Abstract
Genetic mutations, whether they occur within protein-coding or noncoding regions of the genome, can affect various aspects of gene expression by influencing the complex network of intra- and intermolecular interactions that occur between cellular nucleic acids and proteins. One aspect of gene expression control that can be impacted is the intracellular trafficking and translation of mRNA molecules. To study the occurrence and dynamics of translational regulation, researchers have developed approaches such as genome-wide ribosome profiling and artificial reporters that enable single molecule imaging. In this paper, we describe a complementary and optimized approach that combines puromycin labeling with a proximity ligation assay (Puro-PLA) to define sites of translation of specific mRNAs in tissues or cells. This method can be used to study the mechanisms driving the translation of select mRNAs and to access the impact of genetic mutations on local protein synthesis. This approach involves the treatment of cell or tissue specimens with puromycin to label nascently translated peptides, rapid fixation, followed by immunolabeling with appropriate primary and secondary antibodies coupled to PLA oligonucleotide probes, ligation, amplification, and signal detection via fluorescence microscopy. Puro-PLA can be performed at small scale in individual tubes or in chambered slides, or in a high-throughput setup with 96-well plate, for both in situ and in vitro experimentation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Jacob F, Monod J (1961) Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3:318–356
Lehner B (2011) Molecular mechanisms of epistasis within and between genes. Trends Genet 27(8):323–331
Chin A, Lécuyer E (2017) RNA localization: making its way to the center stage, Biochimica et biophysica acta. General Subjects 1861(11 Pt B):2956–2970
Lécuyer E, Yoshida H, Parthasarathy N et al (2007) Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131(1):174–187
Iampietro C, Bergalet J, Wang X et al (2014) Developmentally regulated elimination of damaged nuclei involves a Chk2-dependent mechanism of mRNA nuclear retention. Dev Cell 29(4):468–481
Kugler JM, Lasko P (2009) Localization, anchoring and translational control of oskar, gurken, bicoid and nanos mRNA during drosophila oogenesis. Fly 3(1):15–28
Diot C, Chin A, Lécuyer E (2017) Optimized FISH methods for visualizing RNA localization properties in drosophila and human tissues and cultured cells. Methods (San Diego, Calif.) 126:156–165
Paquin N, Chartrand P (2008) Local regulation of mRNA translation: new insights from the bud. Trends Cell Biol 18(3):105–111
Medioni C, Mowry K, Besse F (2012) Principles and roles of mRNA localization in animal development. Development 139(18):3263–3276
Ham BK, Brandom JL, Xoconostle-Cázares B et al (2009) A polypyrimidine tract binding protein, pumpkin RBP50, forms the basis of a phloem-mobile ribonucleoprotein complex. Plant Cell 21(1):197–215
Kislauskis EH, Zhu X, Singer RH (1997) Beta-actin messenger RNA localization and protein synthesis augment cell motility. J Cell Biol 136(6):1263–1270
Leung KM, van Horck FP, Lin AC et al (2006) Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1. Nat Neurosci 9(10):1247–1256
Liu-Yesucevitz L, Bassell GJ, Gitler AD (2011) Local RNA translation at the synapse and in disease. J Neurosci 31(45):16086–16093
Chouaib R, Safieddine A, Pichon X et al (2020) A dual protein-mRNA localization screen reveals compartmentalized translation and widespread co-translational RNA targeting. Develop Cell 54(6):773–791.e5
Xia C, Fan J, Emanuel G et al (2019) Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression. Proc Natl Acad Sci U S A 116(39):19490–19499
Wilk R, Hu J, Blotsky D et al (2016) Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs. Genes Dev 30(5):594–609
Fazal FM, Han S, Parker KR et al (2019) Atlas of subcellular RNA localization revealed by APEX-Seq. Cell 178(2):473–490.e26
Benoit Bouvrette LP, Cody NAL, Bergalet J et al (2018) CeFra-seq reveals broad asymmetric mRNA and noncoding RNA distribution profiles in drosophila and human cells. RNA (New York, N.Y.) 24(1):98–113
Rodriguez AJ, Shenoy SM, Singer RH et al (2006) Visualization of mRNA translation in living cells. J Cell Biol 175(1):67–76
Xiao Z, Zou Q, Liu Y et al (2016) Genome-wide assessment of differential translations with ribosome profiling data. Nat Commun 7:11194
Weinberg DE, Shah P, Eichhorn SW et al (2016) Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation. Cell Rep 14(7):1787–1799
Ingolia NT, Ghaemmaghami S, Newman JR et al (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science (New York, N.Y.) 324(5924):218–223
Ingolia NT, Brar GA, Rouskin S et al (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7(8):1534–1550
Yan X, Hoek TA, Vale RD et al (2016) Dynamics of translation of single mRNA molecules in vivo. Cell 165(4):976–989
Halstead JM, Wilbertz JH, Wippich F et al (2016) TRICK: a single-molecule method for imaging the first round of translation in living cells and animals. Methods Enzymol 572:123–157
Halstead JM, Lionnet T, Wilbertz JH et al (2015) Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science (New York, N.Y.) 347(6228):1367–1671
Tanenbaum ME, Gilbert LA, Qi LS et al (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159(3):635–646
Boersma S, Rabouw HH, Bruurs LJM et al (2020) Translation and replication dynamics of single RNA viruses. Cell 183(7):1930–1945.e23
Zappulo A, van den Bruck D, Ciolli Mattioli C et al (2017) RNA localization is a key determinant of neurite-enriched proteome. Nat Commun 8(1):583
tom Dieck S, Kochen L, Hanus C et al (2015) Direct visualization of newly synthesized target proteins in situ. Nat Methods 12(5):411–414
Bergalet J, Patel D, Legendre F et al (2020) Inter-dependent Centrosomal co-localization of the cen and ik2 cis-natural antisense mRNAs in drosophila. Cell Rep 30(10):3339–3352.e6
Pestka S (1971) Inhibitors of ribosome functions. Annu Rev Microbiol 25:487–562
Grollman AP (1967) Inhibitors of protein biosynthesis. II. Mode of action of anisomycin. J Biol Chem 242(13):3226–3233
Robb JA (1969) Maintenance of imaginal discs of Drosophila melanogaster in chemically defined media. J Cell Biol 41(3):876–885
Jiang Z, Belforte JE, Lu Y et al (2010) eIF2alpha phosphorylation-dependent translation in CA1 pyramidal cells impairs hippocampal memory consolidation without affecting general translation. J Neurosci 30(7):2582–2594
Wei CM, Hansen BS, Vaughan MH et al (1974) Mechanism of action of the mycotoxin trichodermin, a 12,13-epoxytrichothecene. Proc Natl Acad Sci U S A 71(3):713–717
Acknowledgments
This work was supported by grants to EL from the Canadian Cancer Society Research Institute and from the Canadian Institutes of Health Research (CIHR). AC was supported by the Vanier Canada Graduate Scholarship program from CIHR and EL is a Senior Scholar of the Fonds de Recherche du Québec Santé.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chin, A., Lécuyer, E. (2021). Puromycin Labeling Coupled with Proximity Ligation Assays to Define Sites of mRNA Translation in Drosophila Embryos and Human Cells. In: Vizeacoumar, F.J., Freywald, A. (eds) Mapping Genetic Interactions. Methods in Molecular Biology, vol 2381. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1740-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1740-3_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1739-7
Online ISBN: 978-1-0716-1740-3
eBook Packages: Springer Protocols