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A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes

Nature Protocols volume 11, pages 1309–1325 (2016)Cite this article

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A Publisher Correction to this article was published on 24 January 2024

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Abstract

We describe a cell-free translation system for evaluating the activity of ribosomes stringently purified from human cells. This system is based on in vitro reconstitution of the cellular translation machinery, in which a ribosome-free rabbit reticulocyte lysate (RRL) is reassembled with human ribosomes and in vitro–transcribed reporter mRNAs. The protocol describes the preparation of the RRL-derived fractions, purification of ribosomes devoid of detectable nonribosomal-associated factors, and assembly of the reactions to evaluate ribosomal translational efficiency and fidelity using appropriate reporter transcripts. The whole procedure can be completed in ∼2.5 d (plus 2 weeks for RRL preparation and cell expansion time). This protocol can be applied to study intrinsic functional properties (cis-acting element-mediated translation initiation or translational fidelity) of ribosome populations from different sources (including nonhuman origin). It is therefore useful for the characterization of ribosomal function in ribosomopathies and cancer, and it will be applicable in the emerging fields of ribosome diversity and specialized ribosomes.

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Figure 1: Scheme of the reconstituted cell-free translation system protocol.
Figure 2: Scheme of the experimental setup for evaluation of translation fidelity.
Figure 3: Assessment of the level of purity of the ribosomal preparations.

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Acknowledgements

This work was funded by the Association for International Cancer Research (AICR) (grant 09-0083 to L.M.), the Associazione Italiana per la Ricerca sul Cancro (AIRC) (grant IG-11416 to L.M.) and the Pallotti Legacy for Cancer Research (grants to M.B. and L.M.). M.B. and D.C. thank Lucio Montanaro and S. Sperti, who initiated them into ribosome biochemistry. The authors thank C. Betts for language revision of the manuscript and P. G. Petronini and R. Alfieri for ion determinations.

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Authors and Affiliations

  1. Department of Experimental,

    Marianna Penzo, Domenica Carnicelli, Lorenzo Montanaro & Maurizio Brigotti

  2. , Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy

    Marianna Penzo, Domenica Carnicelli, Lorenzo Montanaro & Maurizio Brigotti

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  1. Marianna Penzo
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  2. Domenica Carnicelli
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  3. Lorenzo Montanaro
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  4. Maurizio Brigotti
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Contributions

M.P., L.M. and M.B. developed the protocol, and wrote and edited the manuscript. D.C. helped to optimize the protocol.

Corresponding authors

Correspondence to Lorenzo Montanaro or Maurizio Brigotti.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ribosomal pellets obtained after centrifugation at different points of the procedure.

Photographs showing the size, shape and color of ribosomal pellets from different sources. (a) Ribosomes pelleted from rabbit reticulocyte lysate (reagent setup, ribosomal salt wash preparation) form a large, red pellet (arrow). (b) Human ribosomes pelleted through a discontinuous sucrose gradient starting from a cellular lysate (step 16) form a small and translucent pellet (arrow).

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Supplementary Figure 1, Supplementary Table 1 (PDF 280 kb)

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Penzo, M., Carnicelli, D., Montanaro, L. et al. A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes. Nat Protoc 11, 1309–1325 (2016). https://doi.org/10.1038/nprot.2016.072

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