Abstract
Ribopuromycylation enables the visualization and quantitation of translation on a cellular level by immunofluorescence or in total using standard western blotting. This technique uses ribosome catalyzed puromycylation of nascent chains followed by immobilization on the ribosome by antibiotic chain elongation inhibitor emetine. Detection of puromycylated ribosome-bound nascent chains can then be achieved using a puromycin-specific antibody.
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
David A, Dolan BP, Hickman HD, Knowlton JJ, Clavarino G, Pierre P, Bennink JR, Yewdell JW (2012) Nuclear translation visualized by ribosome-bound nascent chain puromycylation. J Cell Biol 197(1):45–57. https://doi.org/10.1083/jcb.201112145
Pestka S (1971) Inhibitors of ribosome functions. Annu Rev Microbiol 25:487–562. https://doi.org/10.1146/annurev.mi.25.100171.002415
Eggers DK, Welch WJ, Hansen WJ (1997) Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells. Mol Biol Cell 8(8):1559–1573. https://doi.org/10.1091/mbc.8.8.1559
Schmidt EK, Clavarino G, Ceppi M, Pierre P (2009) SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods 6(4):275–277. https://doi.org/10.1038/nmeth.1314
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(3):501–504. https://doi.org/10.1007/s00418-012-1063-8
Desmet EA, Anguish LJ, Parker JS (2014) Virus-mediated compartmentalization of the host translational machinery. MBio 5(5):e01463-14. https://doi.org/10.1128/mBio.01463-14
Götte B, Panas MD, Hellström K, Liu L, Samreen B, Larsson O, Ahola T, McInerney GM (2019) Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery. PLoS Pathog 15(6):e1007842. https://doi.org/10.1371/journal.ppat.1007842
Shen P, Reineke LC, Knutsen E, Chen M, Pichler M, Ling H, Calin GA (2018) Metformin blocks MYC protein synthesis in colorectal cancer via mTOR-4EBP-eIF4E and MNK1-eIF4G-eIF4E signaling. Mol Oncol 12(11):1856–1870. https://doi.org/10.1002/1878-0261.12384
Brocard M, Iadevaia V, Klein P, Hall B, Lewis G, Lu J, Burke J, Willcocks MM, Parker R, Goodfellow IG, Ruggieri A, Locker N (2020) Norovirus infection results in eIF2α independent host translation shut-off and remodels the G3BP1 interactome evading stress granule formation. PLoS Pathog 16(1):e1008250. https://doi.org/10.1371/journal.ppat.1008250
Casais R, Thiel V, Siddell SG, Cavanagh D, Britton P (2001) Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol 75(24):12359–12369. https://doi.org/10.1128/jvi.75.24.12359-12369.2001
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019
V'Kovski P, Gerber M, Kelly J, Pfaender S, Ebert N, Braga Lagache S, Simillion C, Portmann J, Stalder H, Gaschen V, Bruggmann R, Stoffel MH, Heller M, Dijkman R, Thiel V (2019) Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling. elife 8. https://doi.org/10.7554/eLife.42037
Bastide A, Yewdell JW, David A (2018) The RiboPuromycylation method (RPM): an immunofluorescence technique to map translation sites at the sub-cellular level. Bio Protoc 8(1):e2669. https://doi.org/10.21769/BioProtoc.2669
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Brownsword, M.J., Maier, H.J., Locker, N. (2020). Ribopuromycylation in Coronavirus-Infected Cells. In: Maier, H., Bickerton, E. (eds) Coronaviruses. Methods in Molecular Biology, vol 2203. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0900-2_17
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0900-2_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0899-9
Online ISBN: 978-1-0716-0900-2
eBook Packages: Springer Protocols