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Nucleic Acids Research Advance Access published online on October 8, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn693
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells

Thomas Ohrt1, Jörg Mütze1, Wolfgang Staroske1, Lasse Weinmann2, Julia Höck2, Karin Crell1, Gunter Meister2 and Petra Schwille1,*

1BIOTEC, Biophysics, Dresden University of Technology, Dresden and 2Laboratory of RNA Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

*To whom correspondence should be addressed. Tel: +49 351 46340328; Fax: +49 351 46340342; Email: schwille{at}biotec.tu-dresden.de

Correspondence may also be addressed to Thomas Ohrt. Tel: +49 351 46340324; Fax: +49 351 46340342; Email: thomas.ohrt{at}biotec.tu-dresden.de

Received August 13, 2008. Revised September 25, 2008. Accepted September 25, 2008.

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 ~3 MDa complex in the cytoplasm and a 20-fold smaller complex of ~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.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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