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RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells
使用从HEK-293细胞亲和纯化获得的RNA外泌体复合物进行RNA降解测定   

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Abstract

The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3’-5’ exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al., 2016) and further detailed in the companion bio-protocol to this one (Domanski and LaCava, 2017).

Keywords: RNA exosome(RNA外显子), EXOSC10(EXOSC10), Affinity capture(亲和力捕获), RNA degradation(RNA降解), Ribonuclease(核糖核酸酶)

Background

In our previous work, we established an isogenic HEK-293 cell line expressing C-terminally 3xFLAG-tagged exosome component EXOSC10 (RRP6), under the control of a tetracycline-inducible CMV promoter (HEK-293 Flp-In T-REx – Thermo Fisher Scientific). This system permitted us to express the tagged EXOSC10 protein at a level comparable to the endogenous WT protein, and to explore exosome purification protocols using a magnetic anti-FLAG affinity medium and protein extracts derived from cryomilled cell powder (Domanski et al., 2012). Building on this, we developed protocols for further purifying RNA exosomes by rate-zonal centrifugation, using glycerol density gradients, and assaying their ribonuclease (RNase) activity (Domanski et al., 2016). EXOSC10-containing exosome fractions exhibited apparent exoribonucleolytic activity, consistent with distributive 3’-5’ hydrolysis; the same assay permitted the detection and monitoring of the processive RNase activity of affinity purified DIS3-3xFLAG ([Wasmuth and Lima, 2012] and references therein). The protocol presented here describes the RNase assay. Although this protocol presumes glycerol gradient purified EXOSC10-3xFLAG-containing exosomes as the point of entry into the assay (Domanski and LaCava, 2017), the method should be applicable to any sufficiently pure and concentrated samples.

Materials and Reagents

Note: Catalog numbers are given for most of the reagents listed below; an equivalent quality reagent from an alternative supplier can typically be substituted with comparable results. Due to the potential for artifacts introduced by contaminating RNases, care should be taken to follow best practices, such as the use of RNase-free solutions and reagents and/or using DEPC-treatment where appropriate (Farrell, 2010). Standard materials and reagents for urea-polyacrylamide gel electrophoresis are required; we use the National Diagnostics system but such gels can be prepared using standard methods (Sambrook and Russell, 2006).

  1. Pipette tips  
  2. 1.5 ml microcentrifuge tubes (e.g., Eppendorf, catalog number: 022363204 )
  3. 10-well Gel Combs, 1.5 mm (Thermo Fisher Scientific, NovexTM, catalog number: NC3510 )
  4. Empty Gel Cassettes, mini, 1.5 mm (Thermo Fisher Scientific, NovexTM, catalog number: NC2015 )
  5. Syringe with a bent needle (to wash the residual urea out of the gel wells before sample loading)
  6. HEK-293 Flp-In T-REx EXOSC10-3xFLAG cells ([Domanski et al., 2016]; available upon request) Parental HEK-293 Flp-In T-REx cell line (Thermo Fisher Scientific, InvitrogenTM, catalog number: R78007 )
  7. Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9932 )
  8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  9. 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 54457 )
  10. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  11. Anti-FLAG M2 (Sigma-Aldrich, catalog number: F3165 ) antibody conjugated Dynabeads M-270 Epoxy (Thermo Fisher Scientific, InvitrogenTM, catalog number: 14302D ) (Domanski and LaCava, 2017)
  12. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  13. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  14. RNA substrate with 6-carboxyfluorescein (6-FAM) at the 5’-end (Reconstitute in 10 mM HEPES ~pH 7-7.5, at 0.5 nmol/µl. Store aliquots at -80 °C)
    1. Generic substrate: 5’-(6-FAM)-CCUAU UCUAU AGUGU CACCU AAAUG CUAGA GCU modC(2’-O-Me)-3’
    2. Blocked substrate: 5’-(6-FAM)-CCUAU UCUAU AGUGU CACCU AAAUG CUAGA GCU modC(2’-O-Me, 3’-PO4)-3’
    Note: Both substrates were ordered from the Integrated DNA Technologies at 100 nmole scale, purified by RNase-free HPLC.
  15. RNasin® ribonuclease inhibitors (Promega, catalog number: N2515 )
  16. Formamide (Sigma-Aldrich, catalog number: 47671 )
  17. DNA loading dye (Thermo Fisher Scientific, catalog number: R0611 )
    Note: This consists of 10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA–and so, can be prepared rather than purchased.
  18. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  19. Tris base (Sigma-Aldrich, catalog number: 93362 )
  20. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B7901 )
  21. N,N,N’,N’-tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T7024 )
  22. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678 )
    Note: Prepare 10% solution in H2O and store at 4 °C for several weeks.
  23. UreaGel 29:1 Denaturing Gel System (National Diagnostics, catalog number: EC-829 )
  24. Recapture solution (see Recipes)
  25. Wash solution (see Recipes)
  26. 2x reaction solution (see Recipes)
  27. 2x RNA loading solution (see Recipes)
  28. 1x TBE (5x or 10x stock can be prepared) (see Recipes)

