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Dec 2018

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Organelle-associated rRNA Degradation
附着在细胞器上的rRNA降解   

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Abstract

Cytosolic rRNAs are highly dynamic and can be degraded under conditions such as apoptosis, starvation and magnesium depletion. The degradation is also related to their specific localization, as fractions of cytosolic ribosomes are localized on the surfaces of intracellular organelles, such as endoplasmic reticulum (ER) and mitochondria. Such localized translation facilitates translocation of nascent proteins into these organelles co-translationally, contributing to fast responses to cellular stresses and precise regulations of the organelle. Here, we describe a protocol to establish the in organello system to investigate rRNA degradation on mitochondrial outer membrane or ER. The protocol consists of organelle isolation, rRNA degradation on organelles and agarose gel electrophoresis to examine the remaining rRNAs.

Keywords: In organello (In organello), Ribosomal RNA (核糖体RNA), Cytosolic rRNAs (细胞质rRNAs), rRNA degradation (rRNA降解), Localized translation (局部翻译), Mitochondrion-associated ribosomes (线粒体相关核糖体), ER-associated ribosomes (ER相关核糖体)

Background

Cytosolic ribosomes, where protein translation takes place, have been shown to be localized to specific membranes within the cells, such as ER (Reid and Nicchitta, 2012) and mitochondrial outer membrane (Kellems and Butow, 1972), which couples protein synthesis to protein targeting and translocation (Mukhopadhyay et al., 2004). Such localized translation reduces the protein transportation cost and avoids mistargeting (Lesnik et al., 2015). In addition, localized translation contributes to fast responses to unfolded protein stress on ER (Reid et al., 2014) and modulates protein translation within mitochondria (Dennerlein et al., 2017; Richter-Dennerlein et al., 2016). The binding between cytosolic ribosomes and mitochondrial outer membrane have been previously investigated, and GTP, specific targeting sequences (Crowley and Payne, 1998) and MDI (Zhang et al., 2016) are all shown to be involved. However, how the mitochondrial outer membrane-associated cytosolic rRNAs are regulated by different conditions or mitochondrial proteins remains unclear, and how the ER-associated ribosomes differ from other pools of cytosolic ribosomes is not well studied. To gain a better understanding of these processes and their regulations in mammals, in organello systems are needed. However, no such systems have been established. Here, we describe a protocol to study in organello rRNA degradation on mitochondria or ER. The sample preparation procedures are minimized to reduce the operational errors. However, since this is an in organello system, the rRNA degradation observed may not reflect the real complexity in the cell. Particular caution needs to be taken in interpreting the results.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Quality Scientific Plastics, catalog number: 509-GRD-Q)
  2. 0.22 μm filter (Merk, Millex-GP PES, catalog number: SLGP033RB)
  3. Nuclease-free pipette tips (Quality Scientific Plastics, catalog numbers: T104RLS-Q, T090RLS-Q, and T112NXLRLS-Q)
  4. Mannitol (AMRESCO, catalog number: 0122-500G)
  5. Sucrose (AMRESCO, catalog number: 0335-500G)
  6. HEPES free acid (AMRESCO, catalog number: 0511-1KG)
  7. Sodium dodecyl sulfate (SDS) (AMRESCO, catalog number: 0227-1KG)
  8. EDTA, disodium salt, dihydrate (Na2EDTA·2H2O) (AMRESCO, catalog number: 0105-1KG)
  9. Agarose (BIOWEST, Regular Agarose G-10, catalog number: 111860)
  10. Proteinase K (AMRESCO, catalog number: 0706-100MG)
  11. Bromophenol blue (AMRESCO, ACS grade, catalog number: 0449-25G)
  12. Tris (AMRESCO, catalog number: 0497-5KG)
  13. Glacial acetic acid (Beijing Chemical Works, Analytic Reagent grade)
  14. Nuclease-free water (AMRESCO, catalog number: E476-1L)
  15. Glycerol (AMRESCO, catalog number:0854-1L)
  16. KOH (Sigma-Aldrich, catalog number: P1767-500G)
  17. Double distilled water (ddH2O)
  18. NaOH (sodium hydroxide pellets) (Shanghai Sangon Biotech, catalog number: A100173)
  19. Tween20 (AMRESCO, product code: 0777-1L)
  20. NaCl (AMRESCO, catalog number: 0241-1KG)
  21. PBS (Corning, catalog number: 21-040-CVR)
  22. Luminol/Enhancer solution (Thermo Fisher Scientific, catalog number: 1863096)
  23. Anti-Mortalin antibody (Sigma-Aldrich, catalog number: G4045)
  24. Anti-Calnexin antibody (Cell Signaling Technology, catalog number: 2433S)
  25. Anti-Rabbit IgG (whole molecule)–Peroxidase (Sigma-Aldrich, catalog number: A0545)
  26. Prestained Protein Ladder (Thermo Fisher Scientific, PageRuler, catalog number: 26616)
  27. HEK293 (from Carla M. Koehler’s lab at University of California, Los Angeles)
  28. 10% SDS-PAGE gel (Homemade, refer to He, 2011 for detailed protocol)
  29. Skim milk powder (OXOID, catalog number: LP0031)
  30. BSA (Sigma-Aldrich, catalog number: P3761)
  31. MitoPrep buffer (see Recipes) 
  32. 10% (w/v) SDS (see Recipes)
  33. 0.5 M EDTA (pH 8.0) (see Recipes)
  34. 2x DNA-SDS-EDTA buffer (see Recipes)
  35. Proteinase K (1 mg/ml) (see Recipes)
  36. 50x TAE (see Recipes)
  37. 1x TBS-T (see Recipes)
  38. 2x protein loading buffer (see Recipes)

