Nuclear Export of Heat Shock and Non-Heat-Shock mRNA Occurs via Similar Pathways

In eukaryotic cells, macromolecules are constantly moving between the nucleus and the cytoplasm. Transport occurs through nuclear pore complexes (NPCs), which are imbedded in the double membrane surrounding the nucleus. The NPC is 66 MDa in the yeast Saccharomyces cerevisiae and consists of about 50 different nuclear pore proteins (nucleoporins) (4, 34, 37, 53). Nucleocytoplasmic transport of all macromolecular substrates studied to date is receptor mediated, energy dependent, and saturable (reviewed in references 27 and 33). Accumulating data on protein import and export point to common principles. To access an NPC, transport substrates need to be recognized by soluble receptors. Most well-characterized receptors belong to a family of proteins called β-importins or β-karyopherins, which recognize specific sequences within their respective substrates. The directionality of transport, nucleus to cytoplasm or cytoplasm to nucleus, is determined in large part by a predicted asymmetry in the intracellular nucleotide-bound status of a key transport player, the small GTPase Ran (Gsp1p in yeast) (17, 18, 28; reviewed in reference 9). Due to the nuclear localization of the Ran GTP exchange factor (Prp20p in yeast) and the cytoplasmic localization of the Ran GTPase-activating protein (Rna1p in yeast), nuclear Ran is mostly in the GTP-bound form, whereas cytoplasmic Ran is mostly GDP bound. Nuclear Ran-GTP promotes the assembly of several export complexes that are formed by a cooperative association between export cargo, receptor, and Ran-GTP. Ran-GTP hydrolysis in the cytoplasm promotes the dissociation of such export complexes, whereas Ran-GTP in the nucleus promotes the dissociation of import complexes. Upon transcription and processing, mRNA becomes associated with many different RNA-binding proteins, forming heterogeneous nuclear ribonucleoprotein (hnRNP) particles (6). Some hnRNPs have been shown to remain associated with mRNA during transport to the cytoplasm, leading to the hypothesis that hnRNPs contain export signals and serve as adaptors recognized by export receptors (reviewed in references 29 and 30). This idea is also based on retroviral systems, in which RNA-binding proteins recognize both specific sequences within viral mRNA and the export receptor Crm1p (also called Xpo1p), resulting in the export of unspliced viral mRNA (reviewed in reference 50). Crm1p is a protein exporter with no major role in general mRNA export (32, 50), suggesting that Rev-like nuclear export signals (NESs) do not make a major contribution to mRNA export. Moreover, there are no known functional Rev-like NESs in any hnRNP, and there are no reported interactions between an hnRNP and a known β-karyopherin-like export receptor. Indeed, the few identified mRNA export factors do not fall into the β-karyopherin class of soluble transport receptors (reviewed in references 27 and 50). These factors (human TAP, or yeast Mex67p; human p15, or yeast Mtr2p; yeast Gle1p, or Rss1p; yeast Rip1p; and yeast Dbp5p, or Rat8p) are all characterized by at least transient association with the NPC. Microinjection-competition experiments with Xenopus oocytes pointed to the existence of nonoverlapping export pathways for different classes of RNA molecules (19). This strategy also indicated multiple separable mRNA export pathways (17, 18, 35, 38). Previous studies of mRNA export during stress in the yeast S. cerevisiae strongly supported this notion, in that exposure to 42°C or 10% ethanol resulted in pronounced nuclear accumulation of non-hs mRNA, whereas hs mRNA was efficiently exported (39). In further support of separate transport pathways for hs and non-hs mRNAs, it was suggested that hs mRNA contains cis-acting sequences that allow its preferential export under stress conditions. hs mRNA export was also proposed not to require Ran and its auxiliary proteins, unlike the transport of non-hs mRNA. The discovery that hs mRNA export at 42°C requires the nucleoporinlike protein Rip1p supported this view of a specialized transport route (40, 47). However, our studies indicate that hs mRNA export is mediated by the Ran system and many other factors involved in non-hs mRNA export. Furthermore, we do not observe a block to non-hs mRNA export at 42°C in a wild-type strain. Finally, we show that nuclear export of various non-hs as well as hs mRNAs is severely affected in the absence of Rip1p at 42°C. One can therefore picture mRNA transport under both normal and stress conditions as a competition among different mRNA molecules for common transport factors.