Figure 3

The occlusion of polyadenylation is U1snRNA-dependent. (A) Luciferase activity in BHK-21 cells. Co-transfection with the pGL3LTR derivatives and either wild-type or U1snRNAm2 expression construct complementary to SDm2. The latter restored suppression of polyadenylation in cells transfected with SDm2. An alignment of U1snRNA and splice donor RNA sequences is shown above the diagram (mutated nucleotides are shown in bold and underlined). Bars represent the mean value of three independent transfections, and the error bars represent the standard deviation. The significance of the reduction by the SDm2 mutation or increase by co-transfection of U1snRNAm2 was calculated by the paired two-sample t-test. p-values are indicated. (B) The SDm2 mutant is rescued by U1snRNAm2 co-transfection in a proviral context. Northern blotting analysis of RNA from BHK-21 cells co-transfected with either pHSRV13 or pHSRV13SDm2, and wild-type or SDm2 U1snRNA expression constructs. Viral RNAs were visualised using a tas-specific probe. The positions of the 18S (1.9 kb) and 28S (4.7 kb) rRNAs are indicated. The normalised amounts of gag/pol transcripts are depicted below the lanes. (C) Gag, Tas, and GAPDH were analysed by Western blotting. PFV-infected BHK-21 cells (+) and untransfected cells (−) served as controls.