Equipment

  1. Pipettes (RAININ, Pipet-Lite XLS)
  2. Two heating blocks (Hangzhou Allsheng Instruments, Product Name: dry bath incubator, catalog number: MK200-2)
  3. NanoDrop instrument (Thermo Fisher Scientific, NanoDrop 2000c Spectrophotometer)
  4. Power supply (Tanon, catalog number: EPS 300)
  5. Gel imaging system (Tanon, catalog number: 1600)
  6. Centrifuges (Thermo Fisher Scientific, models: Sorvall Legend Micro 21 and Micro 21R)
  7. pH meter (Sartorius, catalog number: PB-10) 
  8. -80 °C freezer (Thermo Fisher Scientific) 
  9. Nitrocellulose membrane (Merck, catalog number: HATF00010)
  10. Vertical electrophoresis bath (Tanon, catalog number: VE-180)
  11. Glass/Teflon homogenizer (Wheaton, catalog number: 358034)
  12. Incubator
  13. 4 °C refrigerator
  14. -20 °C freezer

Software

  1. Tanon imaging (Tanon MP) and processing (Tanon GIS) software
  2. ImageJ
  3. GraphPad Prism 5
  4. Microsoft PowerPoint 2010

Procedure

Note: Use nuclease-free tubes and tips (Materials and Reagents sections) for all the steps.

  1. Crude mitochondria and ER isolation
    Note: The method for mitochondria isolation was adapted from a previously described protocol (Wang et al., 2015) with minor modifications. During organelle isolation, the pH value of MitoPrep buffer is 7.4 if not specified.
    1. Collect HEK293 cells by centrifuging at 1,000 x g for 2 min at room temperature (RT). Discard the supernatant, and resuspend the cell pellet with 1 ml of PBS. Spin again at 1,000 x g for 2 min at RT. Discard the supernatant.
      Note: HEK293 cells were cultured in a 15-cm cell culture dish. With a density of ~90%, ~3 x 107 cells were harvested.
    2. Resuspend the cells in 1.5 ml ice-cold MitoPrep buffer and transfer the mixture to a 5 ml glass/Teflon homogenizer pre-cooled in an ice bath. Perform 30 strokes on ice.
    3. Transfer the homogenate into a 1.5 ml microcentrifuge tube and centrifuge at 800 x g for 5 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube.
    4. Resuspend the pellet in 1.5 ml MitoPrep buffer. Perform 20 strokes on ice. Transfer the homogenate into a 1.5 ml microcentrifuge tube and centrifuge at 800 x g for 5 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube. 
    5. Spin the supernatants from Steps1c and 1d at 800 x g for 5 min at 4 °C.
    6. Transfer the supernatants to new microcentrifuge tubes and centrifuge at 11,000 x g for 5 min at 4 °C. The resulting pellets are crude mitochondria. 
    7. The supernatants are transferred into two new microcentrifuge tubes and spun at 21,000 x g for 10 min at 4 °C. The pellets are crude ER.
    8. Combine the pellets of mitochondria or ER from the two tubes by resuspending them in 1 ml MitoPrep buffer. Spin at 11,000 x g for 5 min or 21,000 x g for 10 min to spin down mitochondria or ER respectively.
    9. Discard the supernatant and resuspend mitochondria or ER with 30 μl of MitoPrep buffer (pH 7.4).
      Note: Optiprep gradients can be used for further purification, if purer fractions are needed.
    10. Measure protein concentrations with a NanoDrop instrument after diluting 1 μl of protein sample with 19 μl of 0.6% (w/v) SDS. The original protein concentrations are the measured values multiplied by 20.
      Note: After we add 1 μl of protein sample to 19 μl of 0.6% SDS, the total volume becomes 20 μl, that means the concentration of this diluted protein sample is 1/20 of its original concentration. So, we can calculate the original protein concentration by a factor of 20. Usually, 3 x 107 cells can yield 800 μg of mitochondria and 150 μg of ER.
  2. Take out 200 μg of mitochondria (in about 15 μl of MitoPrep buffer, pH 7.4), or 50 μg of ER (in about 10 μl of MitoPrep buffer, pH 7.4), and bring the volume to 60 μl with MitoPrep buffer with a pH value of interest (pH 7.4 and 6.5 are the two values we routinely use). Keep the samples on ice.
    Notes:
    1. Metal ions, metal chelators, or other conditions can be added to the sample before the next step. Some conditions that have effects on the degradation of mitochondrion-associated cytosolic rRNAs have been listed in Table 1.

