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.