Figure 1. NF90ctv recognizes and exports transcripts linked to RRE.

A) Schematic representation of full-length NF90ctv and of the various truncated forms used in this study. The main domains of NF90ctv and their positions are shown. B) Quantification and Western blot results demonstrating that NF90ctv and specifically, the RCN, DRBD1/2 and RG domains bind and export mRNA-Gag linked to RRE. HeLa cells were co-transfected with the construct pCMVGag2RRE in the presence of pNF90ctv-mRFP or each of its truncated forms. pRev-GFP was used as positive control. After 24 h Western blots were performed using pr55Gag antibodies. C) Kinetic and quantification of pr55Gag expression. HeLa cells were co-transfected with pRCN-mRFP in the presence of pCMVGag2RRE. At different times, the cells were lysed and Western blots were performed using pr55Gag antibodies. D) Quantification of the results obtained by Western blots demonstrates that RCN, DRBD1/2 and RG mRNA-Gag linked to RRE export is leptomycin-dependent. HeLa cells were cultured in six-well plates; three wells for each construct of interest were used: one well for leptomycin treatment and two wells as controls at 8 and 12 h. The cells were co-transfected with pRCN-mRFP or with pDRBD1/2-mRFP, with pRG-mRFP in the presence of pCMVGag2RRE. Based on the results shown in D, 8 h later the cells of one well were harvested; in the second well, leptomycin B was added and incubated for 4 h; finally, 12 h later, the cells of the second and the third wells were harvested. Western blots were performed on the cell pellets using pr55Gag antibodies. Each assay was repeated three times. The Western blots were quantified by densitometric scanning using ImageJ and normalized using loading controls (β-tubulin). The data shown are the means and standard errors of the mean of three independent experiments.