Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.
Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.
BIOTEC, Biophysics, Dresden University of Technology, Dresden, Germany.
Thomas
T
Ohrt
Jörg
J
Mütze
Wolfgang
W
Staroske
Lasse
L
Weinmann
Julia
J
Höck
Karin
K
Crell
Gunter
G
Meister
Petra
P
Schwille
D021581:Active Transport, Cell Nucleus; D060565:Argonaute Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000032:analysis; D006801:Humans; D034741:RNA, Small Interfering / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D058727:RNA, Small Untranslated
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D058727:RNA, Small Untranslated