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Peer-reviewed

Organelle-associated rRNA Degradation

附着在细胞器上的rRNA降解

JH Jinliang Huang
Geng Wang Geng Wang

发布: 2019年06月05日第9卷第11期 DOI: 10.21769/BioProtoc.3255 浏览次数: 5030

评审: Vishal S ParekhAnonymous reviewer(s)

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Dec 2018

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

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

English
中文翻译

文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

分子生物学 > RNA > RNA 降解

细胞生物学 > 细胞器分离 > 线粒体

细胞生物学 > 细胞器分离 > 内质网

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