Abstract
Protein translation in the nucleus has been controversial for more than four decades. To take a new look at this potentially important phenomenon, we adapted the RiboPuromycylation Method (RPM) which labels actively translating ribosomes in cells via standard immunofluorescence microscopy. RPM is based on puromycylation of nascent chains trapped on ribosomes by antibiotics which inhibit chain elongation, followed by cell permeabilization/fixation and detection of puromycylated nascent chains using a puromycin-specific monoclonal antibody. To adapt the method to the nucleus, we use NP-40 rather than digitonin to permeabilize cells because NP-40 enables better antibody penetration into the nucleoplasm and particularly the nucleoli, a region of high translation as shown by RPM.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Allfrey VG, Mirsky AE, Osawa S (1955) Protein synthesis in isolated cell nuclei. Nature 176:1042–1049
Allfrey VG (1954) Amino acid incorporation by isolated thymus nuclei. I. The role of desoxyribonucleic acid in protein synthesis. Proc Natl Acad Sci U S A 40:881–885
Iborra FJ, Jackson DA, Cook PR (2001) Coupled transcription and translation within nuclei of mammalian cells. Science 293:1139–1142
Nathanson L, Xia T, Deutscher MP (2003) Nuclear protein synthesis: a re-evaluation. RNA 9:9–13
Dahlberg JE, Lund E, Goodwin EB (2003) Nuclear translation: what is the evidence? RNA 9:1–8
Dolan BP, Knowlton JJ, David A et al (2010) RNA polymerase II inhibitors dissociate antigenic peptide generation from normal viral protein synthesis: a role for nuclear translation in defective ribosomal product synthesis? J Immunol 185:6728–6733
David A, Dolan BP, Hickman HD et al (2012) Nuclear translation visualized by ribosome-bound nascent chain puromycylation. J Cell Biol 197:45–57
Pestka S (1971) Inhibitors of ribosome functions. Annu Rev Microbiol 25:487–562
David A, Bennink JR, Yewdell JW (2013) Emetine optimally facilitates nascent chain puromycylation and potentiates the ribopuromycylation method (RPM) applied to inert cells. Histochem Cell Biol 139:501–504
Graber TE, Hébert-Seropian S, Khoutorsky A et al (2013) Reactivation of stalled polyribosomes in synaptic plasticity. Proc Natl Acad Sci U S A 110:16205–16210
Al-Jubran K, Wen J, Abdullahi A et al (2013) Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus. RNA 19:1669–1683
Apcher S, Millot G, Daskalogianni C et al (2013) Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway. Proc Natl Acad Sci U S A 110:17951–17956
Jiménez A, Carrasco L, Vázquez D (1977) Enzymic and nonenzymic translocation by yeast polysomes. Site of action of a number of inhibitors. Biochemistry 16:4727–4730
Hansen JL, Moore PB, Steitz TA (2003) Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit. J Mol Biol 330:1061–1075
Kedersha N, Chen S, Gilks N et al (2002) Evidence that ternary complex (eIF2-GTP-tRNAiMet) – deficient preinitiation complexes are core constituents of mammalian stress granules. Mol Biol Cell 1:195–210
Fresno M, Jimenez A, Vazquez D (1977) Inhibition of translation in eukaryotic systems by harringtonine. Eur J Biochem 72:323–330
Schmidt EK, Clavarino G, Ceppi M et al (2009) SUnSET, a nonradioactive monitor protein synthesis. Nat Methods 6:275–277
Acknowledgements
J.W.Y. is generously supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
David, A., Yewdell, J.W. (2015). Applying the Ribopuromycylation Method to Detect Nuclear Translation. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 1228. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1680-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1680-1_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1679-5
Online ISBN: 978-1-4939-1680-1
eBook Packages: Springer Protocols