Equipment

Note: Standard equipment for urea-polyacrylamide gel electrophoresis is required, as well as an imager capable of fluorescein detection (absorption λmax = 494 nm, emission λmax = 518 nm).

  1. Pipettes  
  2. Vortexer
  3. Benchtop mini-centrifuge
  4. Neodymium magnet microfuge tube rack (Thermo Fisher Scientific, catalog number: 12321D )
  5. Thermomixer (e.g., Eppendorf, model: ThermoMixer® F , catalog number: 5355000.011; or equivalent)
  6. XCell SureLock Mini (Thermo Fisher Scientific, model: SureLock® Mini-Cell , catalog number: EI0001)
  7. Electrophoresis power supply
  8. Imager with blue light (460 nm) epi-illumination and an Y515-Di (long-pass) filter–i.e., SYBR green settings. E.g., Fujifilm LAS-3000 series or newer (Fujifilm, model: LAS-3000 Series ; or equivalent)

Procedure

Note: Purify EXOSC10-containing RNA exosomes as described in the complementary protocol, Affinity purification of RNA exosomes from HEK-293 cells (Domanski and LaCava, 2017), or by other means. This assay may also be applied effectively to affinity purified DIS3-3xFLAG as previously described (Domanski et al., 2016). Examples of the manipulations associated with the affinity capture aspects of the following protocol can be viewed in our online video protocol (LaCava et al., 2016).

  1. Re-capture of RNA exosomes from the glycerol gradient
    1. After retrieving the fractions from the gradient, pool together up to two fractions constituting the peak of intact EXOSC10-containing exosomes (up to ~450 μl total volume).
      Note: If desired, save an aliquot of up to 50 μl (input) for other analyses.
    2. Add an equal volume of the recapture solution (see Recipe 1).
    3. Pre-wash 10 µl of the anti-FLAG magnetic medium (slurry) twice with 1 ml of recapture solution.
      Note: Combine 1 ml of recapture solution with the beads slurry and briefly vortex. Pulse-spin the tube in a mini-centrifuge to collect all the solution at the bottom and then place the tube on a magnetic tube rack. Wait until beads are collected at the side of the tube and remove the supernatant (using a pipet or an aspirator). Add additional recapture solution to the beads in the tube and repeat the process for the second wash; after removing the supernatant the beads may be held on ice and are ready for use. Both washing steps can be carried out at room temperature.
    4. Transfer diluted fractions from step A2 into a 1.5 ml tube with pre-washed beads.
    5. Incubate for 30 min with rotation at 4 °C (cold room).
    6. Place on a magnet, wait until the beads are collected at the side of the tube, and remove the supernatant.
      Note: The supernatant may be compared to the input by protein staining or Western blot to monitor the extent of exosome depletion.
    7. Wash once with 1 ml wash solution (see Recipe 2).
    8. Resuspend the affinity medium in 25 µl wash solution.
    9. Add 25 µl of the 2x reaction solution (see Recipe 3).
    10. Incubate at 37 °C with mixing at 1,000 rpm.
    11. Produce a time-course for the reaction by taking 10 µl aliquots at different time points e.g., 0, 2.5, 5, 15 and 30 min. Stop the reactions by adding 10 µl 2x RNA loading buffer (see Recipe 4).
      Note: This assay should ideally include two controls set up as independent reactions: a) as above, but using the blocked substrate, and b) a mock reaction with affinity medium and reaction solution containing the generic substrate, but no RNA exosomes. Both controls should reveal the presence of intact substrate across the time course, indicating that the preparation and solutions are free from interfering RNase contamination. DIS3 is capable of degrading the 3’-PO4 blocked substrate.
    12. Place on a magnet and transfer the supernatant to a fresh tube. Keep on ice.

