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PreprintCited by 147RNA and protein synthesis mechanismsRNA Research and SplicingPI3K/AKT/mTOR signaling in cancer

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Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

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Bohlen, Jonathan, Fenzl, Kai, Kramer, Günter, Bukau, Bernd, Teleman, Aurelio A.·2019·Molecular Cell
DOI 10.1101/806364

Preprint — published as 10.1016/j.molcel.2020.06.005

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Abstract

SUMMARY: Translation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro , but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo . It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ∼12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo . HIGHLIGHTS: Selective 40S FPing visualizes regulation of translation initiation on mRNAs in vivo Scanning ribosomes are cap-tethered in human cells Only one ribosome scans a 5’UTR at a time in human cells Ribosomes retain eIFs during early translation, allowing reinitiation after uORFs

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Metadata

Authors
Bohlen, J., Fenzl, K., Kramer, G., Bukau, B., & Teleman, A. A.
Affiliations
German Cancer Research Center (DE)
Corresponding author
Teleman, Aurelio A.
ORCID
Venue
Molecular Cell
Publisher
openRxiv
Published
2019-10-16
Volume
79
Issue
4
Pages
561-574.e5
ISSN
1097-2765 (ISSN-L)
DOI
10.1101/806364
OpenAlex
W3037971445
Cited by
147
References
1
Citations by year
2026: 11 · 2025: 25 · 2024: 28 · 2023: 26 · 2022: 18 · 2021: 29 · 2020: 8 · 2019: 2
FWCI
6.56
Type
journal article
Language
en
Open access
Locations
  • Molecular Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Deutsches Krebsforschungszentrum (NCT3.0_2015.54 DysregPT)
  • Deutsche Forschungsgemeinschaft (EXC 81, SFB 1036, NCT3.0_2015.54 DysregPT, 201348542)
  • Klaus Tschira Stiftung
  • Exzellenzcluster Ozean der Zukunft
  • European Research Council (EXC81)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
Keywords
translation initiation, ribosome scanning, eIF3b, eIF4G1, eIF4E, upstream open reading frames, ribosome reinitiation, initiation factors, 80S ribosome, translation efficiency, human cells
MeSH terms
Animals, HeLa Cells, Humans, Peptide Chain Initiation, Translational, RNA, Messenger, RNA, Transfer, Met, Open Reading Frames, Codon, Initiator, DNA Footprinting, 5' Untranslated Regions, Eukaryotic Initiation Factor-4E, Eukaryotic Initiation Factor-4G, Eukaryotic Initiation Factor-3, NIH 3T3 Cells, Mice, Ribosome Subunits, Ribosome Subunits, Small, Eukaryotic

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