ENERGY-AND TEMPERATURE-DEPENDENT IN-VITRO EXPORT OF RNA FROM SYNTHETIC NUCLEI

We describe a novel assay for the study of RNA export from the nucleus in vitro. Nuclei are assembled in Xenopus egg extract on paramagnetic beads coated with DNA containing a specific template for transcriptio n. T7 RNA polymerase, to which a nuclear localisation signal is attach ed, is added to the nuclei, and after its import into the assembled nu clei, transcription is allowed to proceed. The use of radioactive NTPs coupled with the possibility to purify the nuclei on a magnet and thu s rapidly change the extract in which the nuclei are incubated allows pulse-chase labelling experiments. Using these protocols we show that U1 snRNA-derived templates ave transcribed inside the synthetic nuclei , and that the transcripts leave the intact nuclei in a time-, tempera ture-and energy-dependent way. This offers the possibility of a bioche mical approach to the dissection of RNA export.