Schematic depiction of a proposed model of the relationship between cell stress, translocation of hAgo2 to SGs and loss of RNAi. Active siRNA- and miRNA-induced RNAi occur in the functional compartment (right side), which includes the cytosol, the nucleus and PBs under non-stress conditions. The non-functional compartment includes SGs (left side) to which hAgo2 migrates under stress conditions. Since the hAgo2-related extent of RNAi is reversibly linked with cell stress we assume exchange of hAgo2 between both compartments, i.e. a kind of steady state. The non-functional compartment contains transient structures composed of stalled preinitiation complexes and untranslated mRNA, which are known as SGs. Under cellular stress conditions the equilibrium is perturbed and hAgo2 accumulates in SGs. This may circumvent the functional interactions of hAgo2 with other RNAi involved proteins which induces the exclusion of hAgo2 from the RNAi pathway.

Cell stress-induced decrease of RNAi activity is reversible. Cell stress was transiently induced by the means indicated in the top of each panel [(A)–(D), for detail see the ‘Material and Methods’ section]. One, 2 or 3 days after release of stress, ECV-304 cells were tested for their capability to perform siRNA- or miRNA-mediated RNAi. Briefly, ECV-304 cells were transfected either with 50 pM lamin A/C directed siLAM or with 50 nM Renilla luciferase directed mlet-7A, RL-Hmga2m7 target vector and pGL3 control vector at day 1–3 after induction of cell stress. The extent of siRNA- and miRNA-mediated target inhibition in the absence of cell stress was normalized to Ctrl-RNA transfected ECV-304 cells and set to 1 (indicated by a dashed line in each panel). Higher numbers indicate derepression of target expression, i.e. loss of efficacy of siRNA or miRNA, respectively. Indicated are mean values ± standard deviations.

Translocation of hAgo2 to SGs is accompanied by decreased siRNA- and miRNA-induced RNAi. ECV-304 cells were stressed by different means as indicated below the bars (for detail see the ‘Material and Methods’ section). Subsequently, (A) lamin A/C-directed siRNA (50 pM siLAM) or (B) Renilla luciferase-directed miRNA (50 nM mlet-7A), RL-Hmga2m7 target vector and pGL3 control vector, respectively were transfected. The siLAM-mediated suppression of lamin A/C mRNA was quantified by RT–qPCR. The mlet-7A-mediated inhibition of Renilla luciferase activity was determined by dual-luciferase assay. The siRNA- or miRNA-mediated gene silencing activity in the absence of cellular stress is indicated by a dashed line in each panel. Levels greater than the dashed line indicate loss of RNAi. As control, Ctrl-RNA transfected ECV-304 cells were used and set 1. For each data point three to five independent experiments, each carried out in duplicates, were analyzed using the ANOVA test and the Program SPSS 12.0. Indicated are mean values ± standard error of the mean (SEM); *P < 0.05, ^**P < 0.01.

Different kinds of cell stress direct hAgo2 to SGs. As indicated on the left margin, ECV-304 cells were left untreated (picture 1–4 and 17–20) or stressed by 250 μM NaAsO₂ for 90 min at 37°C (picture 5–8 and 21–24), incubated at 42°C for 16 h (picture 9–12 and 25–28), or transfected with Lipofectamine^TM 2000 and 100 nM PS-modified ON (asAgo2mut^PS) 24 h prior to fixation (picture 13–16 and 29–32). Subsequently, cells were co-stained with a rat anti-hAgo2 antibody (red color, left column in A and B) and (A) a goat anti-TIA-1 antibody (stress granule marker, green color) or (B) a rabbit anti-Dcp1A (P-body marker, green color). All preparations were also co-stained with Hoechst 33342 dye (blue color). Merged images are shown on the right column. Co-staining of hAgo2 and the SG marker TIA1 can be seen in (A) whereas no co-localization could be observed when hAgo2 and the PB-specific marker Dcp1A were co-stained. The white arrows in panel (A) indicate the staining of SGs and in panel (B) the staining of PBs in an exemplary manner. Optical sections are displayed either as merged images or as greyscale images of the respective red, green or blue channel. The white bar in the upper left of panel (A) and (B) represents a 10 µm scale bar.

