FIG. 9.
NXT1 facilitates nuclear export of mRNA in vitro. (A) Radiolabeled Rab11 mRNA was synthesized in vitro and introduced by diffusion into permeabilized nuclei. The nuclear envelope was then repaired (t0), and nuclei were incubated for 90 min with 10% gelatin in transport buffer at 20°C (buffer), with 50% Xenopus extracts in the presence of an energy-regenerating system at 20 or 4°C, or with 50% Xenopus extracts in the presence of apyrase at 20°C, as indicated. mRNAs were then extracted from nuclear fractions (N) and from incubation buffer (S) and analyzed by denaturing gel electrophoresis. (B) Radiolabeled Rab11 mRNA was introduced by diffusion into permeabilized nuclei. The nuclear envelope was then repaired (t0), and nuclei were resuspended either in 50% Xenopus extracts (control) or in 50% Xenopus extracts pretreated with 0.1 or 1 μM LMB (45 min, room temperature). After 60 min of incubation at 20°C, mRNAs were extracted from nuclear fractions (N) and from incubation buffer (S) and analyzed by gel electrophoresis. (C) Radiolabeled Rab11 mRNA was introduced by diffusion into permeabilized nuclei. The nuclear envelope was then repaired (t0), and nuclei were resuspended either in 50% Xenopus extracts alone or supplemented with NXT1 (100 μg/ml) or in 15% Xenopus extracts alone or supplemented with NXT1 (250 μg/ml). After 60 min of incubation at 20°C, mRNAs were extracted from nuclear fractions (N) and from incubation buffer (S) and analyzed by gel electrophoresis.