      Table 1. Conditions that affect the degradation of mitochondrion-associated cytosolic rRNAs


    2. For ER-associated rRNA degradation, the pH value of MitoPrep buffer matters: when the pH is 7.4, degradation is slower; when the pH is lowered to 6.5, the degradation becomes much faster. All these effects have been described in the original paper (Huang et al., 2018).
  3. Divide each sample (60 μl) into 3 microcentrifuge tubes (20 μl per tube). Store one tube at the -80 °C freezer as the first time point (0 min). Transfer the other 2 tubes to 37 °C in a heating block. Incubate the two samples for 30 and 60 min respectively. At each time point, transfer one tube to -80 °C freezer. Store the samples at -80 °C for at least 15 min before the next step.
    Note: For quality control, prepare another set of the samples and perform the same degradation steps. Then mix the samples with an equal volume of 2x protein loading buffer with DTT, and heat them at 95 °C for 5 min. Separate the proteins in the samples on a 10% SDS-PAGE gel. After running the gel at 150 V for 60 min, transfer the proteins in the gel to a nitrocellulose membrane at 450 mA for 70 min and blot the membrane by standard western blotting method. In brief, incubate the membrane at RT for 1 h in 5% skim milk, probe it with primary antibody in 1x TBS-T at RT for 1 h, wash 4 times with 1x TBS-T (5 min/time), incubate with secondary antibody in 1x TBS-T at RT for 1 h, and then wash 4 times with 1x TBS-T (5 min/time). Use Luminol/Enhancer solution for detection of the proteins. Marker proteins used for mitochondria and ER fractions are Mortalin (73.7 kDa) and Calnexin (~68 kDa) respectively. The actual position for Mortalin on a gel is between 70 and 100 kDa, and the position for Calnexin is around 100 kDa. 
  4. Take out samples from the -80 °C freezer and add 20 μl of 2x DNA-SDS-EDTA buffer. Vortex, and incubate the samples at 70 °C for 5 min. Cool the samples to RT.
  5. Add 0.5 μl of 1 mg/ml proteinase K to each sample and incubate it at 37 °C in a heating block for 5 min.
  6. Run the samples on a 1.5% (w/v) agarose gel with ethidium bromide (EB) (0.5 μg/ml) for 15 min at 150 V in 1x TAE buffer. Capture gel images to check RNA degradation pattern with a UV-based gel imaging system. 
  7. The flow sheet of this protocol is shown in Figure 1.


    Figure 1. The flow sheet of this protocol

Data analysis

Representative data (Figures 2-4)


Figure 2. In organello degradation of mitochondrion-associated cytosolic rRNAs. A. Mitochondria were suspended in MitoPrep buffer and incubated for 0, 30 or 60 min. The top panel shows the agarose gel image of mitochondrion-associated cytosolic rRNAs at three time points. The two major bands are cytosolic 28S rRNA and 18S rRNA. The bottom panel is an immunoblot of mitochondrial matrix protein Mortalin (Mw: 73.7 kDa), which is used as a loading control. B. Quantification of the rRNAs in Panel A.


Figure 3. The effects of EDTA on the in organello degradation of mitochondrion-associated cytosolic rRNAs. A. Mitochondria were suspended in MitoPrep buffer, and incubated for 0, 20 or 40 min with or without 2 mM EDTA. The top panel shows the agarose gel image of mitochondrion-associated cytosolic rRNAs at three time points. The two major bands are cytosolic 28S rRNA and 18S rRNA. The bottom panel is an immunoblot of mitochondrial matrix protein Mortalin (Mw: 73.7 kDa), which is used as a loading control. B. Quantification of 28S rRNA in Panel A. C. Antibody specificity confirmation, protein loading control samples from A were separated with SDS-PAGE gels and transferred to nitrocellulose membrane. The entire membrane (upper panel) or the cropped membrane (bottom panel) was probed with anti-Mortalin antibody.


Figure 4. Effects of pH on the in organello degradation of ER-associated cytosolic rRNAs. A. ER was suspended in MitoPrep buffer with a pH of 7.4 or 6.5, and incubated for 0, 30 or 60 min. The top panel shows the agarose gel image of ER-associated cytosolic rRNAs at three time points. The two major bands are cytosolic 28S rRNA and 18S rRNA. The bottom panel is an immunoblot of ER protein Calnexin (the position of the protein on the gel is around 100 kDa), which is used as a loading control. B. Quantification of 28S rRNA in Panel A. C. Antibody specificity confirmation, protein loading control samples from A were separated with SDS-PAGE gels and transferred to nitrocellulose membrane. The entire membrane (upper panel) or the cropped membrane (bottom panel) was probed with Anti-Calnexin antibody.

Data processing
The gel image was captured with Tanon 1600 Gel Image System (Tanon), and cropped using Photoshop.

Data analysis
The intensity of the bands on each gel was measured with ImageJ, normalized to the protein loading control, and analysed with GraphPad Prism.
  For an original image captured on Gel Image System (the background is black, while the bands are white), here is a brief introduction on how to use ImageJ for quantification. Open software ImageJ. Select “Rectangular” tool (this is default choice), and draw a rectangle covering the RNA band at the first time point. Press shortcut “Ctrl + M” to measure the band intensity. Move the same rectangle horizontally to RNA band at the second or third time point, and then press shortcut “Ctrl + M” to measure the band intensity. After all the time points are measured, move the same rectangle to a typical background area, and then press shortcut “Ctrl + M” to measure background intensity.
  Calculation method: Measure the RNA signal by subtracting the background intensity from the band intensity to get the absolute intensity of each band. Set the signal at the first time point as 100, and calculate the second and third ones by dividing the absolute signal of the RNA band to that of the first time point.
  Statistical comparisons were performed using unpaired t-tests (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± standard deviation (S.D.). Data sets at each time point were analyzed individually.

