Figure 1.
Expression of hairpin RNA inhibits the luciferase activity of transiently transfected reporter plasmids.
(A) Schematic composition of pCAGEGFP-MosIR, pCAGEGFP, and pCAGEGFP-MosMos plasmids. (B) Reporter activity is inhibited by hairpin RNA in a concentration-dependent manner. HEK-293 cells were transiently transfected with a constant amount of firefly luciferase (square), Renilla luciferase (triangle) reporter plasmids, and increasing amount of a tested plasmid. Luciferase activities were measured 48 hours post-transfection. pBluescript was added to maintain a constant amount of transfected DNA. Both luciferase activities are shown relative to cells transfected with 0 ng of the pCAGEGFP-MosIR. Data are shown as an average of at least 3 experiments made in triplicates. Error bars = SEM.
Figure 2.
Suppression of co-transfected reporters in mammalian cells is general.
(A, B) Suppression of co-transfected reporters in HeLa cells (A) and mouse 3T3 cells (B). Cells were transiently transfected with a constant amount of firefly luciferase (square), Renilla luciferase (triangle) reporter plasmids, and increasing amounts of pCAGEGFP-MosIR or pCAGEGFP. Luciferase activities were measured 48 hours post-transfection. pBluescript was added to maintain a constant amount of transfected DNA. Both luciferase activities are shown relative to cells transfected with 0 ng of the pCAGEGFP-MosIR. Data are shown as an average of at least 3 experiments performed in triplicates. Error bars = SEM. (C) Suppression of co-transfected reporters is independent of the transfection method. HEK-293 cells were transiently transfected with a constant amount of firefly luciferase (black bars), Renilla luciferase (white bars) reporter plasmids, and 50 ng of pCAGEGFP-MosIR using Nanofectin or calcium phosphate transfection. Luciferase activities were measured 48 hours post-transfection. pBluescript was added to maintain a constant amount of transfected DNA. Both luciferase activities are shown relative to cells transfected with 0 ng of the pCAGEGFP-MosIR. Data show a typical experiment measured in triplicates. Error bars = SEM. (D) Suppression of a co-transfected RFP reporter. HEK-293 were transiently transfected with 150 ng of RFP reporter plasmid and 350 ng of pCAGEGFP or pCAGEGFP-MosIR. Shown is FACS analysis of RFP fluorescence 36 h post-transfection. The experiment was performed three times, a representative result is shown.
Figure 3.
High-throughput sequencing analysis of total RNAs derived from pCAGEGFP and pCAGEGFP-MosIR plasmids.
(A) Distribution of 18–50 nt reads that perfectly map to pCAGEGFP-MosIR. Reads mapping to the sense and antisense strands are shown in the upper and lower half of the graph, respectively. Schematic representation of plasmid features is shown below the histogram. The Y-scale represents normalized read density (counts per million, CPM); y-scale maximum corresponds to 1500 CPM to better visualize RNAs produced from the plasmid backbone. Expression from the CMV/β-actin promoter, which yields normalized read density highly exceeding 1500 CPM, can be seen in the panel G. (B) Distribution of reads 20–24 nt in length with up to 5 adenosine-to-guanosine mismatches that map to pCAGEGFP-MosIR. The Y-scale represents normalized read density (counts per million, CPM); y-scale maximum corresponds to 200 CPM. Schematic representation of plasmid features is shown below the histogram. (C, D) Length distribution of edited (gray) and non-edited (black) reads derived from MosIR (C) or EGFP (D) region of pCAGEGFP-MosIR. (E) Distribution of 18–50 nt reads that perfectly map to pCAGEGFP. Description as in the panel a. (F) Distribution of reads 20–24 nt in length with up to 5 adenosine-to-guanosine mismatches that perfectly to pCAGEGFP. Description as in the panel B. (G) Comparison of normalized read density for pCAGEGFP and pCAGEGFP-MosIR transcripts transcribed from the CMV/β-actin promoter. The Y-scale represents normalized read density (counts per million, CPM); y-scale maximum corresponds to 75000 CPM. Schematic representation each transcript is shown below each histogram.
Figure 4.
Transiently transfected luciferase reporters are inhibited by expressed dsRNA.