  2. RNA PAGE and gel imaging
    1. Cast a 20% urea-polyacrylamide 10-well gel accordingly to the manufacturer’s instructions.
    2. Pre-run the gel in 1x TBE (see Recipe 5) at 15 W for 15-20 min.
      Note: The pre-running step clears excess free ions from the gel which affect the electrical current and heat generation. The cassette will warm during the pre-run and run; typical operating temperatures are between 45-60 °C. The heat produced may help maintain the RNA sample in a denatured, single stranded form; this is not expected to be a major variable for small oligo substrates lacking strong secondary structure.
    3. Heat the samples at 80 °C for 30 sec and then place on ice. Spin down briefly before loading.
      Note: Heating the sample denatures the RNA and dissociates RNA-protein assemblies.
    4. Wash the urea out from the wells using a syringe with a bent needle.
    5. Load on the gel the entire volume of each sample (20 µl) per well.
    6. Run at 15 W until the bromophenol blue tracking dye reaches the first line from the bottom of the plastic gel cassette.
    7. Open the cassette and image the gel directly using an appropriately configured imager (SYBR green on Fuji LAS series).
      Note: The gel does not need to be fixed, washed, or dried prior to imaging. Depending on the level of activity detected, some adjustment of the image brightness/contrast in software, after data collection, may improve visualization.

Data analysis

The distributive, 3’-5’ exoribonucleolytic activity of EXOSC10 can be observed as ‘laddering’ of the substrate degradation intermediates, shifting increasingly over time toward lower molecular mass species; and thus, increasingly toward the bottom of the gel. Such a pattern is depicted in Figure 1. The last step of the ladder (i.e., latest time point) may appear compressed relative to earlier time points because the enzyme may no longer exhibit appreciable activity on a terminally shortened pool of substrates and the rest of the pool converges on this size. A 3’-PO4 blocked substrate will prevent EXOSC10-derived 3’-5’ activity; full length substrate and an absence of breakdown products should be observed across all time points in this case. Note, however, that this expectation presumes the absence of DIS3 or that any DIS3 present in the purified exosome has been inactivated (Domanski et al., 2016; Zinder et al., 2016).


Figure 1. Representative result: RNA degradation assay using affinity-purified human RNA exosome complexes. Schematic diagram of a urea-polyacrylamide gel separation of the reaction products of an exosome RNase assay as described in this protocol, consistent with a distributive activity pattern and the original data. B. The original gel, reproduced from (Domanski et al., 2016): an ExoI preparation was incubated either with a generic or blocked substrate and the indicated time points were collected.

Recipes

  1. Recapture solution
    100 mM NaCl
    20 mM HEPES, pH 7.4
    1% Triton X-100
  2. Wash solution
    100 mM NaCl
    20 mM Tris-HCl, pH 8.0
    0.01% Triton X-100
  3. 2x reaction solution
    20 mM Tris-HCl, pH 8.0
    20 mM DTT
    0.5 mM MgCl2
    5% (v/v) RNasin® ribonuclease inhibitors
    0.4 pmol/µl RNA substrate
  4. 2x RNA loading solution
    95% formamide
    1% DNA loading dye
    20 mM EDTA
  5. 1x TBE (5x or 10x stock can be prepared)
    89 mM Tris base
    89 mM boric acid
    2 mM EDTA

Acknowledgments

We thank Professors Michael P. Rout and Torben Heick Jensen for their invaluable support of our research. We also thank Mr. Artem Serganov and Dr. Zhanna Hakhverdyan for proof reading. This work was supported in part by the National Institutes of Health grants P41GM109824 and P50GM107632, the Lundbeck Foundation, and the Danish National Research Foundation.

