Figure 1.

Continuous expanded poly(A) tract alters the localization of nuclear proteins. A, subcellular localization of Rev(1.4)-poly(A)-EGFP proteins in HEK293 cells. The Rev(1.4)-EGFP control protein localized to the nuclear compartment, whereas the positive control Rev(1.4)-NES-EGFP (NES), which contained a functional HIV-1 Rev NES sequence, localized to the cytoplasm. The Rev(1.4)-poly(A)-EGFP protein with a 6-alanine tract (A6) localized in the nuclear compartment, whereas that with a 37-alanine tract Rev(1.4)-A37-EGFP (A37) showed both nuclear and cytoplasmic localization. Cell nuclei were stained with Hoechst 33342. Scale bar, 10 μm. B, statistical analysis of A. C, subcellular localization of Rev(1.4)-polyAPA-EGFP proteins in HEK293 cells. The Rev(1.4)-A37-EGFP (A37) showed both nuclear and cytoplasmic localization, two Rev(1.4)-polyAPA-EGFP variants (A10(PA9)₃ and A7(PA6)₅) localized in the nuclear compartment. Cell nuclei were stained with Hoechst 33342. Scale bar, 10 μm. D, statistical analysis of C. E, subcellular localization of Rev(1.4)-poly(A)-EGFP protein with A22, A25, A28, A31, and A34. A22, A25, and A28 showed nuclear localization. A31 resulted in 26% of cells with both nuclear and cytoplasmic localization, whereas A34 resulted in up to 78% of cells with both nuclear and cytoplasmic localization. Scale bar, 10 μm. F, statistical analysis (E). N, nuclear; C, cytoplasmic; C + N, cytoplasmic and nuclear localization. Three independent transfection experiments were performed. At least 100 transfected cells were analyzed in each experiment. Error bars, S.E.