Single‐Molecule mRNA Detection in Live Yeast

Tineke L. Lenstra1, Daniel R. Larson1

1 Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, Bethesda
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.24
DOI:  10.1002/0471142727.mb1424s113
Online Posting Date:  January, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub‐second temporal resolution and nanometer spatial resolution. Studies of transcription in single cells indicate that gene activity is heterogeneous between cells and exhibits random variability over time, even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this unit, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loop sequences that are introduced into the gene of interest. Detailed protocols are provided for the construction of the necessary yeast strains, for image acquisition, and for validation. © 2016 by John Wiley & Sons, Inc.

Keywords: single‐molecule; RNA; transcription; live‐cell; microscopy; fluorescence

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

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Visualizing Gene Expression in Live Yeast Using MS2 and PP7 Labeling
  • Support Protocol 1: Coating of Dishes with Concanavalin A
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Visualizing Gene Expression in Live Yeast Using MS2 and PP7 Labeling

  Materials
  • Plasmid containing repeats of stem loop sequences and a selectable integration marker flanked by loxP sites, such as pDZ415 (24MS2SL loxPKanMXloxP), pDZ416 (24PP7SL loxPKanMXloxP), available at Addgene (www.addgene.com), or pTL030 (14MS2SL loxPKanMXloxP), pTL031 (14PP7SL loxPKanMXloxP), available from the authors
  • PCR primers for integration of stem loop repeats
  • PCR primers to check genomic integration of stem loop repeats and to check Cre‐mediated excision of selectable marker
  • Standard materials for PCR (Kramer and Coen, )
  • 0.7% to 1% Agarose gels (Voytas, )
  • PCR cleaning kit to purify the PCR product and remove primers, dNTPs, and enzymes
  • Selective yeast plates and media for growth and transformation (including YPD plates, YPGAL medium, synthetic media minus uracil plates and media, 5‐fluoroorotic acid plates); for recipes, see Guthrie and Fink ( ) and Lundblad and Struhl ( )
  • Standard materials for transformation (Becker and Lundblad, )
  • 50% glycerol
  • URA3 plasmid, such as pSH47‐encoding Cre recombinase under a GAL1 promoter
  • Plasmid encoding the coat protein fused to an NLS and a fluorescent protein, such as pTL041 (pURA P ADE3­‐PCP‐NLS‐2×‐yeGFP) or pTL042 (pLEU P MET17‐MCP‐NLS‐mKate2), available from the authors
  • Concanavalin A‐coated glass‐bottom culture dishes with a coverslip thickness which matches the objective used in the experiment (e.g., MatTek 35 mm, No. 1.5 or NEST 35 mm) (see the Support Protocol)
  • Cryogenic vials
  • Incubator at 30°C
  • Inverted wide‐field microscope (e.g., Zeiss AxioObserver) with:
    • >100 × objective, NA > 1.3 (e.g., Zeiss Plan‐Apochromat 150 × /1.35 Glyc DIC)
  • Laser excitation source (or equivalent strong narrowband excitation)(e.g., Spectra‐Physics Excelsior diode‐pumped solid state lasers)
  • Excitation and emission filters optimized for fluorophore (e.g., for GFP: ET470/30x excitation filter (for non‐laser illumination), T495lpxr dichroic beamsplitter and ET525/50m emission filter, Chroma)
  • EM‐CCD (e.g., Evolve 512, Photometrics)
  • Focusing device (e.g., Definite focus, Zeiss)
  • Microscope stage incubator (e.g., Tokai Hit, INUB‐LPS)