References

  1. Domanski, M. and LaCava, J. (2017). Affinity purification of RNA exosomes from HEK-293 cells. Bio-Protoc 7(08): e2238.
  2. Domanski, M., Molloy, K., Jiang, H., Chait, B. T., Rout, M. P., Jensen, T. H. and LaCava, J. (2012). Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels. Biotechniques 0(0): 1-6.
  3. Domanski, M., Upla, P., Rice, W. J., Molloy, K. R., Ketaren, N. E., Stokes, D. L., Jensen, T. H., Rout, M. P. and LaCava, J. (2016). Purification and analysis of endogenous human RNA exosome complexes. RNA 22(9): 1467-1475.
  4. Farrell, R. E. (2010). RNA Methodologies. 4th edition.
  5. Kilchert, C., Wittmann, S. and Vasiljeva, L. (2016). The regulation and functions of the nuclear RNA exosome complex. Nat Rev Mol Cell Biol 17(4): 227-239.
  6. LaCava, J., Jiang, H. and Rout, M. P. (2016). Protein complex affinity capture from cryomilled mammalian cells. J Vis Exp e54518.
  7. Sambrook, J. and Russell, D. W. (2006). The Condensed Protocols. 1st edition. Cold Spring Harbor Laboratory Press.
  8. Wasmuth, E. V. and Lima, C. D. (2012). Structure and activities of the eukaryotic RNA exosome. Enzymes 31: 53-75.
  9. Zinder, J. C., Wasmuth, E. V. and Lima, C. D. (2016). Nuclear RNA exosome at 3.1 A reveals substrate specificities, RNA paths, and allosteric inhibition of Rrp44/Dis3. Mol Cell 64(4): 734-745.

简介

RNA外植体复合物在RNA加工和调节营养中起核心作用。在细胞质和细胞核中存在,外来体通过与核糖核酸酶和各种衔接蛋白的关联起作用(参见[Kilchert等人,2016])。 RNA外植体相关的EXOSC10蛋白是分布的3'-5'外核糖核酸酶。以下方案描述了监测源自HEK-293细胞的亲和纯化含EXOSC10的RNA外来体的核糖核酸裂解活性的方法,如(Domanski等人,2016)所报道的,并进一步详细描述这个配套生物协议(Domanski和LaCava,2017)。

在我们以前的工作中,我们在四环素诱导型CMV启动子(HEK-293 Flp-In T-REx-Thermo)的控制下,建立了表达C末端3xFLAG标记的外来体成分EXOSC10(RRP6)的同基因HEK-293细胞系费雪科学)。该系统允许我们以与内源WT蛋白质相当的水平表达标记的EXOSC10蛋白质,并且使用磁性抗FLAG亲和介质和来源于冷冻细胞粉末的蛋白质提取物来研究外来体纯化方案(Domanski等, / em>。,2012)。在此基础上,我们开发了通过速率区带离心法,使用甘油浓度梯度和测定其核糖核酸酶(RNase)活性进一步纯化RNA外来体的方案(Domanski等,2016)。含有EXOSC10的外来子部分表现出明显的核糖核酸裂解活性,与分布的3'-5'水解一致;相同的测定允许检测和监测亲和纯化的DIS3-3xFLAG([Wasmuth和Lima,2012]及其中的参考文献)的进程性RNA酶活性。这里介绍的方案描述了RNA酶测定。尽管该方案假定甘油梯度纯化的含有EXOSC10-3xFLAG的外来体作为进入检测点(Domanski和LaCava,2017),该方法应适用于任何足够纯净和浓缩的样品。

关键字:RNA外显子, EXOSC10, 亲和力捕获, RNA降解, 核糖核酸酶

材料和试剂

注意:以下列出的大多数试剂都提供了目录号。来自替代供应商的等效质量试剂通常可以用可比较的结果代替。由于污染RNase引入的人工制品的潜力,应注意遵循最佳实践,例如使用无RNA酶的溶液和试剂和/或酌情使用DEPC处理(Farrell,2010)。需要尿素 - 聚丙烯酰胺凝胶电泳的标准材料和试剂;我们使用国家诊断系统,但是可以使用标准方法制备这种凝胶(Sambrook和Russell,2006)。