Recipes

  1. MitoPrep buffer
    0.225 M mannitol
    0.075 M sucrose
    20 mM HEPES (pH 7.4 or pH 6.5)
    1. Dissolve 2.05 g of mannitol (Mw: 182.17), 1.28 g of sucrose (Mw: 342.3), and 0.2383 g of HEPES (Mw: 238.3) in ~45 ml ddH2O
    2. Adjust pH to 7.4 or 6.5 with 1 M KOH, and add ddH2O to the final volume of 50 ml
    3. Filter through 0.22 μm filter, and store at 4 °C 
  2. 10% (w/v) SDS
    1. Dissolve 10 g of SDS in 100 ml of ddH2O, stir until completely dissolved. Store at RT
    2. Dilute 10% SDS to 0.6% SDS with ddH2O. Store at RT
  3. 0.5 M EDTA (pH 8.0)
    1. Add 9.306 g of EDTA (Mw: 372.24) to ~40 ml of ddH2O, stir and slowly adjust the pH to 8.0 with NaOH, and make up to 50 ml with ddH2O
    2. Filter through a 0.22 μm filter and store at 4 °C 
    Note: EDTA will not fully dissolve until the pH is close to 8.0.
  4. 2x DNA-SDS-EDTA buffer
    2x DNA loading buffer, supplemented with 0.5% SDS and 15 mM EDTA
    First prepare 10x DNA loading buffer:
    For a 10 ml solution, dissolve 0.2 g of SDS and 0.01 g of bromophenol blue in 5 ml of ddH2O, mix with 5 ml of glycerol. Store at RT
    To prepare 100 μl 2x DNA-SDS-EDTA buffer:
    Mix 72 μl of nuclease-free water with 20 μl of 10x DNA loading buffer, add 5 μl of 10% SDS and 3 μl of 0.5 M EDTA (pH 8.0)
  5. Proteinase K (1 mg/ml)
    Dissolve 1 mg of proteinase K in 1 ml of nuclease-free water, aliquot and store at -20 °C
  6. 50x TAE
    2 M Tris
    1 M acetic acid
    50 mM EDTA
    1. Dissolve 242 g of Tris (Mw: 121.14), 37.2 g of Na2EDTA·2H2O (Mw: 372.24) and 57.1 ml of glacial acetic acid (molar concentration: 17.5 M) in ~900 ml of ddH2O, add ddH2O to the final volume of 1 L. Store at RT
    2. Prepare 1x TAE by diluting 50x TAE with ddH2O
  7. 1x TBS-T
    1. Dilute 10x TBS stock with 0.1% (v/v) of Tween20 and ddH2O to make 1x TBS-T 
    2. To prepare 10x TBS (1 L), dissolve 24.2 g of Tris and 80 g of NaCl in ~900 ml of ddH2O, and adjust the pH to 7.6 with HCl 
  8. 2x protein loading buffer
    1. First prepare 100 ml of 0.5 M Tris-HCl (pH 6.8). Dissolve 6.05 g of Tris (Mw: 121.14) in ~90 ml of ddH2O, adjust pH to 6.8 with HCl. Add ddH2O to the final volume of 100 ml. Store at RT
    2. To prepare 10 ml 2x protein loading buffer, mix 1.25 ml of 0.5 M Tris-HCl (pH 6.8), 0.2 ml of 0.5% (w/v) bromophenol blue, 2.5 ml of glycerol, 2 ml of 10% SDS and 3.55 ml of ddH2O, store at RT
    3. A final concentration of 30 mM DTT is added in the 2x protein loading buffer right before use

Acknowledgments

This work was supported by Grant 2017YFA0504600 from the Priority Research Program of the Ministry of Science and Technology of China, by Grants 31371439 and 91649103 from the National Natural Science Foundation of the People’s Republic of China, and by funds from the Ministry of Education of the People’s Republic of China 1000 Talents Youth Program. This protocol was modified from Huang et al. (2018).

Competing interests

The authors declare no conflicts of interest with the contents of this article.

References

  1. Crowley, K. S. and Payne, R. M. (1998). Ribosome binding to mitochondria is regulated by GTP and the transit peptide. J Biol Chem 273(27): 17278-17285.
  2. Dennerlein, S., Wang, C. and Rehling, P. (2017). Plasticity of mitochondrial translation. Trends Cell Biol 27(10): 712-721.
  3. He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol 1(11): e80.
  4. Huang, J., Liu, P. and Wang, G. (2018). Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2). J Biol Chem 293(51): 19633-19644.
  5. Kellems, R. E. and Butow, R. A. (1972). Cytoplasmic-type 80 S ribosomes associated with yeast mitochondria. I. Evidence for ribosome binding sites on yeast mitochondria. J Biol Chem 247(24): 8043-8050.
  6. Lesnik, C., Golani-Armon, A. and Arava, Y. (2015). Localized translation near the mitochondrial outer membrane: An update. RNA Biol 12(8): 801-809.
  7. Mukhopadhyay, A., Ni, L. and Weiner, H. (2004). A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria. Biochem J 382(Pt 1): 385-392.
  8. Reid, D. W., Chen, Q., Tay, A. S., Shenolikar, S. and Nicchitta, C. V. (2014). The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum. Cell 158(6): 1362-1374.
  9. Reid, D. W. and Nicchitta, C. V. (2012). Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling. J Biol Chem 287(8): 5518-5527.
  10. Richter-Dennerlein, R., Oeljeklaus, S., Lorenzi, I., Ronsor, C., Bareth, B., Schendzielorz, A. B., Wang, C., Warscheid, B., Rehling, P. and Dennerlein, S. (2016). Mitochondrial protein synthesis adapts to influx of nuclear-encoded protein. Cell 167(2): 471-483 e410.
  11. Wang, G., Shimada, E., Nili, M., Koehler, C. M. and Teitell, M. A. (2015). Mitochondria-targeted RNA import. Methods Mol Biol 1264: 107-116.
  12. Zhang, Y., Chen, Y., Gucek, M. and Xu, H. (2016). The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication. EMBO J 35(10): 1045-1057.

