Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles

Hedayioglu, F., Mead, E. J., O‘Connor, P. B.F., Skiotys, M., Sansom, O. J. , Mallucci, G. R., Willis, A. E., Baranov, P. V., Smales, C. M. and von der Haar, T. (2022) Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles. Nucleic Acids Research, 50(19), e112. (doi: 10.1093/nar/gkac705) (PMID:35979952) (PMCID:PMC9638929)

Abstract

The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.

• Item Type: Articles
• Additional Information: Funding: Wellcome Trust (UK) Collaborative Award in Science [201487 to T.vdH., G.R.M., C.M.S., O.J.S., A.E.W.); O.J.S. was supported by Cancer Research UK core funding to the CRUK Beatson Institute [A17196, A31287, A21139 to his laboratory]; A.E.W. was supported by MRC core Unit funding [5TR00]; P.V.B. was supported by an SFI-HRB-Wellcome Trust Investigator in Science award [210692]. Funding for open access charge: University of Kent (UK).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Sansom, Professor Owen
• Authors: Hedayioglu, F., Mead, E. J., O‘Connor, P. B.F., Skiotys, M., Sansom, O. J., Mallucci, G. R., Willis, A. E., Baranov, P. V., Smales, C. M., and von der Haar, T.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Published Online: 18 August 2022
• Copyright Holders: Copyright © 2022 The Authors
• First Published: First published in Nucleic Acids Research 50(19): e112
• Publisher Policy: Reproduced under a Creative Commons License

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