(A) The inhibition of reporter activity is independent of the hairpin RNA sequence and it is absent when the inverted repeat is placed downstream of non-active ZP3 promoter. HEK-293 cells were transiently transfected with 100 ng/well of each RL (triangle) and FL (square) reporter plasmids and increasing amount (0–250 ng/well) of either pCAGEGFP-Lin28IR (containing an active promoter) or pZP3EGFP-Lin28IR (containing an inactive promoter). Luciferase activity was analyzed 48 hours after transfection. pBluescript was added to maintain the amount of transfected DNA constant. Both luciferase activities are shown relative to cells transfected with 0 ng of the hairpin-expressing plasmid. (B) Similar to (A) except Elavl2IR-expressing plasmids (pCAGEGFP- Elavl2IR or pZP3EGFP-Elavl2IR) were used. Data are shown as an average of at least 3 experiments made in triplicates. Error bars = SEM. (C, D) A hairpin-expressing pCAGEGFP-MosIR plasmid affects the luciferase activity of transiently transfected but not stably integrated reporter plasmids. HEK-293 cells with stably integrated RL and FL reporters (C), or HEK-293 cells with stably integrated RL reporter only (D) were transiently transfected with an increasing amount of pCAGEGFP-MosIR and a constant amount of FL reporter (if not stably integrated). pBluescript was added to maintain the amount of transfected DNA constant. Both FL (squares) and RL (triangles) luciferase activities were analyzed 48 hours post-transfection. Luciferase activities in cells transfected with 0 ng of the pCAGEGFP-MosIR were set to one. Data show an average of at least 3 experiments done in triplicates. Error bars = SEM.
Figure 5.
dsRNA inhibits translation of transcripts from transiently-transfected plasmids.
(A) Transiently transfected RL and FL reporters are not inhibited at transcript levels. HEK-293 cells were transiently transfected with FL and RL reporters (100 ng/well) and increasing doses of pCAGEGFP-MosIR (0–250 ng/well). Amount of mRNA was analyzed by real-time PCR. Expression was normalized to HPRT1 housekeeping gene and expression levels in cells transfected with 50 ng of MosIR plasmid were set to 1. Error bars = SEM. (B) dsRNA-dependent inhibition of translation affects more transiently transfected plasmids than endogenous genes. HEK-293 cells were transfected with RL, FL, and either pCAGEGFP or pCAGEGFP-MosIR. Distribution of mRNA in fractions collected during polysome profiling was analyzed by real-time PCR. For each sample, a fraction representing monosomes (80S) and early (poly1) and late (poly2) polysomes (depicted on the scheme) was included in the quantification. Expression levels in polysome fractions of pCAGEGFP- (black bars) and pCAGEGFP-MosIR- (white bars) transfected cells are normalized to 80S fraction. Panels show expression profiles for endogenous genes (HPRT1 and B2M), plasmid-expressed transcripts (FL, RL) and either pCAGEGFP or pCAGEGFP-MosIR transcript (pCAG). B2M, β2 microglobulin; HPRT1, hypoxantine phosphoryltransferase; FL, firefly luciferase; RL, Renilla luciferase.
Figure 6.
The inhibition of luciferase reporter activity is partly dependent on PKR protein level.
(A) Stable cell line with shRNA-mediated PKR knock-down. Western blot analysis of HeLa stable cell lines carrying shRNA vector targeting PKR. Ctrl, parental HeLa cells; A1-B6, stably-transfected independent clones carrying antibiotic resistance. Clone B4 showed the highest PKR knock-down and was used for subsequent experiments. (B) Parental HeLa cells (ctrl) or HeLa cells with stably down-regulated PKR expression (KD) were co-transfected with 100 ng/well of each RL (light colors) and FL (dark colors) reporter plasmids and 0 or 50 ng/well of pCAGEGFP-MosIR. Parental plasmid pCAGEGFP was used to maintain a constant amount of transfected DNA. Data are shown as an average of two independent experiments performed in quadruplicates; Error bars = SEM. (C) pCAGEGFP-MosIR transcript associates with PKR. HEK-293 cells were transfected with pCAGEGFP or pCAGEGFP-MosIR. Cell lysates (24 hours post-transfection) were used for immunoprecipitation with anti-PKR antibody or control IgG antibody. Immunoprecipitated RNA was reverse-transcribed and used for real-time PCR analysis. Data are displayed as a percentage of input. Shown is the average of two experiments. Error bars = range of values. (D) pCAGEGFP-MosIR expression activates PKR. Western blotting analysis of HEK-293 cells transfected with increasing amount of pCAGEGFP or pCAGEGFP-MosIR (50–150–250 ng per well). pBluescript was added to maintain the amount of transfected DNA constant. pBS, pBluescript only. UN, untransfected cells; MosIR, pCAGEGFP-MosIR.
Figure 7.
pCAGEGFP-MosIR expression affects the distribution of PKR and its phosphorylated form in polyribosome analysis.
(A) Overlay of polysome profiles from HEK-293 cells transfected with either pCAGEGFP or pCAGEGFP-MosIR. (B) Polysome profile (upper part) and western blotting analysis (lower part) of respective fractions. The alignment of fractions on polysome profile and lanes on western blotting is highlighted by solid red lines flanking fractions 11 and 12. HEK-293 cells transfected with either pCAGEGFP or pCAGEGFP-MosIR were subjected to polysome profiling. Proteins were isolated from each fraction by ethanol precipitation and the same volume aliquots from each fraction were analyzed by polyacrylamide gel electrophoresis and western blotting. The last lane in each sample represents 1% of input. γ, phosphorylated form of protein; RPS14, ribosomal protein S14. The experiment was repeated three times, a representative result is shown.