简介

胞质rRNAs是高度动态的,可以在细胞凋亡、饥饿和镁缺乏等条件下降解。降解也与它们的特异性定位有关,因为部分胞质核糖体定位于细胞内细胞器的表面,如内质网(ER)和线粒体。这种定位翻译有助于新生蛋白在这些细胞器中的共同翻译易位,有助于对细胞压力的快速反应和细胞器的精确调控。在此,我们描述了在organello系统中建立的方案,以研究rRNA在线粒体外膜或ER上的降解。该方案包括细胞器分离、细胞器上rRNA降解和琼脂糖凝胶电泳检测剩余的rRNAs。
【背景】细胞核糖体是蛋白质翻译发生的地方,已被证明定位于细胞内的特定膜,如ER (Reid和Nicchitta, 2012)和线粒体外膜(Kellems和Butow, 1972),它们将蛋白质合成与蛋白质靶向和转位结合(Mukhopadhyay et al., 2004)。这种本地化翻译降低了蛋白质的运输成本,避免了误聚(Lesnik et al., 2015)。此外,定位翻译有助于ER对未展开蛋白应激的快速反应(Reid et al., 2014),并调节线粒体内的蛋白翻译(Dennerlein et al., 2017);Richter-Dennerlein et al., 2016)。细胞核糖体与线粒体外膜的结合已被研究,GTP、特异性靶向序列(Crowley and Payne, 1998)和MDI (Zhang et al., 2016)均参与其中。然而,线粒体外膜相关的胞质rRNAs是如何被不同的条件或线粒体蛋白调控的,以及er相关的核糖体与其他胞质核糖体的区别如何,目前还没有很好的研究。为了更好地理解哺乳动物的这些过程和它们的规则,需要在organello中 系统。但是,还没有建立这种制度。在这里,我们描述了一个研究在奥加纳洛 rRNA降解线粒体或ER的方案。减少了样品制备过程,减少了操作误差。然而,由于这是一个在organello系统,观察到的rRNA降解可能不能反映细胞的真实复杂性。在解释结果时需要特别谨慎。

关键字In organello, 核糖体RNA, 细胞质rRNAs, rRNA降解, 局部翻译, 线粒体相关核糖体, ER相关核糖体

材料和反应

  1. 1.5微腔管(定性科学塑料,目录编号509-GRD-Q)
  2. 0。22过滤器μm (Merk, Millex-GP PES,目录号:SLGP033RB)
  3. 核无核管道tips(优质科学塑料,目录编号:T104RLS-Q, T090RLS-Q,和t112nxlls - q)
  4. 甘露醇(AMRESCO,目录编号:0122-500G)
  5. 琥珀(AMRESCO,目录编号:0335-500G)
  6. HEPES free acid (AMRESCO,目录编号0511-1KG)
  7. 钠十二烷基磺酸(SDS) (AMRESCO,目录编号0227-1KG)
  8. EDTA,去骨盐,二氢盐(Na2EDTA·2H2O) (AMRESCO,目录编号:0105-1KG)
  9. Agarose (BIOWEST, Regular Agarose G-10,目录编号:111860)
  10. 蛋白K (AMRESCO,目录编号:0706-100MG)
  11. 溴苯诺蓝(AMRESCO, ACS等级,目录编号0449-25G)
  12. Tris (AMRESCO,目录编号0497-5KG)
  13. 冰川学酸性(北京化学制品,分析里根等级)
  14. 无核水(AMRESCO,目录编号:E476-1L)
  15. Glycerol (AMRESCO,目录编号0854-1L)
  16. KOH (Sigma-Aldrich,目录编号:P1767-500G)
  17. 双蒸馏水(ddH2O)
  18. NaOH(钠水仙球团)(上海生物科技,目录编号:A100173)
  19. Tween20 (AMRESCO,产品代码:07771 - 1l)
  20. NaCl (AMRESCO,目录编号:0241-1KG)
  21. PBS (Corning,目录编号:21-040-CVR)
  22. 鲁米诺/Enhancer解决方案。
  23. (西格玛-奥尔德里奇,目录编号:G4045)
  24. (细胞信号技术,目录编号2433S)
  25. 抗鼠鼠IgG(全分子)- Peroxidase (Sigma-Aldrich,目录编号:A0545)
  26. 预先确定的蛋白质梯度(热费雪科学,PageRuler,目录编号26616)
  27. HEK293(卡拉·M·科勒在加州大学洛杉矶分校的实验室)
  28. 10%的SDS-PAGE凝胶(Homemade, refer to He, 2011年详细协议)
  29. Skim milk powder (OXOID,目录编号:LP0031)
  30. BSA (Sigma-Aldrich,目录编号:P3761)
  31. 米托普缓冲区(见模式)
  32. 10% (w/v) SDS(见模式)
  33. 0.5 M EDTA (pH 8.0)(见模式)
  34. 2x DNA-SDS-EDTA缓冲区(见模式)
  35. 蛋白K (1 mg/ml)(见模式)
  36. 50x TAE(见配件)
  37. 1x TBS-T(见模式)
  38. 蛋白质负荷缓存(见模式)

