Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
Nov 2023
Nov 2023
Historique:
received:
18
01
2023
accepted:
23
08
2023
medline:
9
11
2023
pubmed:
3
10
2023
entrez:
2
10
2023
Statut:
ppublish
Résumé
Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges.
Identifiants
pubmed: 37783882
doi: 10.1038/s41592-023-02028-1
pii: 10.1038/s41592-023-02028-1
doi:
Substances chimiques
RNA, Messenger
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1704-1715Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM130996
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM007232
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM130315
Pays : United States
Organisme : NHLBI NIH HHS
ID : DP1 HL156819
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM130996
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM007232
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM130315
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM130315
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM130315
Pays : United States
Organisme : NHLBI NIH HHS
ID : DP1 HL156819
Pays : United States
Organisme : NHLBI NIH HHS
ID : DP1 HL156819
Pays : United States
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.
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