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Emetine optimally facilitates nascent chain puromycylation and potentiates the ribopuromycylation method (RPM) applied to inert cells

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Abstract

We previously described the ribopuromyclation method (RPM) to visualize and quantitate translating ribosomes in fixed and permeabilized cells by standard immunofluorescence. RPM is based on puromycylation of nascent chains bound to translating ribosomes followed by detection of puromycylated nascent chains with a puromycin-specific mAb. We now demonstrate that emetine optimally enhances nascent chain puromycylation, and describe a modified RPM protocol for identifying ribosome-bound nascent chains in metabolically inert permeabilized cells.

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References

  • David A, Netzer N, Strader MB, Das SR, Chen CY et al (2011) RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes. J Biol Chem 286:20688–20700

    Article  PubMed  CAS  Google Scholar 

  • David A, Dolan BP, Hickman HD, Knowlton JJ, Clavarino G et al (2012) Nuclear translation visualized by ribosome-bound nascent chain puromycylation. J Cell Biol 197:45–57

    Article  PubMed  CAS  Google Scholar 

  • Eggers DK, Welch WJ, Hansen WJ (1997) Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells. Mol Biol Cell 8:1559–1573

    PubMed  CAS  Google Scholar 

  • Fallon AM, Stollar V (1982) Isolation and characterization of puromycin-resistant clones from cultured mosquito cells. Somatic Cell Genet 8:521–532

    Article  PubMed  CAS  Google Scholar 

  • Grollman AP (1968) Inhibitors of protein biosynthesis. V. Effects of emetine on protein and nucleic acid biosynthesis in HeLa cells. J Biol Chem 243:4089–4094

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Jimenez A, Carrasco L, Vazquez D (1977) Enzymic and nonenzymic translocation by yeast polysomes. Site of action of a number of inhibitors. Biochemistry 16:4727–4730

    Article  PubMed  CAS  Google Scholar 

  • Kappen LS, Suzuki H, Goldberg IH (1973) Inhibition of reticulocyte peptide-chain initiation by pactamycin: accumulation of inactive ribosomal initiation complexes. Proc Natl Acad Sci USA 70:22–26

    Article  PubMed  CAS  Google Scholar 

  • Kedersha N, Chen S, Gilks N, Li W, Miller IJ et al (2002) Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol Biol Cell 13:195–210

    Article  PubMed  CAS  Google Scholar 

  • Pestka S (1971) Inhibitors of ribosome functions. Annu Rev Microbiol 25:487–562

    Article  PubMed  CAS  Google Scholar 

  • Schmidt EK, Clavarino G, Ceppi M, Pierre P (2009) SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods 6:275–277

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Glennys Reynoso provided outstanding technical support. This work was generously supported by the Division of Intramural Research, NIAID.

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Correspondence to Jonathan W. Yewdell.

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David, A., Bennink, J.R. & Yewdell, J.W. Emetine optimally facilitates nascent chain puromycylation and potentiates the ribopuromycylation method (RPM) applied to inert cells. Histochem Cell Biol 139, 501–504 (2013). https://doi.org/10.1007/s00418-012-1063-8

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  • DOI: https://doi.org/10.1007/s00418-012-1063-8

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