Assays for Ribosomal RNA Processing and Ribosome Assembly

Dimitri G. Pestov1, Yevgeniya R. Lapik2, Lester F. Lau2

1 University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Department of Cell Biology, Stratford, New Jersey, 2 University of Illinois at Chicago College of Medicine, Department of Biochemistry and Molecular Genetics, Chicago, Illinois
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.11
DOI:  10.1002/0471143030.cb2211s39
Online Posting Date:  June, 2008
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Abstract

The synthesis of ribosomes is a major metabolic activity critical for cell growth and homeostasis. Understanding the mechanisms of ribosome biogenesis has important implications for studying both protein synthesis and cell cycle control. This unit describes several techniques for the analysis of rRNA maturation and ribosome assembly adapted for mammalian cells. Metabolic labeling of rRNA and hybridization analysis of precursors can be used to assess changes in rRNA processing that occur under experimental conditions of interest. Separation of preribosomal particles by sucrose gradient centrifugation is suitable for the analysis of proteins associated with preribosomes during their assembly and maturation in the cell nucleus. Curr. Protoc. Cell Biol. 39:22.11.1-22.11.16. © 2008 by John Wiley & Sons, Inc.

Keywords: ribosome biogenesis; rRNA; preribosome; pre-rRNA; nucleolus; ribonucleoprotein; RNA processing; mammalian cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Metabolic Labeling of rRNA with [3H]uridine
  • Alternate Protocol 1: Metabolic Labeling of rRNA with 32Pi
  • Alternate Protocol 2: Pulse-Chase Labeling of rRNA with l-[Methyl-3H]methionine
  • Support Protocol: Fluorographic Detection of 3H-Labeled RNA on Nylon Membranes
  • Basic Protocol 2: Hybridization of rRNA Precursors with Oligonucleotide Probes
  • Basic Protocol 3: Sucrose Gradient Analysis of Pre-Ribosomal Ribonucleoprotein Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Metabolic Labeling of rRNA with [3H]uridine

 Materials
  • Adherent cell cultures (e.g., NIH 3T3 cells)
  • Complete cell culture medium (e.g., DMEM supplemented with 10% FBS)
  • 1 mCi/ml [5,6-3H]uridine (specific activity ³35 Ci/mmol)
  • Phenol/guanidine thiocyanate reagent (e.g., TRI Reagent, MRC or TRIzol, Invitrogen)
  • Deionized formamide
  • Scintillation fluid
  • 6-well tissue culture plates
  • 37°C, CO2 humidified cell culture incubator
  • Flask for radioactive waste
  • Microcentrifuge tubes with screw-caps equipped with a rubber O-ring (tubes must withstand centrifugation forces of at least 12,000 × g)
  • 55° to 65°C water bath
  • Liquid scintillation vials and counter
  • Additional reagents and equipment for RNA isolation (see manufacturer's protocol for the isolation reagent used) and RNA gel electrophoresis and nylon membrane transfer (Brown et al., 2004)

Alternate Protocol 1: Metabolic Labeling of rRNA with 32Pi

 Additional Materials (also see Basic Protocol 1)
  • Phosphate-free cell culture medium (e.g., phosphate-free DMEM) supplemented with serum dialyzed against phosphate-free saline, prewarmed to 37°C
  • 32P-Labeled orthophosphate, carrier-free (10 mCi/ml 32Pi)
  • Aerosol-resistant tips
  • Plexiglas shields and other protective equipment for safe work with 32P (appendix 1D)

Alternate Protocol 2: Pulse-Chase Labeling of rRNA with l-[Methyl-3H]methionine

 Additional Materials (also see Basic Protocol)
  • Methionine-free cell culture medium (e.g., methionine-free DMEM) supplemented with dialyzed serum
  • 1 mCi/ml l-[methyl-3H]methionine (specific activity 70 to 85 Ci/mmol)
  • 10× methionine medium: add methionine to culture medium at a final concentration of 0.3 mg/ml, prewarmed to 37°C

NOTE: l-[methyl-3H]Methionine is prone to rapid decomposition if not properly stored. For storage conditions and shelf life, refer to the manufacturer's recommendations.

Support Protocol: Fluorographic Detection of 3H-Labeled RNA on Nylon Membranes

 Materials
  • Nylon membrane with transferred 3H-labeled RNA (see Basic Protocol 1 or Alternate Protocol 2)
  • EN3HANCE Spray (PerkinElmer Life)
  • Carbon tetrachloride (Sigma)
  • Glass or polypropylene tray (a size to fit the membrane)
  • Filter paper
  • High-sensitivity radiography film (e.g., Kodak BioMax MS or equivalent)