  1. 移液器提示
  2. 1.5ml微量离心管(例如,Eppendorf,目录号:022363204)
  3. 10-Gel Gel Combs 1.5mm(Thermo Fisher Scientific,Novex TM,目录号:NC3510)
  4. 空的凝胶盒,迷你,1.5毫米(Thermo Fisher Scientific,Novex TM,目录号:NC2015)
  5. 注射器用弯针(在样品加载前将残留的尿素从凝胶孔中清洗)
  6. HEK-293 Flp-In T-REx EXOSC10-3xFLAG细胞([Domanski等人,2016];可应要求提供)亲代HEK-293 Flp-In T-REx细胞系(Thermo Fisher Scientific ,Invitrogen TM,目录号:R78007)
  7. 无核酸酶的水(Thermo Fisher Scientific,Ambion TM ,目录号:AM9932)
  8. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  9. 2- [4-(2-羟乙基)哌嗪-1-基]乙磺酸(HEPES)(Sigma-Aldrich,目录号:54457)
  10. Triton X-100(Sigma-Aldrich,目录号:T8787)
  11. 抗-FLAG M2(Sigma-Aldrich,目录号:F3165)抗体缀合的Dynabeads M-270环氧树脂(Thermo Fisher Scientific,Invitrogen,目录号:14302D)(Domanski和LaCava,2017) />
  12. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  13. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  14. 在5'端具有6-羧基荧光素(6-FAM)的RNA底物(在10mM HEPES〜pH7-7.5中重构,0.5nmol /μl,储存等分试样于-80℃)
    1. 通用底物:5' - (6-FAM)-CCUAU UCUAU AGUGU CACCU AAAUG CUAGA GCU modC(2'-O-Me)-3'
    2. 封闭的底物:5' - (6-FAM)-CCUAU UCUAU AGUGU CACCU AAAUG CUAGA GCU modC(2'-O-Me,3'-PO 4)-3'
    注意:两种底物均按照100nmole级别从Integrated DNA Technologies订购,通过无RNA酶的HPLC纯化。
  15. RNasin 核糖核酸酶抑制剂(Promega,目录号:N2515)
  16. 甲酰胺(Sigma-Aldrich,目录号:47671)
  17. DNA加载染料(Thermo Fisher Scientific,目录号:R0611)
    注意:由10mM Tris-HCl(pH 7.6),0.03%溴酚蓝,0.03%二甲苯氰醇FF,60%甘油,60mM EDTA组成,因此可以制备而不是购买。 >
  18. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  19. Tris碱(Sigma-Aldrich,目录号:93362)
  20. 硼酸(H 3 O 3 BO 3)(Sigma-Aldrich,目录号:B7901)
  21. 四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:N,/, T7024)
  22. 过硫酸铵(APS)(Sigma-Aldrich,目录号:A3678)
    注意:在H 2 O中准备10%的解决方案,并在4°C下保存数周。
  23. UreaGel 29:1变性凝胶系统(国家诊断,目录号:EC-829)
  24. 回收溶液(参见食谱)
  25. 清洗液(参见食谱)
  26. 2x反应溶液(参见食谱)
  27. 2x RNA加载溶液(参见食谱)
  28. 1x TBE(5x或10x库存可以准备)(见配方)

设备

注意:需要用于尿素 - 聚丙烯酰胺凝胶电泳的标准设备,以及能够进行荧光素检测的成像仪(吸收λmax = 494nm,发射λmax max/= 518nm)

  1. 移液器
  2. Vortexer
  3. 台式微型离心机
  4. 钕磁铁微型管架(Thermo Fisher Scientific,目录号:12321D)
  5. Thermomixer(例如,Eppendorf,型号:ThermoMixer F,目录号:5355000.011;或等效物)
  6. XCell SureLock Mini(Thermo Fisher Scientific,型号:SureLock ® Mini-Cell,目录号:EI0001)
  7. 电泳电源
  8. 具有蓝光(460nm)外照相和Y515-Di(长通)滤光片的成像器,即,SYBR绿色设置。富士胶片LAS-3000系列或更高版本(Fujifilm,型号:LAS-3000系列;或同等品牌)

