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PreprintCited by 9RNA and protein synthesis mechanismsRNA Research and SplicingRNA modifications and cancer

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Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification

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Imai, Hirotatsu, Utsumi, Daisuke, Torihara, Hidetsugu, Takahashi, Kenzo, Kuroyanagi, Hidehito, Yamashita, Akio·2023·Nucleic Acids Research
DOI 10.1101/2023.02.15.525786PMC PMC10415123

Preprint — published as 10.1093/nar/gkad545 · Paper page

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Abstract

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires de novo RNA synthesis and translation. Analysis of the coordinated and circular regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprograming of nascent RNA synthesis and translation. Our method can serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

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Metadata

Authors
Imai, H., Utsumi, D., Torihara, H., Takahashi, K., Kuroyanagi, H., & Yamashita, A.
Affiliations
University of the Ryukyus (JP)
Corresponding author
  • Imai, Hirotatsu
  • Yamashita, Akio
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2023-02-15
Volume
51
Issue
14
Pages
e76-e76
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2023.02.15.525786
PMC
PMC10415123
OpenAlex
W4382345144
Cited by
9
References
1
Citations by year
2026: 2 · 2025: 4 · 2024: 3
FWCI
0.80
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)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by)
  • University oh the Ryukyus Repository (submittedVersion, cc-by-nc-nd)
Funders
  • U.S. Department of Commerce
  • Bristol-Myers Squibb
  • Takeda Science Foundation
  • Bristol-Myers Squibb Canada
  • Japan Society for the Promotion of Science (23K08826, 21K15015, 22H04925, 20H03181, 20H04839, 20H03438, 21K19413, 22K08387, 20J00036)
  • Japan Science and Technology Agency (JPMJMS2022)
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 › RNA modifications and cancer
Keywords
translatome, translating ribosome affinity purification, unfolded protein response, gene expression regulation
MeSH terms
Ribosome Profiling, Animals, Gene Expression Regulation, Genetic Techniques, Mammals, Ribosomes, RNA, Thiouridine, Protein Biosynthesis, Transcriptome

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