Effects of US11 on the stability and immune signaling activity of duplex RNA formed by annealing of partially complementary reporter mRNAs.

Abstract

A) Schematic of dsRNA generation. MCherry expression plasmids with a fragment of the SRPRα ORF (canine signal recognition particle receptor subunit alpha) inserted in the 3’UTR in the forward (fwd) or reverse (rev) orientation. Co-transfection of both plasmids results in formation of dsRNAs with 5’ and 3’ overhangs. B) HeLa cells were transfected with fwd and rev together (fwd/rev) along with either pEGFP or pEGFP-US11 for 20 hours. As a control, cells in separate dishes were transfected with either fwd/pEGFP or rev/pEGFP and combined during Trizol lysis (fwd)+(rev). Total RNA was extracted, digested by DNaseI and RNaseA/T1 (high salt conditions) or DNaseI, RNaseIII and RNaseA/T1 and analyzed by Northern blot with a probe specific for the inserted SRPRα fragment. C) Cotransfection of fwd/rev and pEGFP or pEGFP-US11, respectively. Transfected cells were treated with actinomycin D for 0, 2 or 4 hours prior to RNA extraction and processed as in (B). D) Quantification of the full-length dsRNA SRPRα signal from 3 independent experiments. P-value for pEGFP/4h vs. 0h is 0.010, 4h/pEGFP-US11 is ns/nonsignificant. E) HeLa cells were transfected with fwd or fwd/rev and pcDNA, pEGFP or pEGFP-US11, respectively. After 20 hours cells were treated with actinomycinD for 4 hours. Whole-cell lysates were analyzed by Western blot with the indicated antibodies. Results shown are representative of 3 independent experiments.</p