In vitro RNA cleavage assay for Argonaute-family proteins.
Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage ("Slicing") of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage ("Slicer") activity of the RISC. We describe (in vitro) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli.
Miyoshi
Keita
K
Institute for Genome Research, University of Tokushima, Tokushima, Japan.
Uejima
Hiroshi
H
Nagami-Okada
Tomoko
T
Siomi
Haruhiko
H
Siomi
Mikiko C
MC
D000818:Animals; D060565:Argonaute Proteins; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
C489654:AGO1 protein, Drosophila; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D011993:Recombinant Fusion Proteins; D012313:RNA