设备

  1. Pipettes (RAININ, pipelite XLS)
  2. 两个燃烧块(杭州Allsheng仪器,产品名称:干巴斯incubator,目录编号:MK200-2)
  3. NanoDrop仪器。
  4. 电力供应(Tanon,目录编号:EPS 300)
  5. 凝胶成像系统(Tanon,目录编号1600)
  6. 离心机
  7. pH meter (Sartorius,目录编号:PB-10)
  8. 80度C冰柜
  9. 硝基纤维素膜(默克,目录编号:HATF00010)
  10. 垂直电泳(Tanon,目录编号:v -180)
  11. 玻璃/特氟隆homogenizer(惠顿,目录编号358034)
  12. Incubator
  13. 第四冷却器
  14. -20度

软件

  1. Tanon成像(Tanon MP)和处理(Tanon GIS)软件
  2. ImageJ
  3. 图形板棱镜5
  4. 微软PowerPoint 2010

程序

注意:在所有步骤中使用无核管和tips(材料和反应部分)

  1. Crude mitochondria和ER isolation 注释:米托邦德的隔离方法从先前描述的协议(Wang et al., 2015)中进行了调整,修改较少。在有机分离过程中,线粒体缓冲区的pH值是7.4,如果不指定。
    1. 2分钟房间温度(RT)。卸载supernatant,并用1ml PBS替换细胞。旋转1000 x g 2分钟< / br / > 注释:HEK293细胞是15厘米细胞文化的文化。密度为90% ~3×107细胞是harvested
    2. 将细胞固定在1.5毫升冷冻线粒体缓冲区,并将混合物转移到5毫升玻璃/特氟龙homogenizer在冰浴前冷却。在冰上打30杆。
    3. 将匀浆移入1.5 ml微离心管,800 x g, 4℃离心5 min。将上清液转移到新的微离心管中。
    4. 在1.5 ml MitoPrep缓冲液中复苏小球。在冰上划20下。将匀浆移入1.5 ml微离心管,800 x g, 4℃离心5 min。将上清液转移到新的微离心管中。
    5. 在800 x g温度下,从Steps1c和1d旋转上清5分钟。
    6. 将上清液转移到新的微离心管中,11000 x g, 4℃离心5min。由此产生的微丸是粗糙的线粒体。
    7. 将上清液转移到两个新的微离心管中,在21,000 x g温度下,4℃旋转10分钟。这些球团矿比较粗。
    8. 将线粒体或ER颗粒从两管中分离出来,在1 ml MitoPrep缓冲液中复苏。自旋11000 x g 5 min或21000 x g 10 min,分别自旋线粒体或ER。
    9. 丢弃的上层清液和resuspend线粒体或ER 30μl MitoPrep缓冲区(pH值7.4)。< br / > 注:如果需要更纯净的组分,可以使用Optiprep梯度进一步纯化。
    10. 测定蛋白质浓度稀释后NanoDrop仪器1μl蛋白质样品19μl 0.6% (w / v) SDS。原始蛋白浓度是测量值乘以20。< br / > 注意:加1后的蛋白质样品19μlμl 0.6% SDS,总量成为20μl,这意味着这个稀释蛋白质样品的浓度是原浓度的1/20。所以,我们可以计算原始蛋白质浓度的20倍。通常,3 x 10 <一口> < /一口>细胞能产生800 ?克线粒体和150 ?克。
  2. 拿出200μg线粒体(约15μl MitoPrep缓冲区,pH值7.4),或50μg ER(大约10μl MitoPrep缓冲区,pH值7.4),和带卷60μl MitoPrep缓冲与感兴趣的pH值(pH值7.4和6.5是我们经常使用的两个值)。把样品放在冰上。< br / > 指出:
    1. 金属离子,金属螯合剂,或其他条件可以添加到样品前的下一步。表1列出了一些影响线粒体相关细胞质rRNAs降解的条件。 < br / > < br / > 表1 。影响线粒体相关细胞质rRNAs降解的条件 < br / >
    2. 对于er相关的rRNA降解,MitoPrep缓冲液的pH值很重要:当pH值为7.4时,降解较慢;当pH值降低到6.5时,降解速度加快。所有这些影响都在原始论文中描述过(Huang et al., 2018)。
  3. 将每个样本(60μl)分成3微型离心机管(20μl /管)。在-80°C冰箱中储存一管作为第一个时间点(0分钟)。将另外两根管子转移到37°C的加热块中。将两个样品分别孵育30分钟和60分钟。在每个时间点,转移一管到-80°C冰箱。将样品在-80℃保存至少15分钟,然后进行下一步。< br / > 注意:为了质量控制,准备另一组样品,并执行相同的降解步骤。将等量2x蛋白缓冲液与DTT混合,95℃加热5 min,用10% SDS-PAGE凝胶分离样品中的蛋白。将凝胶以150 V运行60 min后,将凝胶中的蛋白转移到450 mA的硝化纤维素膜上70 min,用标准western blotting方法对膜进行印迹。总之,孵化的膜的1 h RT 5%脱脂牛奶,探针与主要抗体在1 x TBS-T RT 1 h,洗4次1 x TBS-T(5分钟/时间),孵化与二次抗体在1 x TBS-T RT 1 h,然后用1 x TBS-T洗4次(5分钟/时间)。使用鲁米诺/增强剂溶液检测蛋白质。线粒体和ER组分的标记蛋白分别为Mortalin (73.7 kDa)和Calnexin (~68 kDa)。Mortalin在凝胶上的实际位置在70 - 100kda之间,Calnexin的实际位置在100kda左右。
  4. 冰箱取出样本-80°C和添加20μl 2 x DNA-SDS-EDTA缓冲区。涡旋,70℃孵育5 min,冷却至RT。
  5. 添加0.5μl 1毫克/毫升蛋白酶K的每个样本和孵化它在37°C的加热块5分钟。
  6. 运行样本1.5% (w / v)琼脂糖凝胶与溴化乙锭(EB)为15分钟(0.5 ? g / ml)在1 x 150 v TAE缓冲区。利用基于uv的凝胶成像系统捕捉凝胶图像,检查RNA降解模式。
  7. 该协议的流程图如图1所示。< br / > < br / > 图1 。本协议流程表

