VISUALIZING NUCLEAR EXPORT OF DIFFERENT CLASSES OF RNA BY ELECTRON-MICROSCOPY

Export of RNA from the cell nucleus to the cytoplasm occurs through nu clear pore complexes (NPCs). To examine nuclear export of RNA, we have gold-labeled different types of RNA (i.e., mRNA, tRNA, U snRNAs), and followed their export by electron microscopy (EM) after their microin jection into Xenopos oocyte nuclei. By changing the polarity of the ne gatively charged colloidal gold, complexes with mRNA, tRNA, and U1 snR NA can be formed efficiently, and gold-tagged RNAs are exported to the cytoplasm with kinetics and specific saturation behavior similar to t hat of unlabeled RNAs. U6 snRNA conjugates, in contrast, remain in the nucleus, as does naked U6 snRNA. During export, RNA-gold was found di stributed along the central axis of the NPC, within the nuclear basket , or accumulated at the nuclear and cytoplasmic periphery of the centr al gated channel, but not associated with the cytoplasmic fibrils. In an attempt to identify the initial NPC docking site(s) for RNA, we hav e explored various conditions that either yield docking of import liga nds to the NPC or inhibit the export of nuclear RNAs. Surprisingly, we failed to observe docking of RNA destined for export at the nuclear p eriphery of the NPC under any of these conditions. Instead, each condi tion in which export of any of the RNA-gold conjugates was inhibited c aused accumulation of gold particles scattered uniformly throughout th e nucleoplasm. These results point to the existence of steps in export involving mobilization of the export substrate from the nucleoplasm t o the NPC.