程序

注意:如补充方案所述纯化含有EXOSC10的RNA外来体,如HEK-293细胞(Domanski和LaCava,2017)的RNA外来体亲和纯化或其他方式。如先前所述(Domanski等,2016),该测定也可以有效地应用于亲和纯化的DIS3-3xFLAG。可以在我们的在线视频协议(LaCava et al。,2016)中查看与以下协议的亲和力捕获方面相关的操作示例。

  1. 从甘油梯度重新捕获RNA外来体
    1. 从梯度中回收级分后,最多聚集到构成完整的含EXOSC10的外来体的峰的两个级分(总共达到〜450μl)。
      注意:如果需要,请保存最多50μl(输入)的等份用于其他分析。
    2. 添加等体积的重新捕获解决方案(参见配方1)。
    3. 用1 ml的再吸收溶液预洗10μl抗FLAG磁性介质(浆液)两次 注意:将1ml的再吸收溶液与珠浆料混合并短暂涡旋。在小型离心机中旋转管子以收集底部的所有溶液,然后将管放置在磁性管架上。等待珠子收集在管的一侧并除去上清液(使用移液管或吸气器)。向管中的珠添加另外的再捕获溶液,并重复第二次洗涤的过程;除去上清液后,珠可以保持在冰上并准备使用。两种洗涤步骤都可以在室温下进行。
    4. 将来自步骤A2的稀释级分转移到具有预洗珠的1.5ml管中
    5. 孵育30分钟,旋转4°C(冷室)。
    6. 放在磁铁上,等到珠子收集在管子的侧面,然后取出上清液。
      注意:上清可以通过蛋白质染色或蛋白质印迹与输入进行比较,以监测外来体消耗的程度。
    7. 用1ml洗涤溶液洗涤一次(参见方法2)。
    8. 将亲和介质重悬于25μl洗液中
    9. 加入25μl2x反应溶液(参见配方3)。
    10. 在37℃下以1,000rpm混合培育
    11. 通过在不同时间点(例如,0,2.5,5,15和30分钟)取10μl等分试样来产生反应的时间过程。通过加入10μl2x RNA加载缓冲液停止反应(参见配方4)。
      注意:该测定应理想地包括设置为独立反应的两个对照:a)如上所述,但使用封闭的底物,和b)与亲和介质和含有通用底物的反应溶液的模拟反应,但不含RNA外来体。两个对照应该在整个时间过程中显示完整底物的存在,表明制剂和溶液没有干扰RNase污染。 DIS3能够降解3'-PO 4阻断的底物。
    12. 放在磁铁上,并将上清液转移到新鲜管中。保持冰上。

  2. RNA PAGE和凝胶成像
    1. 根据制造商的说明,投入20%的尿素 - 聚丙烯酰胺10孔凝胶。
    2. 将凝胶在1x TBE(参见配方5)中以15 W预处理15-20分钟。
      注意:预运行步骤清除了凝胶中过量的自由离子,影响电流和发热。在预运行和运行期间,磁带将温暖;典型工作温度在45-60°C之间。所产生的热可能有助于将RNA样品维持在变性的单链形式;对于缺乏强大的二级结构的小型寡核苷酸底物,这并不是预期的主要变量。
    3. 将样品在80℃下加热30秒,然后放在冰上。装载之前,请稍后旋转。
      注意:加热样品使RNA变性并解离RNA蛋白组装。
    4. 使用带有弯针的注射器从孔中清洗尿素。
    5. 在凝胶上加载每个样品的每个样品的体积(20μl)。
    6. 以15 W运行,直到溴酚蓝跟踪染料从塑料胶盒底部到达第一行。
    7. 打开盒式磁带并使用适当配置的成像器(富士LAS系列上的SYBR绿色)直接成像。
      注意:凝胶在成像之前不需要固定,洗涤或干燥。根据检测到的活动水平,在数据收集之后,软件中的图像亮度/对比度的一些调整可以改善可视化。

