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 transcription. T7 RNA polymerase, to which a nuclear localisation signal is attached, is added to the nuclei, and after its import into the assembled nuclei, transcription is allowed to proceed. The use of radioactive NTPs coupled with the possibility to purify the nuclei on a magnet and thus 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 are transcribed inside the synthetic nuclei, and that the transcripts leave the intact nuclei in a time-, temperature- and energy-dependent way. This offers the possibility of a biochemical approach to the dissection of RNA export.
Arts
G J
GJ
Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Englmeier
L
L
Mattaj
I W
IW
D000818:Animals; D001693:Biological Transport, Active / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism*; D002474:Cell-Free System; D004247:DNA / Q000138:chemical synthesis; D004734:Energy Metabolism / Q000235:genetics; D008280:Magnetics; D008863:Microspheres; D009865:Oocytes; D012342:RNA, Small Nuclear / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic; D014982:Xenopus laevis
D012342:RNA, Small Nuclear; D004247:DNA