔底部的反应
    6. 对于qPCR使用QuantStudio 5实时PCR系统,在95℃下设置3分钟,在95℃加5秒,60℃加20秒的循环。
    7. 建议分析解离曲线以验证引物组是否为最佳
    8. 使用周期阈值(CT)值,可以使用ΔCT方法(表2和3)计算跨越梯度的mRNA的百分比(%)分布。

      表2.对照细胞中蔗糖梯度的mRNA A分布比例


      表3.处理细胞中蔗糖梯度的mRNA A分布比例

数据分析

密度梯度分馏系统为每个样品产生多聚体谱,其中包括从轻到重级分的无核糖体物质的级分,以及具有40S,60S,80S(单体)和低分子量(LMW)和HMW)多核糖体。由于与图3A中的假想实例的对照相比,这些峰在处理的细胞中似乎并不显示不同,所以治疗似乎对全局mRNA翻译似乎没有重大影响。然而,跨越梯度的mRNA A的%分布显示,在含有对照样品的梯度的活性翻译级分(分数为8至10,跨越高分子量多核糖体)中,该mRNA最丰富(表2和图3B,实线)。在处理的细胞中,mRNA的分布在梯度上显示向左移动,并且在5至7级中最丰富(跨越低分子量多核糖体)(表3和图3B,虚线),表明mRNA A在细胞处理后与较小的多核糖体相关。这些数据与在没有全局mRNA翻译变化的处理细胞中mRNA A的翻译被抑制的概念一致(图3A和3B)。应该测量和编制类似的编码内部蛋白质(例如,/或> GAPDH mRNA或ACTB mRNA)的mRNA的分布(图3C;注10) 。 GAPDH mRNA在分布模式中没有显示显着变化的事实支持下述治疗后mRNA A多核糖体的尺寸减小的概念(图3B和3C)。


图3.处理对蔗糖梯度上mRNAA分布的影响。来自对照和处理的细胞的细胞质裂解物通过蔗糖梯度分级。显示了由密度梯度分馏系统产生的全球RNA多聚体谱。 B.通过RT-qPCR分析RNA中的每一种,研究了在蔗糖梯度上编码持家蛋白质的%mRNA A(左)和GAPDH mRNA(右)的相对分布12个梯度分数。

笔记

  1. 缓慢加入蔗糖梯度层,不引入任何气泡
  2. 对于良好的多聚体峰,使用不超过80%汇合的一个100毫米皿的新鲜裂解物。太小的细胞将导致较小的峰值,也许低于检测器灵敏度设置。过度融合的细胞可能不具有主动翻译的增殖状态,因此多聚体峰不会显示为强壮的。
  3. 使用无核酸酶水或DEPC处理过的水用于所有缓冲液和溶液。
  4. 不要高压蔗糖。
  5. 不含细胞裂解物的10-50%蔗糖梯度的分级应产生一个直线平坦线的吸光度分布。如果轮廓产生斜线,则蔗糖本身有助于吸光度读数,并可能干扰实验裂解物中多聚体峰的分辨率。
  6. 梯度可在16到3天(4°C)使用。
  7. 线性梯度可以在-20°C或-80°C下冷冻2周(或更长时间),并在解冻后立即使用。
  8. 对于分化的细胞(神经元,肌肉)或衰老细胞,最好将PEB直接加入板中并刮擦细胞。
  9. 不要测量多聚体部分中的RNA浓度,因为它们的分数会有差异,有些部分RNA含量非常低
  10. mRNA A与对照mRNA没有正常化RT-qPCR值。编码整理蛋白(例如,/或> GAPDH mRNA或ACTB mRNA)的mRNA被简单地处理以测试通用mRNA是否类似地分布不同治疗条件的梯度。
  11. 为了证实沿着梯度分级的mRNA确实与多核糖体相关,细胞裂解物可以在冰上装载之前与20mM EDTA一起在冰上孵育10分钟。与对照样品不同,EDTA处理的样品应不显示多核糖体峰,并显示单体和核糖体亚基的增加的峰。在EDTA处理的样品中,编码持续性蛋白质的mRNA和感兴趣的mRNA的分布不应该在多聚体部分中达到峰值,而应该在未结合和单体部分中积累,显示出向梯度的较轻部分的整体转移。 />

