Detection of Slicer Activity by Immunopurified Plant ARGONAUTE1.
Arabidopsis
Argonaute
Endonucleolysis
In vitro assay
Slicing
miRNA
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
1
2
2019
pubmed:
1
2
2019
medline:
30
6
2019
Statut:
ppublish
Résumé
Small RNA-guided endonucleolysis ("slicing") of target mRNA is the signature biochemical activity underlying many RNA silencing phenomena. The catalytic slicer activity resides in Argonaute (AGO) proteins. Here, we present two protocols to detect microRNA-guided slicer activity of AGO1 immunopurified from Arabidopsis tissues. The first uses radioactive, cap-labeled RNA substrates produced by in vitro transcription of RNA fragments corresponding to endogenous target sites flanked by 100-200 nucleotides of target sequence. The second protocol uses similarly designed but shorter (around 50 nt) fluorescently labeled RNA. Advantages and disadvantages of the two setups are also discussed.
Identifiants
pubmed: 30701509
doi: 10.1007/978-1-4939-9042-9_22
doi:
Substances chimiques
AGO1 protein, Arabidopsis
0
Arabidopsis Proteins
0
Argonaute Proteins
0
MicroRNAs
0
RNA, Messenger
0
RNA, Plant
0
RNA, Small Interfering
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM