Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Selective ribosome profiling to study interactions of translating ribosomes in yeast

Abstract

A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor’s nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8–10 working days, and initial data analysis can be completed within 1–2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Schematic overview of SeRP, exemplified for the yeast Hsp70 chaperone Ssb.
Fig. 2: Scheme of the experimental workflow for SeRP in eukaryotic cells.
Fig. 3: Detected Ssb-GFP in the sucrose gradient reflects its association to ribosomes.
Fig. 4: Detected Ssb–RNC complex interactions reflect the in vivo binding properties of Ssb.
Fig. 5: Mixing with controls to analyze the extent of ex vivo interactions in Ssb-SeRP.
Fig. 6: Analysis of eukaryotic selective RP data.

Data availability

The datasets analyzed with the current protocol are available in the GEO repository with the identifiers GSE93830 (primary Ssb dataset) and GSE123166 (rebinding control experiments).

Code availability

Scripts provided in this protocol and a demo dataset are available in the repository under the GNU General Public License: https://github.com/gfkramer/SeRP_yeast and https://doi.org/10.5281/zenodo.2602493.

References

  1. 1.

    Kramer, G., Shiber, A. & Bukau, B. Mechanisms of cotranslational maturation of newly synthesized proteins. Annu. Rev. Biochem. 88, 1–28 (2018).

    Google Scholar 

  2. 2.

    Pechmann, S., Willmund, F. & Frydman, J. The ribosome as a hub for protein quality control. Mol. Cell 49, 411–421 (2013).

    CAS  Article  Google Scholar 

  3. 3.

    Preissler, S. & Deuerling, E. Ribosome-associated chaperones as key players in proteostasis. Trends Biochem. Sci. 37, 274–283 (2012).

    CAS  Article  Google Scholar 

  4. 4.

    Kramer, G., Boehringer, D., Ban, N. & Bukau, B. The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nat. Struct. Mol. Biol. 16, 589–597 (2009).

    CAS  Article  Google Scholar 

  5. 5.

    Shieh, Y. W. et al. Operon structure and cotranslational subunit association direct protein assembly in bacteria. Science 350, 678–680 (2015).

    CAS  Article  Google Scholar 

  6. 6.

    Shiber, A. et al. Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling. Nature 561, 268–272 (2018).

    CAS  Article  Google Scholar 

  7. 7.

    Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).

    CAS  Article  Google Scholar 

  8. 8.

    Oh, E. et al. Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147, 1295–1308 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    Becker, A. H., Oh, E., Weissman, J. S., Kramer, G. & Bukau, B. Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes. Nat. Protoc. 8, 2212–2239 (2013).

    CAS  Article  Google Scholar 

  10. 10.

    Schibich, D. et al. Global profiling of SRP interaction with nascent polypeptides. Nature 536, 219–223 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Döring, K. et al. Profiling Ssb-nascent chain interactions reveals principles of Hsp70-assisted folding. Cell 170, 298–311 e220 (2017).

    Article  Google Scholar 

  12. 12.

    Chartron, J. W., Hunt, K. C. & Frydman, J. Cotranslational signal-independent SRP preloading during membrane targeting. Nature 536, 224–228 (2016).

    CAS  Article  Google Scholar 

  13. 13.

    Albanese, V., Yam, A. Y., Baughman, J., Parnot, C. & Frydman, J. Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells. Cell 124, 75–88 (2006).

    CAS  Article  Google Scholar 

  14. 14.

    Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353, aac4354 (2016).

    Article  Google Scholar 

  15. 15.

    Huang, P., Gautschi, M., Walter, W., Rospert, S. & Craig, E. A. The Hsp70 Ssz1 modulates the function of the ribosome-associated J-protein Zuo1. Nat. Struct. Mol. Biol. 12, 497–504 (2005).

    CAS  Article  Google Scholar 

  16. 16.

    Craig, E. A. & Jacobsen, K. Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures. Mol. Cell. Biol. 5, 3517–3524 (1985).

    CAS  Article  Google Scholar 

  17. 17.

    Nelson, R. J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M. & Craig, E. A. The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell 71, 97–105 (1992).

    CAS  Article  Google Scholar 

  18. 18.

    Koplin, A. et al. A dual function for chaperones SSB-RAC and the NAC nascent polypeptide-associated complex on ribosomes. J. Cell Biol. 189, 57–68 (2010).

    CAS  Article  Google Scholar 

  19. 19.