Support Protocol 1: Coating of Dishes with Concanavalin A

  Materials
  • Concanavalin A from Canavalia ensiformis (Jack bean)
  • Phosphate‐buffered saline (PBS; e.g., Gibco PBS, pH 7.4)
  • 0.22‐μm filter
  • Glass‐bottom culture dishes with a coverslip thickness that matches the objective used in the experiment (e.g., MatTek 35 mm, no. 1.5 or NEST 35 mm)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Becker, D. M. and Lundblad, V. 2001. Introduction of DNA into yeast cells. Curr. Protoc. Mol. Biol. 27:13.7.1‐13.7.10.
  Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S.M., Singer, R.H., and Long, R.M. 1998. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2:437‐445. doi: 10.1016/S1097-2765(00)80143-4.
  Burke, D.J., Amberg, D.C., and Strathern, J.N. 2005. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition, 2005 Edition First Printing edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Chao, J.A., Patskovsky, Y., Almo, S.C., and Singer, R.H. 2008. Structural basis for the coevolution of a viral RNA‐protein complex. Nat. Struct. Mol. Biol. 15:103‐105. doi: 10.1038/nsmb1327.
  Coulon, A., Ferguson, M.L., de Turris, V., Palangat, M., Chow, C.C., and Larson, D.R. 2014. Kinetic competition during the transcription cycle results in stochastic RNA processing. eLife 3:e03939.
  Darzacq, X., Shav‐Tal, Y., de Turris, V., Brody, Y., Shenoy, S.M., Phair, R.D., and Singer, R.H. 2007. In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14:796‐806. doi: 10.1038/nsmb1280.
  Forrest, K.M. and Gavis, E.R. 2003. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr. Biol. 13:1159‐1168. doi: 10.1016/S0960-9822(03)00451-2.
  Fusco, D., Accornero, N., Lavoie, B., Shenoy, S.M., Blanchard, J.M., Singer, R.H., and Bertrand, E. 2003. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13:161‐167. doi: 10.1016/S0960-9822(02)01436-7.
  Golding, I., Paulsson, J., Zawilski, S.M., and Cox, E.C. 2005. Real‐time kinetics of gene activity in individual bacteria. Cell 123:1025‐1036. doi: 10.1016/j.cell.2005.09.031.
  Guthrie, C. and Fink, G.R. 2004. Guide to Yeast Genetics and Molecular and Cell Biology. Gulf Professional Publishing.
  Haim, L., Zipor, G., Aronov, S., and Gerst, J.E. 2007. A genomic integration method to visualize localization of endogenous mRNAs in living yeast. Nat. Methods 4:409‐412.
  Hocine, S., Raymond, P., Zenklusen, D., Chao, J.A., and Singer, R.H. 2013. Single‐molecule analysis of gene expression using two‐color RNA labeling in live yeast. Nat. Methods 10:119‐121. doi: 10.1038/nmeth.2305.
  Janicki, S.M., Tsukamoto, T., Salghetti, S.E., Tansey, W.P., Sachidanandam, R., Prasanth, K.V., Ried, T., Shav‐Tal, Y., Bertrand, E., Singer, R.H., and Spector, D.L. 2004. From silencing to gene expression: Real‐time analysis in single cells. Cell 116:683‐698. doi: 10.1016/S0092-8674(04)00171-0.
  Jensen, T.H., Patricio, K., McCarthy, T., and Rosbash, M. 2001. A block to mRNA nuclear export in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription. Mol. Cell 7:887‐898. doi: 10.1016/S1097-2765(01)00232-5.
  Johnston, G.C., Ehrhardt, C.W., Lorincz, A., and Carter, B.L. 1979. Regulation of cell size in the yeast Saccharomyces cerevisiae. J. Bacteriol. 137:1‐5.
  Kramer, M. F. and Coen, D. M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
  Larson, D.R. 2010. The economy of photons. Nat. Methods 7:357‐359. doi: 10.1038/nmeth0510-357.
  Larson, D.R., Singer, R.H., and Zenklusen, D. 2009. A single molecule view of gene expression. Trends Cell Biol. 19:630‐637. doi: 10.1016/j.tcb.2009.08.008.
  Larson, D.R., Zenklusen, D., Wu, B., Chao, J.A., and Singer, R.H. 2011. Real‐time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332:475‐478. doi: 10.1126/science.1202142.
