Identifying Translationally Regulated Genes During Stem Cell Differentiation

Prabha Sampath1, Qian Yi Lee2, Vivek Tanavde2

1 Institute of Medical Biology, Agency for Science, Technology, and Research (A*STAR), Singapore, 2 Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), Singapore
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.8
DOI:  10.1002/9780470151808.sc01b08s18
Online Posting Date:  September, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a protocol for genome‐wide identification of translationally regulated genes during embryonic stem cell differentiation using integrated transcriptome and translation state profiling. Actively translated mRNAs associated with multiple ribosomes (known as polysomes) and translationally inactive mRNAs sequestered in messenger ribonucleoprotein particles (mRNPs), can be separated by sucrose gradient fractionation based on size. Because the number of ribosomes on a transcript correlates with the rate of synthesis of its encoded protein, this allows an operational distinction between well‐translated and poorly translated mRNA molecules. In this analysis, fractionated mRNA and total RNA are used to probe microarrays to identify differentially translated genes. Curr. Protoc. Stem Cell Biol. 18:1B.8.1‐1B.8.13. © 2011 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; translational control; polysome analysis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Total and Fractionated RNA for Microarray Analysis
  • Support Protocol 1: RNA Quality Control by Bioanalysis
  • Basic Protocol 2: Microarray Analysis of Fractionated and Total RNA
  • Basic Protocol 3: Analysis of Microarray Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Total and Fractionated RNA for Microarray Analysis

  Materials
  • Sucrose gradient solutions (see recipe)
  • Mouse ESCs grown to ∼5 × 106 cells/dish in two 10‐cm dishes per experimental condition (undifferentiated control and RA‐treated)
  • Cycloheximide
  • Sterile PBS
  • Lysis buffer (see recipe), ice cold
  • Nuclease‐free water
  • 10% (w/v) SDS
  • Synthetic poly(A) RNA (Affymetrix, cat. no. 900433)
  • Luciferase RNA (Promega)
  • RNeasy RNA isolation minikit (Qiagen, cat. no. 74104)
  • 14‐ml conical polypropylene centrifuge tubes (Falcon, cat. no. 331372)
  • Cell scrapers
  • 1.5‐ml microcentrifuge tubes
  • Spectrophotometer
  • Ultracentrifuge (Beckman) with SW40‐Ti swinging bucket rotor
  • Gradient fraction collector (ISCO)
  • UV detector (ISCO UA‐6 or equivalent)
  • NanoDrop 8000 spectrophotometer v3.7 (Thermoscientific)
  • Additional reagents and equipment for bioanalysis (see protocol 2)
NOTE: Cell culture procedures should be performed in a Class II biological hazard flow hood or a laminar‐flow hood. Cell culture incubations are performed in a humidified 37°C, 5% CO 2 incubator.NOTE: All solutions and equipment must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 1: RNA Quality Control by Bioanalysis

  Materials
  • Purified RNA, pools 1 and 2 (see protocol 1)
  • RNA 6000 Nano Kit (Agilent Technologies, cat. no. 5067‐1512)
  • 0.5‐ml RNase‐free microcentrifuge tube
  • RNA Nano Chip (Agilent Technologies, cat. no. 5067‐1511)
  • BioAnalyzer 2100 (Agilent Technologies)

Basic Protocol 2: Microarray Analysis of Fractionated and Total RNA

  Materials
  • Pool 1, pool 2, and total RNA from control and treated cell cultures (see protocol 1)
  • TargetAmp‐Nano Labeling Kit for Illumina Expression BeadChip (Epicentre, cat. no. TAN07924)
  • RNeasy RNA isolation minikit (Qiagen, cat. no. 74104)
  • Absolute molecular‐grade ethanol
  • Streptavidin Cy3 (GE Healthcare, cat. no. PA43001)
  • NanoDrop 8000 spectrophotometer v3.7 (Thermoscientific)
  • RNA microarray: HumanWG‐6 v3.0 Whole‐Genome Expression BeadChip (Illumina)
  • BeadArray Reader (Illumina)

Basic Protocol 3: Analysis of Microarray Data

  Materials
  • Illumina BeadChip results (see protocol 3)
  • Partek Genomics Suite (http://www.partek.com/)
  • Partek Gene Expression Plug‐In
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 1.B0.1 (A) Polysome fractionation in sucrose density gradient. Actively translated, polysome‐bound mRNAs are denser and settle at the bottom of the sucrose gradient. Translationally inactive mRNAs that are not bound to ribosomes are less dense and settle at the top of the gradient. (B) Collected RNA fractions (F) are pooled before RNA is isolated for microarray analysis. (C) Absorbance profile ( A254) across the sucrose gradient.
  •   FigureFigure 1.B0.2 Experimental design. Cultured ESCs are treated with retinoic acid (RA) to induce differentiation. Cellular lysates from control (untreated) and treated ESCs are then fractionated to separate poorly translated (pool 1) and highly translated (pool 2) mRNA for translational state profiling. The unfractionated cellular lysate (total RNA), pool 1, and pool 2 samples are split into three replicates each and loaded onto microarrays in a randomized manner.
  •   FigureFigure 1.B0.3 Quality control by principal components analysis or hierarchical clustering. Technical and/or biological replicates should cluster more tightly together compared to biologically distinct samples.

