Letter | Published:

Translation readthrough mitigation

Nature 534, 719–723 (30 June 2016) | Download Citation

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Abstract

A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance1 are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic ‘Constant Spring’ haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

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Acknowledgements

We thank the Fire Laboratory for critical reading of the manuscript, C. Frøkjær-Jensen and K. Artiles for technical expertise, and T. Schedl, T. Inada, C. Joazeiro, L. Ling, A. Nager, and N. Spies for discussions. A. Sapiro and B. Li were instrumental in developing the RNA-seq2 protocol. This work was supported by grants from NIH R01GM37706, T32HG000044 (G.T.H.), 1DP2HD084069-01 (M.C.B.), 5F32GM112474-02 (J.A.A.), Walter and Idun Berry Foundation (E.S.C.), and NSF DGE-114747 (C.H.L.).

Author information

Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Joshua A. Arribere
    • , Elif S. Cenik
    •  & Andrew Z. Fire

  2. Department of Bioengineering, Stanford University, Stanford, California 94305, USA

    • Nimit Jain

  3. Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA

    • Gaelen T. Hess
    • , Cameron H. Lee
    • , Michael C. Bassik
    •  & Andrew Z. Fire

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Contributions

J.A.A., E.S.C., and A.Z.F. designed C. elegans experiments. J.A.A. and E.S.C. conducted C. elegans experiments. N.J. developed the RNA-seq2 protocol. J.A.A. performed computational analyses. J.A.A. conducted experiments in human cell lines, as designed and aided by J.A.A., G.T.H., C.H.L., M.C.B., and A.Z.F. J.A.A. and A.Z.F. wrote the paper with help from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andrew Z. Fire.

Sequencing data are available at Sequence Read Archive (SRP064516).

Reviewer Information Nature thanks J. S. Butler, M. Yarus and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Extended data figures

  1. 1.

    Distribution of C-terminal extensions upon stop codon readthrough.

  2. 2.

    Example quantification of the GFP:mCherry fluorescence ratios of images.

  3. 3.

    Readthrough regions confer a loss of superfolder GFP fluorescence.

  4. 4.

    Explanation of ‘shuffle’ sequences.

  5. 5.

    RNA-seq and ribo-seq from unc-54 mutants.

  6. 6.

    Ribo-seq of unc-54(cc3389) shows an unexceptional progression of ribosomes in the readthrough region.

  7. 7.

    Lack of general conservation of coding potential downstream of stop codons in Caenorhabditis.

  8. 8.

    Nucleotide and amino acid composition of readthrough regions (C. elegans).

  9. 9.

    Nucleotide and amino acid composition of readthrough regions (H. sapiens).

Extended data tables

  1. 1.

    Translation into 3′ UTRs at endogenous loci tends to yield hypomorphs

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This table contains a list of strains used in the study.

  2. 2.

    Supplementary Table 2

    This table contains a list of plasmids used in the study.

  3. 3.

    Supplementary Table 3

    This table contains DNA oligos for rRNA digestion by RNaseH.

PDF files

  1. 1.

    Supplementary Information

    This file contains supplementary text and gel source data for figure 3d.

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DOI

https://doi.org/10.1038/nature18308

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