数据分析

代表性数据(图2-4) < br / > 图2 。对线粒体相关的胞质rRNAs的降解。 A.线粒体悬浮于MitoPrep缓冲液中,孵育0,30或60min。顶部面板为线粒体相关胞质rRNAs在三个时间点的琼脂糖凝胶图像。两个主要的谱带是胞质28S rRNA和18S rRNA。下面板为线粒体基质蛋白Mortalin免疫印迹(Mw: 73.7 kDa),作为加载对照。B. A图中rRNAs的定量。< br / > < br / > 图3 。EDTA对线粒体相关细胞质rRNAs 降解的影响。 A.线粒体悬浮于MitoPrep缓冲液中,加入或不加入2mm EDTA孵育0、20或40min。顶部面板显示了线粒体相关的胞质rRNAs在三个时间点的琼脂糖凝胶图像。两个主要的谱带是胞质28S rRNA和18S rRNA。下面板为线粒体基质蛋白Mortalin免疫印迹(Mw: 73.7 kDa),作为加载对照。B.定量28S rRNA在a.c.抗体特异性确证中,用SDS-PAGE凝胶分离A蛋白加载对照样品,转移到硝基膜上。用抗死亡蛋白抗体检测全膜(上膜)或切短膜(下膜)。< br / > < br / >图4 。pH对er相关胞质rRNAs 降解的影响 a . ER悬浮在pH为7.4或6.5的MitoPrep缓冲液中,孵育0、30或60 min。顶部面板为三个时间点ER相关胞质rRNAs的琼脂糖凝胶图像。两个主要的谱带是胞质28S rRNA和18S rRNA。底板为ER蛋白Calnexin免疫印迹(蛋白在凝胶上的位置约为100 kDa),作为加载对照。B.定量28S rRNA在a.c.抗体特异性确证中,用SDS-PAGE凝胶分离A蛋白加载对照样品,转移到硝基膜上。用抗钙黏蛋白抗体检测全膜(上膜)或切短膜(下膜)。< br / > < br / > < >强数据处理 < br / > 凝胶图像采用Tanon 1600凝胶图像系统(Tanon)拍摄,Photoshop裁剪。< br / > < br / > 数据分析 用ImageJ法测定凝胶上的条带强度,归一化为蛋白加载控制,用GraphPad棱镜分析条带强度。< br / > ,对于在凝胶图像系统上捕获的原始图像(背景为黑色,条带为白色),下面简要介绍如何使用ImageJ进行量化。开放软件ImageJ。选择“矩形”工具(这是默认选项),在第一个时间点绘制一个覆盖RNA带的矩形。按快捷键Ctrl + M测量光带强度。在第二个或第三个时间点将相同的矩形水平移动到RNA波段,然后按快捷键Ctrl + M测量波段强度。测量完所有的时间点后,将相同的矩形移动到一个典型的背景区域,然后按快捷键“Ctrl + M”来测量背景强度。< br / > ,计算方法:用带强度减去背景强度来测量RNA信号,得到每个带的绝对强度。将第一个时间点的信号设为100,将RNA带的绝对信号除以第一个时间点的绝对信号,计算第二个和第三个时间点的信号。< br / > ,采用非配对t-test (n = 3)进行统计学比较;* P & lt;0.05,* * P & lt;0.01 * * * P & lt;0.001 * * * * P & lt;0.0001。数据以均数±标准差(S.D.)表示。对每个时间点的数据集分别进行分析。

