Ligation of 2', 3'-cyclic phosphate RNAs for the identification of microRNA binding sites.
2′,3′-cyclic phosphate
CLIP
RtcB
ligation
microRNA
Journal
FEBS letters
ISSN: 1873-3468
Titre abrégé: FEBS Lett
Pays: England
ID NLM: 0155157
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
07
07
2020
revised:
27
09
2020
accepted:
08
10
2020
pubmed:
29
10
2020
medline:
16
7
2021
entrez:
28
10
2020
Statut:
ppublish
Résumé
Identifying the targetome of a microRNA is key for understanding its functions. Cross-linking and immunoprecipitation (CLIP) methods capture native miRNA-mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA-mRNA sequences via ligation of 5'-phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2'-, 3'-cyclic phosphate groups, as part of a new CLIP method that does not require 5'-phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3'-phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes.
Identifiants
pubmed: 33113149
doi: 10.1002/1873-3468.13976
pmc: PMC7894349
doi:
Substances chimiques
Bacterial Proteins
0
Cross-Linking Reagents
0
MicroRNAs
0
RNA, Messenger
0
Recombinant Proteins
0
Ligases
EC 6.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
230-240Informations de copyright
© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
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