    Archer, S. K., Shirokikh, N. E., Beilharz, T. H. & Preiss, T. Dynamics of ribosome scanning and recycling revealed by translation complex profiling. Nature 535, 570–574 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Simsek, D. et al. The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity. Cell 169, 1051–1065.e18 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Raue, U., Oellerer, S. & Rospert, S. Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence. J. Biol. Chem. 282, 7809–7816 (2007).

    CAS  Article  Google Scholar 

  22. 22.

    Merz, F. et al. Molecular mechanism and structure of trigger factor bound to the translating ribosome. EMBO J. 27, 1622–1632 (2008).

    CAS  Article  Google Scholar 

  23. 23.

    Rutkowska, A. et al. Dynamics of trigger factor interaction with translating ribosomes. J. Biol. Chem. 283, 4124–4132 (2008).

    CAS  Article  Google Scholar 

  24. 24.

    Zhang, Y. et al. NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum. Mol. Biol. Cell 23, 3027–3040 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    del Alamo, M. et al. Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes. PLoS Biol. 9, e1001100 (2011).

    Article  Google Scholar 

  26. 26.

    Willmund, F. et al. The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis. Cell 152, 196–209 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Jan, C. H., Williams, C. C. & Weissman, J. S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling. Science 346, 1257521 (2014).

    Article  Google Scholar 

  28. 28.

    Williams, C. C., Jan, C. H. & Weissman, J. S. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling. Science 346, 748–751 (2014).

    CAS  Article  Google Scholar 

  29. 29.

    Costa, E. A., Subramanian, K., Nunnari, J. & Weissman, J. S. Defining the physiological role of SRP in protein-targeting efficiency and specificity. Science 359, 689–692 (2018).

    CAS  Article  Google Scholar 

  30. 30.

    Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7, 282–289 (2008).

    CAS  Article  Google Scholar 

  31. 31.

    Pech, M., Spreter, T., Beckmann, R. & Beatrix, B. Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome. J. Biol. Chem. 285, 19679–19687 (2010).

    CAS  Article  Google Scholar 

  32. 32.

    Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

    CAS  Article  Google Scholar 

  33. 33.

    Marks, J. et al. Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center. Proc. Natl. Acad. Sci. USA 113, 12150–12155 (2016).

    CAS  Article  Google Scholar 

  34. 34.

    Gerashchenko, M. V. & Gladyshev, V. N. Translation inhibitors cause abnormalities in ribosome profiling experiments. Nucleic Acids Res. 42, e134 (2014).

    Article  Google Scholar 

  35. 35.

    Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protoc. 7, 1534–1550 (2012).

    CAS  Article  Google Scholar 

  36. 36.

    Teter, S. A. et al. Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains. Cell 97, 755–765 (1999).

    CAS  Article  Google Scholar 

  37. 37.

    Blobel, G. & Sabatini, D. Dissociation of mammalian polyribosomes into subunits by puromycin. Proc. Natl. Acad. Sci. USA 68, 390–394 (1971).

    CAS  Article  Google Scholar 

  38. 38.

    McGlincy, N. J. & Ingolia, N. T. Transcriptome-wide measurement of translation by ribosome profiling. Methods 126, 112–129 (2017).

    Article  Google Scholar 

  39. 39.

    Gerashchenko, M. V. & Gladyshev, V. N. Ribonuclease selection for ribosome profiling. Nucleic Acids Res. 45, e6 (2017).

    Article  Google Scholar 

  40. 40.

    Diament, A. et al. The extent of ribosome queuing in budding yeast. PLoS Computat. Biol. 14, e1005951 (2018).

    Article  Google Scholar 

  41. 41.

    Mohammad, F., Woolstenhulme, C. J., Green, R. & Buskirk, A. R. Clarifying the translational pausing landscape in bacteria by ribosome profiling. Cell Rep. 14, 686–694 (2016).

    CAS  Article  Google Scholar 

  42. 42.

    Wu, C. C. C., Zinshteyn, B., Wehner, K. A. & Green, R. High-resolution ribosome profiling defines discrete ribosome elongation states and translational regulation during cellular stress. Mol. Cell 73, 959–970.e5 (2019).

    CAS  Article  Google Scholar 

  43. 43.

    Wang, H., Wang, Y. & Xie, Z. Computational resources for ribosome profiling: from database to Web server and software. Brief Bioinform. 20, 144–155 (2019).

    Article  Google Scholar 

  44. 44.

    Calviello, L. & Ohler, U. Beyond read-counts: Ribo-seq data analysis to understand the functions of the transcriptome. Trends Genet. 33, 728–744 (2017).

