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Selective ribosome profiling to study interactions of translating ribosomes in yeast

Nature Protocols volume 14, pages 2279–2317 (2019)Cite this article

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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.

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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.

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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.

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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).

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Author notes

  1. These authors contributed equally: Carla V. Galmozzi, Dorina Merker.

Authors and Affiliations

  1. Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

    Carla V. Galmozzi, Dorina Merker, Ulrike A. Friedrich, Kristina Döring & Günter Kramer

  2. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Carla V. Galmozzi, Dorina Merker, Ulrike A. Friedrich, Kristina Döring & Günter Kramer

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  1. Carla V. Galmozzi
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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.

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Correspondence to Günter Kramer.

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The authors declare no competing interests.

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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.

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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.

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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

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