Revealing nascent RNA processing dynamics with nano-COP.
Animals
Exons
/ genetics
Humans
Introns
/ genetics
Protein Modification, Translational
/ genetics
RNA
/ genetics
RNA Polymerase II
/ metabolism
RNA Precursors
/ analysis
RNA Processing, Post-Transcriptional
/ genetics
RNA Splicing
/ genetics
RNA, Messenger
/ genetics
Sequence Analysis, RNA
/ methods
Transcription, Genetic
/ genetics
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
03 2021
03 2021
Historique:
received:
03
07
2020
accepted:
20
11
2020
pubmed:
31
1
2021
medline:
7
4
2021
entrez:
30
1
2021
Statut:
ppublish
Résumé
During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.
Identifiants
pubmed: 33514943
doi: 10.1038/s41596-020-00469-y
pii: 10.1038/s41596-020-00469-y
pmc: PMC8713461
mid: NIHMS1765166
doi:
Substances chimiques
RNA Precursors
0
RNA, Messenger
0
RNA
63231-63-0
RNA Polymerase II
EC 2.7.7.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
1343-1375Subventions
Organisme : NIGMS NIH HHS
ID : F31 GM122133
Pays : United States
Organisme : CIHR
Pays : Canada
Organisme : NHGRI NIH HHS
ID : R01 HG010538
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM136794
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM117333
Pays : United States
Organisme : NHGRI NIH HHS
ID : R21 HG009264
Pays : United States
Organisme : NHGRI NIH HHS
ID : R01 HG007173
Pays : United States
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