Letter

Dynamics of ribosome scanning and recycling revealed by translation complex profiling

  • Nature volume 535, pages 570574 (28 July 2016)
  • doi:10.1038/nature18647
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Abstract

Regulation of messenger RNA translation is central to eukaryotic gene expression control1. Regulatory inputs are specified by the mRNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs) near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU)2,3,4,5 to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling6 approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to ~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU ‘lingering’ at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development7. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.

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Accessions

Data deposits

The sequences of reads with poly(A) tracts were deposited to the Sequence Read Archive, under accession code SRP074093. An online interface for browsing and analysing the TCP-seq data is available at (http://bioapps.erc.monash.edu/TCP/) along with (http://dx.doi.org/10.6084/m9.figshare.3206725). The underlying mapped dataset is also available (http://dx.doi.org/10.6084/m9.figshare.3206698).

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Acknowledgements

This work was supported by an ARC Discovery Grant (DP1300101928) and an NHMRC Senior Research Fellowship (514904) awarded to T.P. N.E.S. was supported by a Go8 European Fellowship. We are grateful to A. G. Hinnebusch, C. G. Proud and R. D. Hannan for discussions and suggestions for this work. We acknowledge technical support from the Australian Cancer Research Foundation Biomolecular Resource Facility (JCSMR, ANU), D. Powell and S. Androulakis at the Monash Bioinformatics Platform.

Author information

Author notes

    • Stuart K. Archer
    •  & Nikolay E. Shirokikh

    These authors contributed equally to this work.

Affiliations

  1. EMBL–Australia Collaborating Group, Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 2601, Australia

    • Stuart K. Archer
    • , Nikolay E. Shirokikh
    •  & Thomas Preiss
  2. Monash Bioinformatics Platform, Monash University, Melbourne, Victoria 3800, Australia

    • Stuart K. Archer
  3. Moscow Regional State Institute of Humanities and Social Studies, Kolomna 140410, Russia

    • Nikolay E. Shirokikh
  4. Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia

    • Traude H. Beilharz
  5. Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia

    • Thomas Preiss

Authors

  1. Search for Stuart K. Archer in:

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Contributions

S.K.A., T.H.B. and T.P. designed the research, S.K.A. and N.E.S. performed the experiments, S.K.A, N.E.S., T.H.B. and T.P. analysed the data, discussed the results and wrote the paper.

Corresponding author

Correspondence to Thomas Preiss.

Reviewer Information Nature thanks E. Alkalaeva, P. Baranov and Y. Mechulam for their contribution to the peer review of this work.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    Summary of sequencing read counts for each of the RNA fragment libraries, mapped step-wise to different RNA biotypes (rRNA, other non-coding RNAs, mRNAs). The libraries included RNA fragments from either ribosomal small subunit (SSU), full ribosome (RS) and unseparated total input (TI) fractions of the wild-type or SYO1-TAP yeast strains.

  2. 2.

    Supplementary Table 2

    Ranking of Saccharomyces cerevisiae mRNAs by the degree of disrupted ribosomal scanning. To derive a measure for scanning disruption, for each mRNA a ratio was calculated of SSU peak coverage in the 5'UTR over SSU peak coverage in the start codon region.

  3. 3.

    Supplementary Table 3

    List of sequences of DNA probes used to deplete libraries for cDNA molecules containing ribosomal RNA (rRNA) sequences by the probe-directed degradation method.

PDF files

  1. 1.

    Supplementary Discussion

    The introduction provides an in-depth and referenced account of eukaryotic translation initiation focusing on the major questions addressed in this work. Results and discussion further integrate particulars of the TCP-Seq method, and the findings obtained with it, into the current knowledge of eukaryotic protein synthesis.

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