Coleopteran-specific StaufenC functions like Drosophila melanogaster Loquacious-PD in dsRNA processing

Abstract

In Drosophila melanogaster, PD isoform of the double-stranded RNA binding protein (dsRBP) Loquacious (Loqs-PD) facilitates dsRNA cleavage to siRNA by Dicer-2. StaufenC (StauC) was discovered as a coleopteran-specific dsRBP required for dsRNA processing in coleopteran insects. Here, we show that StauC is essential for the high RNAi efficiency observed in coleopterans. Knockdown of StauC but not the homologs of Loqs-PD and R2D2 evoked a long-lasting insensitivity to RNAi in the coleopteran cell line, Ledp-SL1. The dsRNA insensitivity induced by StauC knockdown could not be overcome merely by an increase in dose or time of exposure to dsRNA or expression of Loquacious or R2D2. Furthermore, StauC but not Loqs and R2D2 are required for processing of dsRNA into siRNA. StauC overexpression also partly restored the impaired RNAi caused by the knockdown of Loqs-PD in D. melanogaster Kc cells. However, StauC was unable to compensate for the loss-of-the function of Dcr-2 or R2D2. Overall, these data suggest that StauC functions like Lops-PD in processing dsRNA to siRNA.

Keywords: Dicer-2; Loqs-PD; RNAi; StaufenC; dsRNA; siRNA.

Figure 1.

Exposure to dsStauC significantly knockdowns StauC protein in Lepd-SL1 cells. (A) Lepd-SL1 cells were exposed to 300 ng/ml dsStauC for 72 h before western blot analysis using StauC antibody. (B) Lepd-SL1 cells pre-exposed to dsStauC for 72 h were incubated for addition 24 and 48 h in the presence or absence of dsStauC before subjected to western blot analysis using StauC antibody. 50 µg of total cell lysate was utilized to measure endogenous StauC level. (C) Apoptosis phenotype was observed after exposing Lepd-SL1 cells to dsIAP, but not dsGFP. Lepd-SL1 cells pre-exposed to dsStauC or dsGFP were incubated with dsIAP (a final concentration of 30 ng/ml) and photographed 24 after treatment. (Scale bar = 100 µm). (D) TUNEL staining of dsRNA-treated Lepd-SL1 cells to examine the apoptotic cell death. (Scale bar = 200 µm) Images are representative of three replicates shown in

Figure 2.

Effect of StauC expression on the RNAi efficiency in Lepd-SL1 and Sf9 cells. (A) StauC depletion induces high resistance to RNAi in Lepd-SL1 cells. Lepd-SL1 cells acquire high resistance to apoptosis induced by dsIAP. Lepd-SL1 cells pre-exposed to dsStauC were incubated with different concentrations of dsIAP for 24 h (1 to 1075 ng/ml)) and 48 h (0.3 to 1000 ng/ml). (B) StauC facilitates dsRNA-mediated gene silencing in the Spodoptera Sf9 system. Expression of genes coding for StauC and Dcr-2a decreased the luciferase gene expression in Sf9 cells exposed to dsLuc compared to the dsGFP treatment control. The measured luciferase activity was normalized to total protein concentration. Each dot refers to the average of triplicates, and its values were expressed as the mean ± standard deviation. Sf9 cells were transiently co-transfected with a luciferase reporter vector and gene expression construct encoding L. decemlineata StauC and Dcr-2a protein. The cells were then re-seeded into a 48-well culture plate (1 x 10⁵ cells per well) at 4 h post-transfection, followed by incubation with 4 µg/ml dsLuc or dsGFP for an additional 60 h. The measured luciferase activity was normalized to total protein concentration. (C) Heterologous expression of StauC and Dcr-2 in Sf9 cells. These recombinant proteins were detected by western blot analysis using the antibodies specific to S tag and V5 tag at 60 h post-transfection.

Figure 3.

Heterologous expression of StauC in part functionally compensates for the loss-of-function of Loqs-PD in Kc cells. Kc cells were pre-incubated with dsRNA corresponding to (A) GFP or D. melanogaster (B) Loqs-PD, (C) R2D2, or (D) DCR2 for 3 days to allow for a significant reduction in the target protein. A luciferase expression construct was then transfected into the cells along with pIEx4 or pIEx4/StauC. 4 h after transfection; the cells were exposed to different concentrations of dsLuc (3.9 ng/ml – 7000 ng/ml) for 48 h. Incubation with 3.9 ng/ml dsLuc induced no significant reduction in luciferase activity in all the tested groups, compared to no dsRNA treatment (Fig. S7). The measured luciferase activity was normalized to total protein concentration. Mean ± standard deviation (n=3) are shown. Asterisks, significant differences of the mean values between two groups. p values were determined using the Student’s t test: *, p < 0.05; **, p < 0.01

Figure 4.

StauC forms a protein complex with Dcr-2a in cells. (A) Schematic representation for the recombinant StauC-S and Dcr-2a-V5-8xHis. (B) Sf9 cells were co-transfected with the gene expression constructs encoding L. decemlineata StauC-S and Dcr-2a-V5-8xHis. The transfected cells were incubated with or without dsGFP for 4 h before harvesting them at 48 h post-transfection. The proteins were etracted and used to perform western blot analysis using S and V5 tag-specific antibodies. 50 µg of total cell lysate was utilized to measure the levels of target proteins. (C) The same cells but exposed to DSP, a cell-permeable and reversible crosslinking agent, were analysed by western blot against S and V5 tags. 50 µg of total cell lysate was used for analysis. (D) The His-tagged protein and protein complexes extracted from the DSP-treated cells were subjected to immunoblotting using S and V5 tag-specific antibodies. (E) L. decemlineata StauC-S was expressed in Sf9 cells in the presence or absence of Dcr-2a. The protein complexes isolated from the DSP-treated cells by immortalized metal affinity chromatography were subjected to SDS-PAGE and western blot analysis

Figure 5.

Effect of the knockdown of StauC, Loquacious, and R2D2 on the RNAi susceptibility of Lepd-SL1 cells. (A-D) The Lepd-SL1 cells were pre-treated with dsRNA targeting L. decemlineata Loquacious (A), R2D2 (B), GFP (C, control) StauC, (D) for 3 days. The cells were exposed to different concentrations of dsIAP (3.9 ng/ml – 4000 ng/ml) for 48 h. The cell viability was determined by Cell Counting Kit 8. Mean ± standard deviation (n = 3) are shown. (E) Processing of dsRNA to siRNA in Lepd-SL1 cells treated with dsLoqs, dsR2D2, dsStauC, or dsmalE (control). The Lepd-SL1 cells were seeded in six-well plates and exposed to dsLoqs, dsR2D2, dsStauC, or dsmalE for 3 days. The cells were then exposed to 1.6 million CPM []³²P-labelled dsGFP. At 48 h after treatment with the second dsRNA, the total RNA was isolated from the cells and resolved on 16% acrylamide-urea gel. The dried gel was then analysed using the phosphorImager. The first lane shows dsGFP used as a marker. (F) Schematic representation of protein domain organization for StauC, Loquacious and R2D2 orthologs in L. decemlineata (Ld) and D. melanogaster (Dm)

Figure 6.

Tissue-specific expression of StauC in L. decemlineata. (A) qRT-PCR analysis of StauC mRNA levels in different tissues of L. decemlineata 4^th instar larvae. (B) qRT-PCR analysis of StauC mRNA levels in various tissues of L. decemlineata adults