Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing.
Small RNAs triggering RNA silencing are loaded onto Argonautes and then sequence-specifically guide them to target transcripts. Epitope-tagged human Argonautes (hAgo1, hAgo2, hAgo3, and hAgo4) are associated with siRNAs and miRNAs, but only epitope-tagged hAgo2 has been shown to have Slicer activity. Contrarily, how endogenous hAgos behave with respect to small RNA association and target RNA destruction has remained unclear. Here, we produced monoclonal antibodies for individual hAgos. High-throughput pyrosequencing revealed that immunopurified endogenous hAgo2 and hAgo3 associated mostly with miRNAs. Endogenous hAgo3 did not show Slicer function but localized in P-bodies, suggesting that hAgo3 endogenously expressed is, like hAgo2, involved in the miRNA pathway but antagonizes the RNAi activity of hAgo2. Sequence variations of miRNAs were found at both 5' and 3' ends, suggesting that multiple mature miRNAs containing different "seed" sequences can arise from one miRNA precursor. The hAgo antibodies we raised are valuable tools for ascertaining the functional behavior of endogenous Argonautes and miRNAs in RNA silencing.
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan.
Asuka
A
Azuma-Mukai
Hideo
H
Oguri
Toutai
T
Mituyama
Zhi Rong
ZR
Qian
Kiyoshi
K
Asai
Haruhiko
H
Siomi
Mikiko C
MC
Siomi
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D035683:MicroRNAs / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D034741:RNA, Small Interfering