Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress

Abstract

Short interfering RNAs (siRNAs) target specific mRNAs for their degradation mediated by RNA-induced silencing complex (RISC). Persistent activation of siRNA-RISC frequently leads to non-targeting toxicity. However, how cells mediate this stress remains elusive. In this communication, we found that the presence of non-targeting siRNA selectively induced the expression of an endoplasmic reticulum (ER)-resident protein, non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), but not other ER-stress proteins including GRP78, Calnexin and XBP1. Cells suffering from constant non-targeting siRNA stress grew slower and prolonged G1 phase, while NPGPx-depleted cells accumulated mature non-targeting siRNA and underwent apoptosis. Upon the stress, NPGPx covalently bound to exoribonuclease XRN2, facilitating XRN2 to remove accumulated non-targeting siRNA. These results suggest that NPGPx serves as a novel responder to non-targeting siRNA-induced stress in facilitating XRN2 to release the non-targeting siRNA accumulation.

Figure 1.

NPGPx expression was induced by non-targeting shRNA/RNAi. (a and b) Western blot analysis of NPGPx, p84 and α-tubulin proteins from WI38 cells infected with lentivirus carrying shLuc, shRFP, shLacZ, GFP, shEmpty (shRNA cloning vector) or uninfected as control. Specific antibodies against each protein were used as probes. p84 and α-tubulin served as internal loading controls. (c) Western blot analysis of NPGPx protein level in WI38 cells transfected with various amount of non-targeting siRNA (CTRL siRNA). Relative NPGPx/p84 ratio was shown in below. (d) NPGPx mRNA expression analyzed by RT–qPCR from WI38 cells infected with lentivirus carrying with shLuc, GFP or control vector (shEmpty) or uninfected as control (mock). Relative NPGPx mRNA level (normalized with β-actin and compare with Mock) was shown. (e and f) RT–qPCR analysis. WI38 cells transfected with non-targeting siRNA (Ctrl siRNA, 160 nM) or PPIB-1 siRNA were used in this assay. The PPIB-1 mRNA amount (e) and NPGPx mRNA expression level (f) were shown. Mock: WI38 cells without siRNA transfection. (g) Western blot analysis of WI38 cells infected with vector control (shEmpty) or shLuc, and then transfected with either pBSK or Luciferase expression vector (pGL3-Luc). Cell lysates were harvested 48 h after transfection for western blot analysis. Relative expression ratio of NPGPx/α-tubulin (Rel. NP/α-tu) was calculated. (h) Expressions of stress-related proteins including NPGPx, Calnexin, GRP78, eIF2α, phospho-eIF2α (p-eIF2α) in WI38 cells infected with lentivirus carrying shLuc, GFP, shRNA cloning vector (shEmpty) or uninfected as control (mock). α-Tubulin served as an internal loading control. (i) Expression of unspliced (uXBP1) or spliced (sXBP1) XBP1 mRNA analyzed by RT–PCR from WI38 cells infected with lentivirus carrying with shLuc, shRFP, shEmpty or uninfected as the control (mock). Cells treated with tunicamycin (2 µg/ml for 8 h) served as a positive control, where spliced XBP1 (sXBP1) was detected. Ribosomal RNA S26 served as an internal loading control. Each experiment has been repeated at least twice, and the representative data from one of these experiments were shown.

Figure 2.

Cells suffering from non-targeting siRNA stress grew slowly with prolonged G1 phase. (a) Western blot analysis using MEFs (WT or NPGPx^−/− MEFs) transduced with shLuc or control vector (shEmpty). (b) Cell growth assay. MEFs (WT or NPGPx^−/− MEFs) at early passages transduced with shLuc or control vector (shEmpty) were seeded by equal amount (1 × 10⁵ cell/dish) in 6 cm dishes, and cell numbers were counted every day. (c) Cell cycle analysis by FACS was performed at 0, 16, 20 or 24 h after cells were released from starvation. The percentages of cells in G1, S or G2/M phase were shown individually by plot. (d) Cell cycle profiles of WI38 cells transduced with shLuc or control vector (shEmpty). WI38 cells were synchronized with serum starvation, and the cell cycle analysis was performed at 0, 16, 24, 44 h after cells released from starvation. The percentage of cells in G1 phase was shown on each plot. These experiments have been repeated three times with similar results.

Figure 3.

Persistent exposure to non-targeting siRNA stress led to ROS production, DNA damage and apoptosis in NPGPx^−/− MEFs. (a) Cell growth patterns of MEFs at passage 5. (b) Annexin V staining. NPGPx^−/− MEFs at the fourth day from (a) were stained with Annexin V and PI. Percentage of the apoptotic (Annexin V+PI−) cell was illustrated. **P = 0.005. (c) Endogenous ROS measurement. NPGPx^−/− MEFs [same as in (b)] were stained with CM-H₂DCFDA (29) which converted into a green florescence when encounters intracellular ROS, and analyzed by FACS. (d) A representative γH2Ax IF staining with green florescence of NPGPx^−/− MEFs [same with (b)]. Blue: DAPI. (e). The percentage of γH2Ax positive staining cells from (d). These experiments have been repeated three times.