Basic Protocol 2: Hybridization of rRNA Precursors with Oligonucleotide Probes

 Materials
  • Oligonucleotides (see Critical Parameters for details)
  • 10× T4 polynucleotide kinase buffer (supplied with enzyme)
  • [-32P]ATP (10 mCi/ml, specific activity ³3000 Ci/mmol)
  • T4 polynucleotide kinase
  • 0.5 M EDTA (appendix 2A)
  • TE buffer (appendix 2A)
  • Liquid scintillation fluid, optional
  • Nylon membranes containing total cellular RNA
  • 2× and 5× SSC (see recipe)
  • Hybridization solution (see recipe)
  • 2× SSC/0.1% SDS, room temperature and 70°C
  • 0.1× SSC/0.1% SDS
  • 1.5-ml microcentrifuge tubes
  • 37°C and 60° to 75°C water baths or hybridization ovens
  • Spin columns (e.g., Micro Bio-Spin P-6 columns, BioRad) or equivalent
  • Liquid scintillation counter and vials, optional
  • Plastic boxes with tight-fitting lids or hybridization tubes suitable for the hybridization equipment used
  • Additional reagents and equipment for autoradiography or phosphorimaging (unit 6.3)

Basic Protocol 3: Sucrose Gradient Analysis of Pre-Ribosomal Ribonucleoprotein Complexes

 Materials
  • 10% and 30% (w/w) sucrose solutions (see recipe)
  • Three 150-mm plates of subconfluent NIH 3T3 cells (~2 × 107 cells)
  • Phosphate buffered saline (PBS; appendix 2A), ice cold
  • LSB (see recipe)
  • LSB+ (see recipe)
  • 10% (w/v) Igepal CA-630 (Sigma)
  • 10% (w/v) sodium deoxycholate
  • Sonication buffer (see recipe)
  • 0.1% (w/v) BSA (appendix 2A)
  • 100% TCA, 4°C
  • 100% ethanol
  • Urea sample buffer (see recipe)
  • Polyallomer ultracentrifuge tubes for the SW41Ti rotor (9/16 × 3½– in., Beckman)
  • Plastic cell scraper
  • 15-ml conical-bottom plastic centrifuge tubes
  • Refrigerated centrifuge with a swinging bucket rotor accommodating 15-ml conical-bottom tubes
  • 1.5-ml microcentrifuge tubes
  • Ultrasonic processor equipped with a microtip
  • Refrigerated microcentrifuge
  • 1-ml syringes and 20-G needles
  • Ultracentrifuge and SW41Ti rotor (Beckman)
  • 50° and 95° to 100°C heating blocks
  • Additional reagents and equipment for gradient formation and fractionation (unit 5.3) and SDS-PAGE and immunoblotting (units 6.1 & 6.2)

NOTE: This protocol was developed for NIH 3T3 cells. For other cell types, the amounts of detergents in steps 11 to 12 may require adjustment to isolate nuclei.

NOTE: Unless otherwise indicated, all steps in the protocol are performed at 4°C.
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Figures

  •  FigureFigure 22.11.1 Gel analysis of labeled rRNA precursors from mouse cells. (A) An example of metabolic labeling with [3H]uridine. (B) An example of pulse-chase labeling with l-[methyl-3H]methionine. In the experiments shown, maturation of pre-rRNA in normal cells (– lanes) is compared with cells inducibly expressing Pes1-tn12 (+ lanes), a dominant-negative mutant of the processing factor Pes1 (Lapik et al., 2004). The positions of the major labeled RNA species are indicated on the left of each panel.
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  •  FigureFigure 22.11.2 A typical profile of UV absorbance obtained upon fractionation of nuclear extracts on a sucrose gradient. The two major peaks sedimenting at ~30S and 50S contain small and large ribosomal subunits, respectively, and their precursors. Earlier preribosomal particles are found in fractions sedimenting ahead of 50S (fractions 6 through 9).
    View Image
  •  FigureFigure 22.11.3 Structure of the primary transcript (47S) and major rRNA processing intermediates in mammals. The 47S pre-rRNA contains the sequences of mature 18S, 5.8S, and 28S rRNAs separated by the internal transcribed spacers (ITS1 and 2) and flanked by the external transcribed sequences (5'ETS, 3'ETS). Processing sites involved in the formation of each precursor are indicated. Adapted from Bowman et al. (1981) and Eichler and Craig (1994), with modifications.
    View Image

Videos

Literature Cited

Literature Cited
    Bowman, L.H., Rabin, B., and Schlessinger, D. 1981. Multiple ribosomal RNA cleavage pathways in mammalian cells. Nucleic Acids Res. 9:4951-4966.
    Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blot hybridization. Curr. Protoc. Mol. Biol. 67:4.9.1-4.9.19.
    Eichler, D.C. and Craig, N. 1994. Processing of eukaryotic ribosomal RNA. Prog. Nucl. Acid Res. Mol. Biol. 49:197-239.
    Lapik, Y.R., Fernandes, C.J., Lau, L.F., and Pestov, D.G. 2004. Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis. Mol. Cell 15:17-29.
    Strezoska, Z., Pestov, D.G., and Lau, L.F. 2000. Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5 8S rRNA processing and 60S ribosome biogenesis. Mol. Cell Biol. 20:5516-5528.
    Strezoska, Z., Pestov, D.G., and Lau, L.F. 2002. Functional inactivation of the mouse nucleolar protein Bop1 inhibits multiple steps in pre-rRNA processing and blocks cell cycle progression. J. Biol. Chem. 277:29617-29625.
    Warner, J.R. and Soeiro, R. 1967. Nascent ribosomes from HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 58:1984-1990.
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