  Lee, S., Lim, W.A., and Thorn, K.S. 2013. Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae. PloS One 8:e67902.
  Lundblad, V. and Struhl, K. 2010. Yeast. Curr. Protoc. Mol. Biol. 92:13.01‐13.0.4.
  Ozawa, T., Natori, Y., Sato, M., and Umezawa, Y. 2007. Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat. Methods 4:413‐419.
  Paige, J.S., Wu, K.Y., and Jaffrey, S.R. 2011. RNA mimics of green fluorescent protein. Science 333:642‐646. doi: 10.1126/science.1207339.
  Pelechano, V., Wei, W., and Steinmetz, L.M. 2013. Extensive transcriptional heterogeneity revealed by isoform profiling. Nature 497:127‐131. doi: 10.1038/nature12121.
  Powrie, E.A., Zenklusen, D., and Singer, R.H. 2011. A nucleoporin, Nup60p, affects the nuclear and cytoplasmic localization of ASH1 mRNA in S. cerevisiae. RNA 17:134‐144. doi: 10.1261/rna.1210411.
  Rahman, S. and Zenklusen, D. 2013. Single‐molecule resolution fluorescent in situ hybridization (smFISH) in the yeast S. cerevisiae. Methods Mol. Biol. 1042:33‐46.
  Rines, D.R., Thomann, D., Dorn, J.F., Goodwin, P., and Sorger, P.K. 2011. Live cell imaging of yeast. Cold Spring Harb. Protoc. 2011:pii: pdb.top065482.
  Rook, M.S., Lu, M., and Kosik, K.S. 2000. CaMKIIalpha 3′ untranslated region‐directed mRNA translocation in living neurons: Visualization by GFP linkage. J. Neurosci. 20:6385‐6393.
  Shav‐Tal, Y., Darzacq, X., Shenoy, S.M., Fusco, D., Janicki, S.M., Spector, D.L., and Singer, R.H. 2004. Dynamics of single mRNPs in nuclei of living cells. Science 304:1797‐1800. doi: 10.1126/science.1099754.
  Sheth, U. and Parker, R. 2003. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300:805‐808. doi: 10.1126/science.1082320.
  Shin, I., Ray, J., Gupta, V., Ilgu, M., Beasley, J., Bendickson, L., Mehanovic, S., Kraus, G.A., and Nilsen‐Hamilton, M. 2014. Live‐cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters. Nucleic Acids Res. 42:e90.
  Trcek, T., Larson, D.R., Moldón, A., Query, C.C., and Singer, R.H. 2011. Single‐molecule mRNA decay measurements reveal promoter‐regulated mRNA stability in yeast. Cell 147:1484‐1497. doi: 10.1016/j.cell.2011.11.051.
  Trcek, T., Chao, J.A., Larson, D.R., Park, H.Y., Zenklusen, D., Shenoy, S.M., and Singer, R.H. 2012. Single‐mRNA counting using fluorescent in situ hybridization in budding yeast. Nat. Protoc. 7:408‐419. doi: 10.1038/nprot.2011.451.
  Valencia‐Burton, M., McCullough, R.M., Cantor, C.R., and Broude, N.E. 2007. RNA visualization in live bacterial cells using fluorescent protein complementation. Nat. Methods 4:421‐427.
  Voytas, D. 2001. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
  Wu, B., Chao, J.A., and Singer, R.H. 2012. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys. J. 102:2936‐2944. doi: 10.1016/j.bpj.2012.05.017.
  Wu, B., Chen, J., and Singer, R.H. 2014. Background free imaging of single mRNAs in live cells using split fluorescent proteins. Sci. Rep. 4:3615.
  Zenklusen, D., Larson, D.R., and Singer, R.H. 2008. Single‐RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15:1263‐1271. doi: 10.1038/nsmb.1514.
  Zid, B.M. and O'Shea, E.K. 2014. Promoter sequences direct cytoplasmic localization and translation of mRNAs during starvation in yeast. Nature 514:117‐121. doi: 10.1038/nature13578.
Internet Resources
  http://www.yeastgenome.org
  The Saccharomyces genome database (SGD) Web site is a great repository for information on all yeast genes.
  http://www.micro‐manager.org
  μManager is an open source microscopy software package.
  http://fiji.sc/Fiji
  Fiji is an imaging‐processing software package.
  http://www.larsonlab.net
  Software downloads for RNA tracking.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library