Figure 5.

W45-dependent effect of Rev on the biogenesis of RRE-containing mRNA in HeLa cells. (A) Schematic representation of pDM128. The intronic sequence is represented as a dashed line, SD4 – splice donor 4, SA7 – spliced acceptor 7. The location of qPCR amplicons detecting either spliced or unspliced mRNA is shown. (B, C) HeLa cells were transiently transfected with pDM128 and Rev WT or W45A (+ pRLSV40 to control for transfection efficiency and pBSIISK+ as plasmid DNA for dilution). Total RNA was isolated 48 hours after transfection, reverse-transcribed with an oligo(dT) primer. (B) The relative abundance of spliced to unspliced poly(A)+ RNA was assessed by qPCR (left graph). (C) In order to look at the levels of spliced and unspliced mRNAs separately instead of as a ratio, the same qPCR data were plotted comparing changes in the levels of spliced and unspliced mRNA species in cells transfected with RevW45A versus Rev WT (right graph). (D) HeLa cells were transiently transfected with GFP-tagged Rev WT or W45A and assayed 48 hours after transfection. Extracts from HeLa cells were incubated with or without RNase A and subjected to immunoprecipitation with α-GFP antibody. 1% of input and 50% of the immunoprecipitate was analyzed by Western blot, using α-GFP and α-U2AF65 antibodies. The experiments were performed three times, a representative gel is shown.