数据分析

EXOSC10的分布式3'-5'exoribonucleolytic活性可以看作是底物降解中间体的"梯度",随着时间的推移越来越多地转向较低的分子量物种;并因此越来越多地朝向凝胶的底部。这种模式如图1所示。梯子的最后一步( ie ,最新时间点)可能会相对于较早的时间点出现压缩,因为酶可能不会在末端显示明显的活动缩短的底物池和池的其余部分收敛于这个大小。 3'-PO 4封闭的底物将阻止EXOSC10衍生的3'-5'活性;在这种情况下,应在所有时间点观察全长底物和不存在分解产物。但是,请注意,这个期望假定不存在DIS3,或者纯化的外来体中存在的任何DIS3已被灭活(Domanski等人,2016; Zinder等人)。 ,2016)

图1.代表性的结果:使用亲和纯化的人类RNA外源体复合物的RNA降解测定。如本方案所述,外源核糖核酸酶测定的反应产物的尿素 - 聚丙烯酰胺凝胶分离示意图,一致具有分布式活动模式和原始数据。 B.从(Domanski等人,2016)转载的原始凝胶:将ExoI制剂与普通或封闭的底物一起温育,并收集指定的时间点。

食谱

  1. 回收方案
    100 mM NaCl
    20 mM HEPES,pH 7.4
    1%Triton X-100
  2. 清洗液
    100 mM NaCl
    20mM Tris-HCl,pH8.0
    0.01%Triton X-100
  3. 2x反应液
    2x反应液
    20mM Tris-HCl,pH8.0
    20 mM DTT
    0.5mM MgCl 2
    5%(v/v)RNasin 核糖核酸酶抑制剂
    0.4pmol/μlRNA底物
  4. 2x RNA加载溶液
    95%甲酰胺
    1%DNA负载染料
    20 mM EDTA
  5. 1×TBE(5x或10x股票可以准备)
    89 mM Tris碱基
    89 mM硼酸
    2 mM EDTA

致谢

感谢Michael P. Rout教授和Torben Heick Jensen教授对我们研究的宝贵支持。我们还感谢Artem Serganov先生和Zhanna Hakhverdyan博士作证。这项工作得到了美国国家卫生研究院授予的P41GM109824和P50GM107632,Lundbeck基金会和丹麦国家研究基金会的支持。

参考文献

  1. Domanski,M.和LaCava,J.(2017)。亲和纯化来自HEK-293细胞的RNA外来体。 7(08):e2238。
  2. Domanski,M.,Molloy,K.,Jiang,H.,Chait,BT,Rout,MP,Jensen,TH and LaCava,J.(2012)。  改进在近内源水平表达的人类蛋白质复合物的亲和力分离的方法。生物技术 0(0):1-6。
  3. Domanski,M.,Upla,P.,Rice,WJ,Molloy,KR,Ketaren,NE,Stokes,DL,Jensen,TH,Rout,MP and LaCava,J.(2016)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27402899"target ="_ blank">内源性人RNA外显子复合物的纯化和分析。 em> 22(9):1467-1475。
  4. Farrell,R.E。(2010)。  RNA Methodologies。第4版
  5. Kilchert,C.,Wittmann,S。和Vasiljeva,L。(2016)。核RNA外显子复合物的调节和功能。 Nat Rev Mol Cell Biol 17(4):227-239。
  6. LaCava,J.,Jiang,H.and Rout,MP(2016)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226390 /"target ="_ blank">来自低温哺乳动物细胞的蛋白质复合物亲和力捕获。 J Vis Exp e54518。
  7. Sambrook,J。和Russell,DW(2006)。< a class ="ke-insertfile"href ="https://www.amazon.com/Condensed-Protocols-Molecular-Cloning-Laboratory/dp/0879697717 "target ="_ blank">缩写协议。第一版冷泉港实验室出版社。
  8. Wasmuth,EV和Lima,CD(2012)。  结构和真核RNA外来体的活性。 酶:31:53-75。
  9. Zinder,JC,Wasmuth,EV和Lima,CD(2016)。 
  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Domanski, M. and LaCava, J. (2017). RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells. Bio-protocol 7(8): e2239. DOI: 10.21769/BioProtoc.2239.
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