Density gradient fractionation indicates a NaAsO₂-induced re-distribution of hAgo2 to different complexes or organelles. (A and B) ECV-304 cells were either left untreated or incubated with 250 μM NaAsO₂ for 90 min at 37°C. The post-nuclear supernatant of total cell extracts was separated on a continuous 10–25% (w/v) iodixanol gradient and fractions were analyzed by western analysis with rat anti-Ago2 [(A), lower panels] (B) A chart of the hAgo2 content of fractions of untreated cells shows three distinct populations (I–III) with the main peak in fraction 8 and 9. In NaAsO₂-treated cells hAgo2 is primarily located in one fraction (II*) with the main peak in fraction 6 and 7. The distribution of hAgo2 relative to the sedimentation density is expressed as percentage of the respective signal in each fraction to the sum of all fractions. (C and D) ECV-304 cells were transfected with 50 nM siLAM and then left untreated or stressed by incubation with 250 μM NaAsO₂ for 90 min at 37°C. (C) Cell fractionation was performed as described above and total RNA was isolated from each fraction. The siLAM content in each fraction was determined via hybridization to a ³²P-labeled siLAM-specific probe. As a further control naked siLAM was separated in the absence of a total cell extract on a continuous 10–25% (w/v) iodixanol gradient (lower panel). A comparison of the distribution of transfected siRNA in untretaed versus NaAsO₂-stressed cells with signals normalized to the distribution of naked siRNA is shown (D) and indicates a ‘shoulder’ in fraction 8/9 of NaAsO₂-stressed cells compared to untreated cells.

Transfection of cells with Lipofectamine^TM 2000 and PS-modified ON shows stress characteristics. First, ECV-304 cells were transfected with 100 nM of hAgo2-directed asON (asAgo2) or control ON (asAgo2mut) carrying the indicated modifications. The effect of the above mentioned pre-treatment of cells with asON on the hAgo2 expression, localization and RNAi activity was determined after 24 h. (A) The ratio of hAgo2 mRNA and hAgo2 protein indicates cell stress in case of PS-ON but not for 2′-OMe-modified ON. (B) Pre-treatment of cells with 2′-OMe-modified ON (asAgo2mut^OMe) is not related to co-localization of hAgo2 (upper left panel) and TIA1 (upper right panel) in large irregularly shaped SGs. The lack of co-localization (lower panel right) indicates the absence of stress and was interpreted as a prerequisite for the capability of a hAgo2-directed asON to suppress hAgo2 effectively at the protein level. The white bar in the upper left panel represents a 10 µm scale bar. (C and D) The effect of asAgo2^OMe-mediated knockdown of hAgo2 on siRNA- or miRNA-induced gene silencing activity was investigated. (C) To test the siRNA pathway ECV-304 cells were transfected with 50 pM lamin A/C directed siLAM. (D) The miRNA pathway was studied with 50 nM Renilla luciferase directed mlet-7A, RL-Hmga2m7 target vector, and pGL3 control vector, respectively. The siLAM-mediated suppression of lamin A/C mRNA was quantified by RT–qPCR. The mlet-7A-mediated inhibition of Renilla luciferase activity was determined by dual-luciferase assay. In both cases (C and D) as controls, Ctrl-RNA transfected ECV-304 cells were used and set 1. Indicated are mean values ± standard deviations. A dashed line in each panel indicates the full siRNA- or miRNA-mediated gene silencing activity in the absence of hAgo2 knockdown.

Translocation of hAgo2 to SGs is accompanied by its increased metabolic stability and by resistance to its siRNA-mediated knockdown. (A) CHX treatment (20 µg/ml) of ECV-GL3 cells, which blocks translation, shows suppression of stable firefly luciferase activity (left panel) as well as firefly luciferase protein expression (middle panel) whereas hAgo2 protein was not affected for up to 48 h of treatment (right panel). As control, ECV-GL3 cells, which were cultured in the absence of CHX were used and set 1. Indicated are mean values ± standard deviations. (B) ECV-304 cells were first stressed by different means as indicated below the bars (for details see the ‘Material and Methods’ section) and then transfected with 100 nM hAgo2-directed siAgo2 in order to test whether the hAgo2 gene expression can be suppressed under cellular stress conditions. After 24 h the gene silencing activity of siAgo2 was determined on the level of hAgo2 mRNA (RT–qPCR) and on the level of Ago2 protein (western analysis). The results are depicted in the left (hAgo2 mRNA) and the middle (hAgo2 protein) panel. As control, Ctrl-RNA transfected ECV-304 cells were used and set 1. The ratio of the hAgo2 protein and mRNA upon siAgo2 treatment is shown on the right panel. A value of 1 (dashed line) indicates a comparable suppression at the mRNA and protein level whereas a value greater than 1 means that less suppression at the protein level was observed. For each data point three to five independent experiments, each carried out in duplicates, were analyzed using the ANOVA test and the Program SPSS 12.0. Indicated are mean values ± standard error of the mean (SEM); *P < 0.05, ^**P < 0.01.