Figure 4.

NPGPx was required for releasing non-targeting siRNA stress. (a) Schematic of primers designed for siRNA detection. (b) Steady amount of siLuc in MEFs transduced with shLuc. MEFs transduced with shLuc were used for measuring siLuc/U6 ratio by RT–qPCR at 4, 5, 7 days after shLuc-lentivirus infection. siLuc RNA accumulation was observed in NPGPx^−/− MEFs. (c) Northern blot analysis of RNA from WT or NPGPx^−/− MEFs transduced with shLuc probed with biotinylated-RNA against siLuc. tRNA was used as a loading control. The relative siLuc/tRNA ratio (Rel. siLuc/tRNA) was shown. (d) Western blot analysis of NPGPx protein using NPGPx^−/− MEFs restored with WT or mutant NPGPx (C2A2-NPGPx). α-Tubulin served as an internal control. Mock: MEFs without retroviral transduction. (e) Steady amount of siLuc in MEFs transduced with shLuc. MEFs transduced with shLuc were used for measuring siLuc/U6 ratio by RT–qPCR as descrived in (b). siLuc accumulation was found in C2A2-NPGPx restored MEFs compared with control MEFs (WT or WT-NPGPx restored MEFs). (f) NPGPx protein expression level of MEFs transfected with 160 nM control non-targeting siRNA (CTRL) or siNPGPx. (g) siLuc RNA level in NPGPx-depleted MEFs. WT MEFs expressing shLuc were transfected with increasing amount of NPGPx siRNA. The ratios of cellular siLuc and U6 RNA were measured by RT–qPCR. CTRL: MEFs cells transfected with 160 nM non-targeting siRNA. (h and i) Stability of siLuc measured by RT–qPCR using DRB-treated MEFs. Equal amount of reverse-transcribed RNA was used in the RT–qPCR analysis. Relative siLuc level (relative to 0 h) was shown. The kinetic plot indicated that the siLuc had a slower turnover rate in NPGPx^−/− MEFs (h) and C2A2-restored MEFs (i) when compared with control MEFs. Each experiment has been repeated more than three times, and the representative data from one of these experiments were shown.

Figure 5.

NPGPx and XRN2 were associated for releasing non-targeting siRNA. (a) Western blot analysis of XRN2 protein expression in shLuc-expressing WI38 cells infected with lentiviruses carrying with XRN2 shRNAs (two clones as indicated) or shEmpty as control. (b) RT–qPCR analysis of relative siLuc expression level normalized to U6 in XRN2-depleted WI38 cells. (c and e) Western blot analysis of XRN2 from WT, NPGPx^−/− MEFs (c) or NPGPx^−/− MEFs expressing WT or C2A2 mutant (e). MEFs were transfected with either 160 nM non-targeting siRNA (CTRL) or XRN2 siRNA (siXRN2), and analyzed XRN2 protein level by western blot. α-Tubulin serves as an internal control. (d and f) RT–qPCR analysis of relative siLuc expression level in XRN2-depleted MEFs. siLuc RNA level was normalized with U6 RNA level. This experiment has been repeated twice.

Figure 6.

NPGPx and XRN2 interacted through covalent bonding. (a and b) NPGPx was reciprocally coimmunoprecited with XRN2. Cell lysates prepared from WI38 cells transduced with shLuc were used in this experiment. Asterisk: heavy chain; arrow: NPGPx. (c) NPGPx and XRN2 were covalently associated. Immunocomplexes of NPGPx and XRN2 were analyzed by SDS–PAGE before adding or without reducing agent (+DTT or −DTT). NPGPx and XRN2 were then blotted as indicated. (d) Coimmunoprecipitation analysis of XRN2 with WT NPGPx and C2A2 mutant in shLuc-expressing MEFs. Cell lysates prepared from NPGPx −/− MEFs expressing WT or C2A2 mutant were used. Immunoprecipitated complexs were separated by SDS–PAGE and probed by XRN2- or NPGPx-specific antibodies. (e) Immunofluorescence staining of WI38 cells transduced with shLuc or control vector (shEmpty). Green: NPGPx; red: XRN2; blue: DAPI. (f) NPGPx and XRN2 interacted directly in vitro. Purified XRN2 (rXRN2) were immobilized on nickel column, and incubated with purified NPGPx (oxidized form, oNPGPx) or C2AS mutant (C57AC86S). NPGPx and XRN2 complex were eluted by imidazole and analyzed by western blot. These experiments have been repeated three times. (g) Model for NPGPx responding to non-targeting siRNA stress. When cells experienced non-targeting siRNA stress, NPGPx expression was induced (1) and in turn activated XRN2 (2) to release/reduce non-targeting siRNA (3). In contrast, cells without NPGPx will accumulate non-targeting siRNA stress (4) and result in DNA damage, elevated ROS and apoptosis (5).