    CAS  Article  Google Scholar 

  45. 45.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  46. 46.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    Article  Google Scholar 

  47. 47.

    Michel, A. M. et al. RiboGalaxy: a browser based platform for the alignment, analysis and visualization of ribosome profiling data. RNA Biol. 13, 316–319 (2016).

    Article  Google Scholar 

  48. 48.

    Kiniry, S. J., O’Connor, P. B. F., Michel, A. M. & Baranov, P. V. Trips-Viz: a transcriptome browser for exploring Ribo-Seq data. Nucleic Acids Res. 47, D847–D852 (2019).

    Article  Google Scholar 

  49. 49.

    Martens, A. T., Taylor, J., Hilser, V. J. & Ribosome, A. and P sites revealed by length analysis of ribosome profiling data. Nucleic Acids Res. 43, 3680–3687 (2015).

    CAS  Article  Google Scholar 

  50. 50.

    Knorr, A. G. et al. Ribosome-NatA architecture reveals that rRNA expansion segments coordinate N-terminal acetylation. Nat. Struct. Mol. Biol. 26, 35–39 (2019).

    CAS  Article  Google Scholar 

  51. 51.

    Pfund, C., Huang, P., Lopez-Hoyo, N. & Craig, E. A. Divergent functional properties of the ribosome-associated molecular chaperone Ssb compared with other Hsp70s. Mol. Biol. Cell 12, 3773–3782 (2001).

    CAS  Article  Google Scholar 

  52. 52.

    Dunn, J. G. & Weissman, J. S. Plastid: nucleotide-resolution analysis of next-generation sequencing and genomics data. BMC Genomics 17, 958 (2016).

    Article  Google Scholar 

  53. 53.

    Malone, B. et al. Bayesian prediction of RNA translation from ribosome profiling. Nucleic Acids Res. 45, 2960–2972 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L. Eismann and other members of the Bukau laboratory at ZMBH for valuable comments on the manuscript. This work was supported by the ERC (advanced grant 743118) and the DFG (KR3593/2-1, SFB1036 and FOR1805).

Author information

Affiliations

Authors

Contributions

G.K. designed the study. K.D. and D.M. performed experiments. K.D., D.M. and C.V.G. set up the protocol for general RP in yeast. K.D. and G.K. established the protocol for selective RP. U.A.F. and K.D. generated the Python scripts, and performed data analysis. C.V.G., D.M. and G.K. wrote the manuscript.

Corresponding author

Correspondence to Günter Kramer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nature Protocols thanks Pavel Baranov, Gary Loughran and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Döring, K. et al. Cell 170, 298–311 (2017): https://doi.org/10.1016/j.cell.2017.06.038

Oh, E. et al. Cell 147, 1295–1308 (2011): https://doi.org/10.1016/j.cell.2011.10.044

Becker, A. H., Oh, E., Weissman, J. S., Kramer, G. & Bukau, B. Nat. Protoc. 8, 2212–2239 (2013): https://doi.org/10.1038/nprot.2013.133

Shiber, A. et al. Nature 561, 268–272 (2018): https://doi.org/10.1038/s41586-018-0462-y

Protocol to which this paper is an extension

Becker, A. H., Oh, E., Weissman, J. S., Kramer, G. & Bukau, B. Nat. Protoc. 8, 2212–2239 (2013): https://doi.org/10.1038/nprot.2013.133

This protocol is an extension to: Nat. Protoc. 8, 2212–2239 (2013), doi:10.1038/nprot.2013.133

Integrated supplementary information

Supplementary Fig. 1 Purification of Ssb-bound ribosomes depends on the presence of nascent chains.

(a) Western blot analysis of three Ssb1-GFP purifications performed under low salt (LS: 140 mM KCL) or high salt conditions (HS: 500 mM KCL) and in presence of cycloheximide (CHX) or puromycin (Puro). The upper panel shows a western blot developed using Ssb antibodies whereas the lower western blot was developed with antibodies detecting ribosomal protein Rpl35. (L: lysate, P: resuspended ribosomes used as input for AP, U: unbound, supernatant of AP, W: first wash fraction, B: bound fraction of AP.) (b) Bioanalyzer results of a Nano chip to measure the co-purified RNA in the bound fractions of the APs. The Figure was published previously as FigureS2 (c) in11.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Galmozzi, C.V., Merker, D., Friedrich, U.A. et al. Selective ribosome profiling to study interactions of translating ribosomes in yeast. Nat Protoc 14, 2279–2317 (2019). https://doi.org/10.1038/s41596-019-0185-z

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing