We investigated the mechanisms of siRNA-induced TGS during retrovirus infection by direct visualization of critical components of RITS-like complex. We describe both distinct subcellular distribution of RITS-like complex components and a novel nuclear transport mechanism for RITS-like components mediated by the actin filament (F-actin) component of the cytoskeleton. Ago1 and promoter-targeted siRNAs were found preferentially in the nucleus of retrovirus-infected cells, while Ago2 and promoter-targeted siRNAs were found in the inner nuclear envelope of retrovirus-infected cells. The distinct nuclear compartmentalization of these RITS-like components was observed only in infected cultures, suggesting accumulation of RITS-like components in the nucleus of infected cells during TGS. The clear association between Ago1 and promoter-targeted siRNAs in the nucleus is consistent with the site of TGS being nuclear and supports previous studies suggesting Ago1 and siRNAs are RITS-like complex components (2
). Our interesting finding that Ago2 co-localized with promoter-targeted siRNAs in the inner nuclear lamin membrane indicated the association alone between Ago2 and promoter-targeted siRNAs was not sufficient for nuclear transport. This may be due to Ago2 possibly requiring Ago1 for nuclear trafficking as part of the RITS-like complex. Our experiments, which separately expressed exogenous FLAG-tagged Ago1 or Ago2, but not both combined, may indicate the low endogenous levels of Ago1 are not adequate to facilitate nuclear transport of exogenous FLAG-tagged Ago2 protein. It is tempting to speculate that Ago2 could also be retained in the nuclear pore complex (NPC), further regulating traffic of protein complexes between the nucleus and cytoplasm (25
); however, additional co-localization studies are required for confirmation.
This study demonstrated the distinct distribution of Ago:siRNAs using two different retroviruses; SIVmac251 and two different HIV-1 subtype B strains: CXCR4-tropic HIV-1SF162
and CCR5-tropic HIV-1LAV
. Observing the same phenomenon in all three retrovirus infection TGS models highlights that the findings are compelling. Moreover, it is important to appreciate that the si2A sequence, targeting a region well upstream of the SIV NF-κB binding motif, is quite different from the siPromA sequence, targeting the tandem NF-κB binding motifs in the HIV-1 5` LTR promoter region. To provide important specificity controls for our experiments, we showed that scrambled siRNAs and siPromA-M2, which varies from HIV-1 promoter-targeted siPromA by only two bp, had no effect on nuclear co-localization of RITS-like components and were instead found in the cytoplasm. We also confirmed the previously reported finding that the antisense siRNA strand participates in the formation of the RITS-like complex in human cells (26
), as shown by our experiments using two different HIV-1 subtype B strains, where only the antisense strand of siPromA was labeled.
The distinct subcellular compartmentalization of Ago:siRNA led us to investigate the mechanism regulating nuclear transport of these RITS-like components. Studies in S. pombe
have found RNAi machinery proteins and RNAi-associated P bodies are transported via cytoskeleton microtubule networks in the cytoplasm (28–30
). A recent report further describes the microtubule-associated kinesin motor protein Cut7 regulates cytoplasmic RNAi effector complexes during PTGS, but not TGS (31
). Our study focused on elucidating mechanisms of the TGS pathway and reports the novel finding of cytoskeleton polymeric actin being linked to transport of promoter-targeted siRNAs as part of RNAi machinery in the nucleus of mammalian cells. Although both promoter-targeted siRNAs and F-actin were found to localize to the nucleus of retrovirus-infected cells, there was no significant co-localization of these two molecules within the nucleus as quantified by PCC values. This indicates that while F-actin may play a role in nuclear trafficking of promoter-targeted siRNAs, it may not necessarily directly interact with siRNA:Ago1 in the nucleus during transcriptional suppression, although Ago1 was observed to directly associated with β-actin by immunoprecipiation. Furthermore, when we depolymerized actin using cytD treatment, we did find significant co-localization between F-actin and promoter-targeted siRNAs in the cytoplasm, demonstrating that they are spatially related in this compartment and could interact. Alternately, Abps are known to be involved in nucleocytoplasmic transport and may provide the bridging link between the long filamentous F-actin structure and promoter-targeted siRNAs. This may also account for the biochemical association observed between F-actin and Ago1, although the effect could also be a direct protein interaction but fine delineation of this requires further investigation. Abps proteins associate with actin to continuously regulate actin polymerization, cross-link actin filaments and move cargo along the filaments. The TGS pathway may utilize this association to load RITS-like complex components onto actin filaments in the cytoplasm, traffic them into the nuclear compartment, then disassociate from the actin filament once in the nucleus.
Cofilin, is one such Abp essential for chaperoning actin into the nucleus, as shown by an associated block in nuclear actin accumulation upon inhibiting cofilin (32
). Another Abp of interest is the giant NUANCE (NUcleus and ActiN Connecting Element) protein, also termed Nesprin-2 Giant (33
). This mammalian nesprin protein has an N-terminal region comprising of an α-actinin-type actin-binding domain, which facilitates the physical connection between the nucleus and actin cytoskeleton. Nesprins also interact with emerin in the inner nuclear membrane (34
), where emerins have been shown to bind F-actin (35
). Interestingly, Arps have also been found in the nucleus and physically interact with chromatin-modifying complexes, specifically Arp4 and 6, which co-localize with heterchromatin protein 1 (HP1) in Drosophila
). The Arp BAF53 also associates with actin as part of the BAF chromatin-modifying complex in humans (38
). Many proteins have also been identified to associate with Ago1 and Ago2 as part of the RNAi machinery. These include TBRC6B, MOV10, nuclear DNA helicase II (also termed RNA helicase A), Importin 8, Gemin3, Gemin4, HDAC-1 and EZH2 (1
). Furthermore, nuclear DNA helicase II has been shown to bind F-actin in the nucleus of human cells (41
). All of these proteins are further candidates worthy of investigation using our model to dissect the mechanisms of mammalian TGS.
Nuclear actin and myosin are also reported to regulate nucleo-cytoplasmic transport, possibly as NPC components (42
). This nucleo-cytoplasmic transport is an ATP-dependent process and is inhibited by cytD treatment (42
), suggesting that our observations of siRNA nuclear import inhibition during cytD treatment also indicates nuclear import of RITS-like components is an ATP-dependent process. Nuclear import of some Ago proteins also requires association with siRNAs, as demonstrated for the C. elegans
Ago protein NRDE-3 (43
). Moreover, in mice the Ago protein MIWI2 binds Piwi-associated (pi) RNAs and is localized to the nucleus (44
). Another study shows the protozoan Tetrahymena
Ago protein Twi1p associated with siRNA is transported into the nucleus via Giw1p (45
). These studies suggest a mechanism that selectively transports only Ago proteins that are associated with siRNAs. Our findings support this selection mechanism, which would explain why we only observed co-localized FLAG-tagged Ago and siRNA in the nucleus of retrovirus-infected cells and not Ago or siRNA alone.
Our study shows a distinct subcellular distribution of Ago proteins and promoter-targeted siRNAs in the nuclear compartment of retrovirus-infected cells and directly links the actin cytoskeleton with nuclear delivery. We propose a model for the nuclear import of RITS-like components whereby F-actin translocation into the nuclear compartment, primarily the inner nuclear membrane, corresponds to nuclear delivery of promoter-targeted siRNAs, which then recruit other proteins to form the nuclear RITS-like complex (). Although we have shown both co-localization and a direct biochemical interaction between F-actin and FLAG-tagged Ago1 protein in the nucleus of retrovirus-infected cells, it is important to understand that other factors may also interact with actin filaments in the cytoplasm to facilitate the mechanical delivery of promoter-targeted siRNAs and Ago proteins into the nuclear compartment. It is likely that other factors also contribute to nuclear transport and regulation of this process during TGS warrants further investigation. The findings of our study present direct visualization of siRNA, Ago1 and Ago2 in the mammalian TGS pathway. The study also provides valuable insight into the fundamental mechanism underlying nuclear trafficking of RITS-like complex components by the actin cytoskeleton, which may also be functional in lower eukaryotes.
Figure 10. Model of the actin cytoskeleton role in nuclear trafficking of mammalian RITS-like complex components. Our results show that Ago1 and promoter-targeted siRNA co-localize in the nucleus of retrovirus-infected cells as part of the RITS-like complex during (more ...)