Videos

Literature Cited

Literature Cited
   Bolstad, B.M., Irizarry, R.A., Astrand, M., and Speed, T.P. 2003. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185‐193.
   Bushell, M., Stoneley, M., Kong, Y.W., Hamilton, T.L., Spriggs, K.A., Dobbyn, H.C., Qin, X., Sarnow, P., and Willis, A.E. 2006. Polypyrimidine tract binding protein regulates IRES‐mediated gene expression during apoptosis. Mol. Cell 23:401‐412.
   Dinkova, T.D., Keiper, B.D., Korneeva, N.L., Aamodt, E.J., and Rhoads, R.E. 2005. Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform. Mol. Cell Biol. 25:100‐113.
   Downey, T. 2006. Analysis of a multifactor microarray study using Partek genomics solution. Methods Enzymol. 411:256‐270.
   Dvash, T., Mayshar, Y., Darr, H., McElhaney, M., Barker, D., Yanuka, O., Kotkow, K.J., Rubin, L.L., Benvenisty, N., and Eiges, R. 2004. Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies. Hum. Reprod. 19:2875‐2883.
   Gebauer, F. and Hentze, M.W. 2004. Molecular mechanisms of translational control. Nat. Rev. Mol. Cell Biol. 5:827‐835.
   Guan, K., Chang, H., Rolletschek, A., and Wobus, A.M. 2001. Embryonic stem cell‐derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell Tissue Res. 305:171‐176.
   Gunji, W., Kai, T., Sameshima, E., Iizuka, N., Katagi, H., Utsugi, T., Fujimori, F., and Murakami, Y. 2004. Global analysis of the expression patterns of transcriptional regulatory factors in formation of embryoid bodies using sensitive oligonucleotide microarray systems. Biochem. Biophys. Res. Commun. 325:265‐275.
   Iguchi, N., Tobias, J.W., and Hecht, N.B. 2006. Expression profiling reveals meiotic male germ cell mRNAs that are translationally up‐ and down‐regulated. Proc. Natl. Acad. Sci. U.S.A. 103:7712‐7717.
   Ingolia, N.T., Ghaemmaghami, S., Newman, J.R., and Weissman, J.S. 2009. Genome‐wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218‐223.
   Kuersten, S. and Goodwin, E.B. 2003. The power of the 3′ UTR: Translational control and development. Nat. Rev. Genet. 4:626‐637.
   Laflamme, M.A., Chen, K.Y., Naumova, A.V., Muskheli, V., Fugate, J.A., Dupras, S.K., Reinecke, H., Xu, C., Hassanipour, M., Police, S., O'Sullivan, C., Collins, L., Chen, Y., Minami, E., Gill, E.A., Ueno, S., Yuan, C., Gold, J., and Murry, C.E. 2007. Cardiomyocytes derived from human embryonic stem cells in pro‐survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25:1015‐1024.
   Lee, Q.Y., Koh, W., Sampath, P., and Tanavde, V. 2011. Integrating transcriptome and translational state profiling to identify genes under translational control during embryonic stem cell differentiation. In Computational Biology of Embryonic Stem Cells ( M. Zhan, ed.), in press. Bentham Scientific Publishers, Oak Park, Ill.
   Pradet‐Balade, B., Boulme, F., Beug, H., Mullner, E.W., and Garcia‐Sanz, J.A. 2001. Translation control: Bridging the gap between genomics and proteomics? Trends Biochem. Sci. 26:225‐229.
   Pritsker, M., Ford, N.R., Jenq, H.T., and Lemischka, I.R. 2006. Genomewide gain‐of‐function genetic screen identifies functionally active genes in mouse embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 103:6946‐6951.
   Qin, X., Ahn, S., Speed, T.P., and Rubin, G.M. 2007. Global analyses of mRNA translational control during early Drosophila embryogenesis. Genome Biol. 8:R63.
   Ramalho‐Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., and Melton, D.A. 2002. “Stemness”: Transcriptional profiling of embryonic and adult stem cells. Science 298:597‐600.
   Richter, J.D. and Theurkauf, W.E. 2001. Development. The message is in the translation. Science 293:60‐62.
   Sampath, P., Pritchard, D.K., Pabon, L., Reinecke, H., Schwartz, S.M., Morris, D.R., and Murry, C.E. 2008. A hierarchical network controls protein translation during murine embryonic stem cell self‐renewal and differentiation. Cell Stem Cell 2:448‐460.
   Sanz, E., Yang, L., Su, T., Morris, D.R., McKnight, G.S., and Amieux, P.S. 2009. Cell‐type‐specific isolation of ribosome‐associated mRNA from complex tissues. Proc. Natl. Acad. Sci. U.S.A. 106:13939‐13944.
   Stavridis, M.P. and Smith, A.G. 2003. Neural differentiation of mouse embryonic stem cells. Biochem. Soc. Trans. 31:45‐49.
   Wobus, A.M. and Boheler, K.R. 2005. Embryonic stem cells: Prospects for developmental biology and cell therapy. Physiol. Rev. 85:635‐678.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library