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PreprintCited by 12RNA Research and SplicingBiotin and Related StudiesAdvanced Proteomics Techniques and Applications

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SHIFTR enables the unbiased and multiplexed identification of proteins bound to specific RNA regions in live cells

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Aydin, Jens, Gabel, Alexander, Zielinski, Sebastian, Ganskih, Sabina, Schmidt, Nora, Hartigan, Christina R., Schenone, Monica, Carr, Steven A., Munschauer, Mathias·2023·Nucleic Acids Research
DOI 10.1101/2023.10.09.561498

Preprint — published as 10.1093/nar/gkae038 · Paper page

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Abstract

RNA-protein interactions determine the cellular fate of RNA and are central to regulating gene expression outcomes in health and disease. To date, no method exists that is able to identify proteins that interact with specific regions within endogenous RNAs in live cells. Here, we develop SHIFTR ( S elective RNase H -mediated interactome framing for target RNA regions), an efficient and scalable approach to identify proteins bound to selected regions within endogenous RNAs using mass spectrometry. Compared to state-of-the-art techniques, SHIFTR is superior in accuracy, captures close to zero background interactions and requires orders of magnitude lower input material. We establish SHIFTR workflows for targeting RNA classes of different length and abundance, including short and long non-coding RNAs, as well as mRNAs and demonstrate that SHIFTR is compatible with sequentially mapping interactomes for multiple target RNAs in a single experiment. Using SHIFTR, we comprehensively identify interactions of cis -regulatory elements located at the 5ʹ and 3ʹ- terminal regions of the authentic SARS-CoV-2 RNA genome in infected cells and accurately recover known and novel interactions linked to the function of these viral RNA elements. SHIFTR enables the systematic mapping of region-resolved RNA interactomes for any RNA in any cell type and has the potential to revolutionize our understanding of transcriptomes and their regulation.

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Metadata

Authors
Aydin, J., Gabel, A., Zielinski, S., Ganskih, S., Schmidt, N., Hartigan, C. R., Schenone, M., Carr, S. A., & Munschauer, M.
Affiliations
  • Helmholtz Centre for Infection Research (DE)
  • Broad Institute (US)
  • University of Würzburg (DE)
Corresponding author
Munschauer, Mathias
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2023-10-10
Volume
52
Issue
5
Pages
e26-e26
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2023.10.09.561498
OpenAlex
W4391291215
Cited by
12
References
1
Citations by year
2026: 5 · 2025: 5 · 2024: 2
FWCI
1.63
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • European Molecular Biology Organization (ALTF 1260-2020)
  • H2020 European Research Council (101040914 to M.M)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
Keywords
RNA-protein interactions, RNase H, SARS-CoV-2 RNA, cis-regulatory elements, non-coding RNA, mRNA, transcriptome regulation, viral RNA
MeSH terms
RNA, RNA, Messenger, RNA, Viral, Software, RNA-Binding Proteins, Proteomics, Transcriptome, RNA, Long Noncoding

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