Figure 12

SRp20 promotes gag mRNA association with polysomes in an NXF1-dependent manner. HEK293T cells were transfected with expression plasmids for gag and either SRp20 wild-type, SRp20ΔRRM, or SRp20R3A. 48 h post-transfection cell lysate was loaded onto a 15% to 55% linear sucrose gradient. After acceleration for 3 hrs at 210,000 × g, RNA content across the gradient was assessed by reading absorbance at 254 nM (upper panel). The position of migration of the various ribosomal components and polysomes is indicated. Fractions were collected from the gradient (lower panel) and relative gag mRNA in fractions 3 to 10 was quantified by qRT-PCR using GAPDH as a control (left Y axis) and displayed against the percent sucrose (right Y axis). The ratio of gag RNA signal expressed in the presence of SRp20 wild-type, versus the gag RNA signal in the absence of SRp20 plasmid is shown for each fraction. This experiment was repeated on three separate occasions with the same result. Similar results were obtained with identical constructs from MLV and XMRV. A representative experiment with MLV-derived constructs is shown.