食谱

  1. MitoPrep缓冲< br / > 0.225 M甘露醇 0.075 M蔗糖 20mm HEPES (ph7.4或ph6.5)
    1. 将2.05 g甘露醇(分子量:182.17)、1.28 g蔗糖(分子量:342.3)、0.2383 g HEPES(分子量:238.3)溶于~45 ml ddH2O
    2. 将pH值调整到7.4或6.5,并加入ddH2O到最终体积为50 ml
    3. 通过0.22μm过滤器过滤,储存在4°C
  2. 10% (w / v) SDS
    1. 将10克SDS溶解于100毫升ddH2O中,搅拌至完全溶解。在RT商店
    2. 用ddH2O稀释10% SDS至0.6% SDS。在RT商店
  3. 0.5 M EDTA (pH 8.0)
    1. 加入9.306 g EDTA (Mw: 372.24)至~ 40ml ddH2O,用氢氧化钠搅拌并缓慢调节pH至8.0,用ddH2O配制至50ml
    2. 透过0.22μm过滤和储存在4°C
    注:EDTA在pH值接近8.0时才会完全溶解
  4. 2x DNA-SDS-EDTA缓冲器 2倍DNA缓冲液,加入0.5% SDS和15mm EDTA 首先准备10x DNA缓冲液: 取10ml溶液,将0.2 g SDS和0.01 g溴酚蓝溶于5ml ddH2O中,与5ml甘油混合。存储在RT 准备100μl 2 x DNA-SDS-EDTA缓冲区: < br / > 混合72μl nuclease-free水20μl 10 x DNA加载缓冲区,加5μl 0.5的10% SDS和3μl EDTA (pH值8.0)
  5. 蛋白酶K (1mg /ml) 将1mg蛋白酶K溶解于1ml无核酸酶的水中,取下保存于-20℃
  6. 50 x TAE < br / > 2 M 1 M醋酸 50 mM EDTA
    1. 溶解242克三羟甲基氨基甲烷(Mw: 121.14),液37.2 g的Na <子> < /订阅> EDTA·2 h <子> 2 < /订阅> O (Mw: 372.24)和57.1毫升的冰醋酸(摩尔浓度:17.5米)~ 900毫升ddH 2 <子> < /订阅> O,添加ddH <子> 2 < /订阅> O的最终体积1 l .存储在RT
    2. 用ddH2O稀释50x TAE制备1x TAE
  7. 1 x TBS-T
    1. 用0.1% (v/v)的Tween20和ddH2O稀释10x TBS原料药,制成1x TBS- t
    2. 制备10x TBS (1 L),将24.2 g Tris和80 g NaCl溶解在~900 ml ddH2O中,用盐酸调pH至7.6
  8. 2x蛋白缓冲液
    1. 首先准备100毫升0.5 M三氯化氢(pH 6.8)。将6.05 g Tris(分子量:121.14)溶于ddH2O ~ 90ml中,用盐酸调pH至6.8。将ddH2O添加到最终100ml的体积中
    2. 制备10 ml 2x蛋白缓冲液,将1.25 ml 0.5 M Tris-HCl (pH 6.8)、0.2 ml 0.5%溴酚蓝(w/v)、2.5 ml甘油、2 ml 10% SDS和3.55 ml ddH2O混合保存于RT
    3. 在使用前,将最终浓度为30mm的DTT添加到2x蛋白加载缓冲液中

致谢

这项工作是2017年由格兰特yfa0504600优先研究项目的中国的科学技术部,由国家自然科学基金会的拨款31371439和31371439的中华人民共和国教育部基金从1000年中华人民共和国的青年人才项目。本协议由Huang 等人(2018)修改而成。

相互竞争的利益

作者声明与本文内容没有利益冲突。

参考文献

  1. 克罗利,K. S.和佩恩,R. M.(1998)。jbiol Chem 273(27): 17278-17285。
  2. 丹纳雷,S。, Wang, C.和Rehling, P.(2017)。线粒体翻译可塑性。 趋势细胞生物学 27(10): 712-721。
  3. 他,f(2011)。< a href = " https://en.bio-protocol.org/bio101/e80 " target = "平等" > Laemmli-SDS-PAGE。 生物协议 1(11): e80。
  4. 黄,J。刘,P.和王,G.(2018)。通过哺乳动物线粒体核糖核酸酶T2 (RNASET2)调控线粒体相关的细胞核糖体。jol Chem 293(51): 19633-19644。
  5. 凯莱姆斯,r.e.和布托,r.a.(1972)。核糖体结合在酵母线粒体上的证据。jbiol Chem 247(24): 8043-8050。
  6. Lesnik C。, Golani-Armon, A.和Arava, Y.(2015)。线粒体外膜定位翻译:更新。 RNA Biol 12(8): 801-809。
  7. Mukhopadhyay, A。, Ni, L.和Weiner, H.(2004)。一种解释在体内蛋白导入HeLa细胞线粒体的协同翻译模型。生物化学J 382(Pt 1): 385-392。
  8. 里德,d . W。陈问。, Tay, a。,申诺里卡尔,S.和尼奇塔,C. V.(2014)。未折叠蛋白反应触发内质网选择性mRNA释放。 Cell 158(6): 1362-1374。
  9. 里德,d.w.和Nicchitta, c.v.(2012)。 J生化 287(8): 5518-5527。
  10. Richter-Dennerlein, R。- = ytet -伊甸园字幕组= -翻译:洛伦兹,我。Ronsor, C。巴里斯,B。schendzilorz, a.b.。王,C。沃沙伊德,B。(2016)Mitochondrial protein synthesis适应了核编码protein的影响。 Cell 167(2): 471-483 e410。
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  12. 张,Y。陈,Y。古切克,M.和徐,H.(2016)。 MDI推广本地蛋白质合成和mtDNA复制膜。 EMBO 35(10): 1045-1057。< / br / >
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Huang, J. and Wang, G. (2019). Organelle-associated rRNA Degradation. Bio-protocol 9(11): e3255. DOI: 10.21769/BioProtoc.3255.
  2. Huang, J., Liu, P. and Wang, G. (2018). Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2). J Biol Chem 293(51): 19633-19644.
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