食谱

  1. 2.2 M蔗糖(MW 342.3)
    753.06克蔗糖在1000毫升无核酸酶水中(注3和4)
    储存于4°C
  2. 10x盐溶液
    100 mM NaCl
    20mM Tris-HCl(pH7.5)
    5mM MgCl 2
    储存于4°C
  3. 追逐溶液(60%蔗糖)
    40 ml 2.2 M蔗糖 5ml H 2 O O
    5 ml 10x盐溶液
    溴酚蓝粉(BPB) - 可选
    储存于4°C
  4. 环己酰亚胺(CHX)(1,000x)
    储存溶液在二甲基亚砜(DMSO)中为100mg/ml 等分并储存于-20°C
  5. 25x蛋白酶抑制剂
    将1片蛋白酶抑制剂溶解于2毫升无RNase的水中 等分并储存于-20°C
  6. 多聚赖氨酸提取缓冲液(PEB)
    20mM Tris-HCl(pH7.5)
    100 mM KCl
    5mM MgCl 2
    0.5%Nonidet P-40
    储存于4°C
    在使用前将蛋白酶抑制剂,RNase抑制剂和100μg/ml CHX加入到PEB的等分试样中

致谢

这项工作得到了国立卫生研究院老龄化研究方案的支持。

参考文献

  1. Abdelmohsen,K.,Panda,AC,Kang,MJ,Guo,R.,Kim,J.,Grammatikakis,I.,Yoon,JH,Dudekula,DB,Noh,JH,Yang,X.,Martindale,JL and Gorospe ,M.(2014)。 7SL RNA represses p53 translation通过与HuR竞争。 Nucleic Acids Res 42(15):10099-10111。
  2. 熊猫,AC,Abdelmohsen,K.,Martindale,JL,Di Germanio,C.,Yang,X.,Grammatikakis,I.,Noh,JH,Zhang,Y.,Lehrmann,E.,Dudekula,DB,De,S 。,Becker,KG,White,EJ,Wilson,GM,de Cabo,R.and Gorospe,M。(2016)。 MYF5的新型RNA结合活性增强肌发生期间的Ccnd1 /细胞周期蛋白D1 mRNA翻译。核酸Res 44(5 ):2393-2408。
  3. 熊猫,AC,Abdelmohsen,K.,Yoon,JH,Martindale,JL,Yang,X.,Curtis,J.,Mercken,EM,Chenette,DM,Zhang,Y.,Schneider,RJ,Becker,KG,de Cabo ,R.和Gorospe,M.(2014a)。 RNA结合蛋白AUF1通过调节MEF2C表达水平来促进肌细胞生成。分子细胞生物学34(16):3106-3119。
  4. 熊猫,AC,Sahu,I.,Kulkarni,SD,Martindale,JL,Abdelmohsen,K.,Vindu,A.,Joseph,J.,Gorospe,M。和Seshadri,V.(2014b)。 ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25003985"target ="_ blank">通过5'UTR的小鼠胰岛素2的miR-196b介导的翻译调节。/a> PLoS One 9(7):e101084。
  5. Schwanhausser,B.,Busse,D.,Li,N.,Dittmar,G.,Schuchhardt,J.,Wolf,J.,Chen,W.,Selbach,M。(2011)。< a class = ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/21593866"target ="_ blank">哺乳动物基因表达控制的全球定量。 自然 473; 337-342。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Panda, A. C., Martindale, J. L. and Gorospe, M. (2017). Polysome Fractionation to Analyze mRNA Distribution Profiles. Bio-protocol 7(3): e2126. DOI: 10.21769/BioProtoc.2126.
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