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Evaluating ribosomal frameshifting in CCR5 mRNA decoding

Nature volume 604, pages E16–E23 (2022)Cite this article

Subjects

The Original Article was published on 09 July 2014

arising from A. T. Belew et al. Nature https://doi.org/10.1038/nature13429 (2014)

During the decoding of a subset of mRNAs, a proportion of ribosomes productively shift to the −1 reading frame at specific slippage-prone sites in a phenomenon known as programmed −1 ribosomal frameshifting1 to generate a frameshifted, C-terminally unique protein. The first experimentally verified occurrence of functionally utilized non-retroelement derived programmed −1 ribosomal frameshifting in humans has been reported in the mRNA encoding the immune-functioning C-C chemokine receptor 5 (CCR5)2. Here we show that frameshifting does not occur during CCR5 decoding, in contrast to the findings of Belew et al.2. Apart from its importance in understanding expression of a gene relevant to cancer, an HIV-1 receptor (and the associated claimed rationale for generating the first humans derived from genetically modified embryos), the findings imply that caution is appropriate in assessing suggestions of widespread regulated mRNA decay by advantageous frameshifting in cellular gene expression3.

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Fig. 1: Evaluation of the original evidence for CCR5 frameshifting.
Fig. 2: Additional approaches to CCR5 −1 PRF detection.

Data availability

The data used to support our findings are either publicly available or were generated in this study and are included in the source data. Source data are provided with this paper.

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Acknowledgements

We thank A. T. Belew for his courage in alerting key outside individuals in May 2017 to problems with his work and recently in providing us with crucial information about the original plasmids. This work was supported by grants from the NIH (R35GM118070 and R01AI133348) to J.S.K., the Wellcome Trust (106207) to A.E.F., a SFI-HRB-Wellcome Trust Investigator in Science award (210692/Z/18) to P.V.B., and the Science Foundation Ireland and the Irish Research Council (13/IA/1853 and IRCLA/2019/74n) to J.F.A. A.-L.S. was supported by DFG STE 2509/2−1. S.J.K. was supported by an Irish Research Council studentship. Y.A.K. was supported by an award of the Winston Churchill Foundation of the USA, a Graduate Research Fellowship from the National Science Foundation and the Knight-Hennessy Scholarship. Y.A.K. thanks A. S. Khan for support.

Author information

Author notes

  1. These authors contributed equally: Yousuf A. Khan, Gary Loughran

Authors and Affiliations

  1. Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK

    Yousuf A. Khan, Katherine Brown, Hazel Stewart & Andrew E. Firth

  2. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Yousuf A. Khan

  3. School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland

    Gary Loughran, Stephen J. Kiniry, Pavel V. Baranov & John F. Atkins

  4. Department of Biochemistry and Molecular Genetics, Denver School of Medicine, Aurora, CO, USA

    Anna-Lena Steckelberg & Jeffrey S. Kieft

  5. Department of Human Genetics, University of Utah, Salt Lake City, UT, USA

    John F. Atkins

  6. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Anna-Lena Steckelberg

Authors
  1. Yousuf A. Khan
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Contributions

The splicing work, which was initiated in 2016, was performed by A.-L.S. and J.S.K. Two teams—one involving G.L. and J.F.A., and the other involving Y.A.K., K.B., H.S. and A.E.F.—semi-independently performed the frameshifting analysis. S.J.K. and P.V.B., with input from A.E.F., performed the ribosome profiling analysis. After the initial independent work of the teams involved, Y.A.K. and J.F.A. coordinated their combination. Y.A.K. and G.L. had major roles in preparing the manuscript with contributions and refinements from all other authors.

Corresponding authors

Correspondence to Jeffrey S. Kieft, Andrew E. Firth or John F. Atkins.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Alignment of mammalian CCR5 sequences for the putative −1 PRF region in human.

Sequence alignment of CCR5 CDS regions obtained from tblastn hits to human CCR5, trimmed to only include the frameshift region. The red box indicates the putative slippery site. See Supplementary Table 1b for a list of the accession numbers used.

Extended Data Fig. 2 Luciferase values for each experiment.

a, Absolute Renilla luciferase values for CCR5 DNA transfection experiments performed in HeLa cells. b, Absolute firefly luciferase values for CCR5 DNA transfection experiments performed in HeLa cells. c, Absolute Renilla luciferase values for CCR5 DNA transfection experiments performed in HEK293T cells. d, Absolute firefly luciferase values for CCR5 DNA transfection experiments performed in HEK293T cells. e, Absolute Renilla luciferase values for CCR5 DNA transfection experiments performed in HeLa cells. f, Absolute firefly luciferase values for CCR5 DNA transfection experiments performed in HeLa cells. g, Absolute Renilla luciferase values for CCR5 DNA transfection experiments performed in HEK293T cells. h, Absolute firefly luciferase values for CCR5 DNA transfection experiments performed in HEK293T cells i, Absolute Renilla luciferase values for retrotransposon transfection experiments performed in HeLa cells. j, Absolute firefly luciferase values for retrotransposon transfection experiments performed in HeLa cells. k, Absolute Renilla luciferase values for retrotransposon transfection experiments performed in HEK293T cells. l, Absolute firefly luciferase values for retrotransposon transfection experiments performed in HEK293T cells. m, Absolute Renilla luciferase values for CCR5 RNA transfection experiments performed in HeLa cells. n, Absolute firefly luciferase values for CCR5 RNA transfection experiments performed in HeLa cells.

Supplementary information

Supplementary Information

This file contains Supplementary Methods.

Reporting Summary

Supplementary Figure

This file contains the uncropped gels.

Supplementary Table 1

Oligonucleotides and accession numbers.

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Khan, Y.A., Loughran, G., Steckelberg, AL. et al. Evaluating ribosomal frameshifting in CCR5 mRNA decoding. Nature 604, E16–E23 (2022). https://doi.org/10.1038/s41586-022-04627-y

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