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Fig. 5. Nuclear localization does not restore full splicing activity in the absence of the C terminus. (A) Incorporation of the SV40 large T antigen NLS at the C-termini of the GFPcETR3 and GFPDD.4 fusion proteins (GFPcETR3NLS and GFPDD.4NLS, respectively) conferred nuclear localization in COS-M6 cells. Scale bar: 10 µm. (B) RT-PCR analysis was performed (upper panel) to determine the extent of exon inclusion (lower panel) in cotransfection assays with a cTNT minigene. While all of the GFP-ETR3 fusion constructs gave levels of exon inclusion that were statistically different from that of the minigene alone (P0.05), GFPcETR3 and GFPDD.4 activated exon inclusion only slightly above the basal level. The activity of the C-terminal deletion mutant was not enhanced by the addition of an NLS, as GFPcETR3NLS did not activate levels of exon inclusion significantly higher than those observed for GFPcETR3. When the divergent domain deletion was restored to the nucleus, however, GFPDD.4NLS enhanced exon inclusion significantly above the level observed for GFPDD.4 and close to the level observed for the full-length protein. The average percentages of total mRNAs containing the alternative exon plus or minus the standard error of the mean are shown for each (n4).

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