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Translation complex profile sequencing to study the in vivo dynamics of mRNA–ribosome interactions during translation initiation, elongation and termination

Nature Protocols volume 12, pages 697–731 (2017)Cite this article

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Abstract

Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome–mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes ∼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.

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Figure 1: Overview of the eukaryotic translation cycle of mRNA.
Figure 2: Comparative overview of the TCP-seq and ribosome profiling methods.
Figure 3: Overview of the TCP-seq data browser functions.
Figure 4: Underrepresentation of the SSU footprints in mRNAs with short 5′ UTRs.
Figure 5: Analysis of sucrose gradient UV absorbance profiles.
Figure 6: Flowchart of the complete TCP-seq method.
Figure 7: Fast staining of sucrose density gradients to optimize sedimentation.
Figure 8: Examples of TCP-seq data analysis and visualization.

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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 acknowledge technical support from the Australian Cancer Research Foundation Biomolecular Resource Facility (John Curtin School of Medical Research, Australian National University) and S. Androulakis at the Monash Bioinformatics Platform.

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Author notes

  1. Nikolay E Shirokikh and Stuart K Archer: These authors contributed equally to this work.

Authors and Affiliations

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

    Nikolay E Shirokikh & Thomas Preiss

  2. State Socio-Humanitarian University, Kolomna, Russia

    Nikolay E Shirokikh

  3. Monash Bioinformatics Platform, Monash University, Melbourne, Victoria, Australia

    Stuart K Archer & David Powell

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

    Traude H Beilharz

  5. Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia

    Thomas Preiss

Authors
  1. Nikolay E Shirokikh
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  2. Stuart K Archer
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  3. Traude H Beilharz
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  4. David Powell
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  5. Thomas Preiss
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Contributions

N.E.S., S.K.A. and T.P. developed the protocol; N.E.S. and S.K.A. performed the biochemical experiments; S.K.A. and D.P. developed the bioinformatics analysis framework and analyzed the data; and N.E.S., S.K.A., T.H.B. and T.P. discussed and interpreted results. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Thomas Preiss.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Library construction approach used in TCP-seq.

Overview of the library construction steps used in TCP-seq, starting from the de-blocked RNA fragments isolated from the SSU, complete ribosome or total translated RNA fractions (Step 73), and ending with the amplified, size-selected strand-specific library (Step 128).

Supplementary Figure 2 Schematic of algorithm for inferring footprint lengths from alignments.

Representative alignments of reads (top sequences) to a reference (bottom sequences) are given and the nucleotides or gaps counted towards the footprint lengths is indicated in red. To the right of each alignment is its corresponding CIGAR representation, output by the aligner. 3′ poly(A) tracts that were mismatched with the reference (but not trimmed away in earlier steps due to an erroneous internal G or T base-call) were trimmed in this step also.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1 and 2, and Supplementary Table 1. (PDF 532 kb)

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Shirokikh, N., Archer, S., Beilharz, T. et al. Translation complex profile sequencing to study the in vivo dynamics of mRNA–ribosome interactions during translation initiation, elongation and termination. Nat Protoc 12, 697–731 (2017). https://doi.org/10.1038/nprot.2016.189

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