Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.
Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.
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DOI：
10.1093/nar/gkn693
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发表时间：
2008-11
影响因子：
14.9
通讯作者：
Schwille P
中科院分区：
文献类型：
--
作者：
Ohrt T;Mütze J;Staroske W;Weinmann L;Höck J;Crell K;Meister G;Schwille P
RNA干扰（RNAi）的研究提供的证据表明，除了明确的细胞质机制，核机制也存在。哺乳动物细胞中形成核RNA诱导沉默复合物（RISC）的机制以及核和细胞质区室中RNA沉默途径之间的关系仍然未知。在这里，我们表明，通过应用荧光相关和互相关光谱（FCS/FCCS）在体内，存在两个不同的RISC：一个大的103 MDa复杂的细胞质和20倍较小的复杂的158 kDa的细胞核中。我们进一步表明，核RISC，仅由Ago 2和一个短RNA，被加载在细胞质中，并输入到细胞核。基于与同源RNA的受损切割的miRNA样相互作用，加载的RISC在细胞核中积累，这取决于靶标的存在。总之，这些结果表明，一个新的RISC穿梭机制之间的细胞核和细胞质，确保伴随基因调控的小RNA在两个区室。
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.
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DOI：
10.1073/pnas.0600148103
发表时间：
2006-05-16
影响因子：
11.1
作者：
Berezhna, Svitlana Y.;Supekova, Lubica;Deniz, Ashok A.
通讯作者：
Deniz, Ashok A.
影响因子：
10.5
作者：
Ketting, RF;Fischer, SEJ;Plasterk, RHA
通讯作者：
Plasterk, RHA
影响因子：
16.8
作者：
Janowski, Bethany A.;Huffman, Kenneth E.;Corey, David R.
通讯作者：
Corey, David R.
影响因子：
10.5
作者：
Martinez, J;Tuschl, T
通讯作者：
Tuschl, T
DOI：
10.1073/pnas.0710869105
发表时间：
2008-01-15
影响因子：
11.1
作者：
MacRae, Ian J.;Ma, Enbo;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.