1. Transport across NPCs is driven by diffusion. The importin  -cargo complex diffuses from the cytoplasmic side of the NPC to the nuclear side of the NPC because its concentration is higher in the cytoplasm where the complex forms and lower in the nucleus where the complex is dissociated when RanGTP binds the importin . The RanGTP- importin  complex formed in the nucleus diffuses into the cytoplasm where its concentration is low because Ran-GAP in the NPC cytoplasmic filaments converts RanGTP to RanGDP, dissociating the RanGTP- importin  complex.

2. The same arguments apply to the transport of the NTF2- RanGDP complex from the cytoplasm into the nucleus and of free NTF2 from the nucleus into the cytoplasm.

3. Cell fusion experiments revealed that several hnRNP proteins that appear to be confined to nuclei actually shuttle between the nucleus and cytoplasm. Cultured Xenopus and human cells were fused to each other using polyethylene glycol. Cycloheximide was added to prevent further protein synthesis. After 2 h, the cells were fixed and stained with fluorescent antibodies specific for human hnRNP A or human hnRNP C.

4. Construction of several hnRNP A deletion mutants and analysis of the ability of these mutants to shuttle between the nucleus and cytoplasm using cell fusion experiments revealed that deletion of an ~10 aa region caused the mutant hnRNP A to be retained in nuclei and not shuttle into the cytoplasm. The sequence required for shuttling into the cytoplasm is called a nuclear export sequence or NES. Several NESs have been defined in different shutlling hnRNP proteins.

5. NESs are bound by proteins that are homologous to importin . These proteins required for nuclear export are called exportins. The importin  -related proteins, different importins and exportins, are referred to as karyopharins. All of these proteins have the ability to interact with the FG-domains of FG-nucleoporins and consequently can diffuse through the molecular meshwork that fills the central transporter of NPCs.

6. The direction of transport is dependent on the localization of Ran-GAP in the cytoplasm and Ran-GEF in the nucleus. This maintains Ran in the GTP form in the nucleus and in the GDP form in the cytoplasm. Ran-GAP is bound to the cytoplasmic filaments of the NPC. Ran-GEF is bound to chromosomes.

7. The export of most mRNPs requires a heterodimeric mRNA transporter. The small subunit of the mRNA transporter and the middle and C-terminal domains of the large subunit interact with FG-repeats of the FG-nucleoporins. The large subunit binds to mRNAs cooperatively with SR-proteins bound to exonic enhancers. Thus, SR-proteins both specify exons during RNA- splicing and assist in transporting the resulting mRNA into the cytoplasm, distinguishing exons from other RNAs in the nucleus.

9. Other proteins that assist in binding of the mRNA exporter to mRNAs include: The nuclear cap binding protein. PABPII bound to the polyA tail. Proteins retained at spliced exon junctions following RNA splicing that form exon-junction complexes. Thus, mRNP export depends on the additive effects of multiple weak protein-RNA and protein-protein interactions that bind to mRNAs cooperatively through interactions with structures and sequences that distinguish mRNAs including the 5’ cap, 3’ polyA tail, and exonic splicing enhancers.

10. Fig. 12-25

12. The association of snRNPs involved in RNA-splicing with a pre-mRNA prevents its nuclear export. This prevents the export of an mRNA until all of its introns have been spliced out.

13. Fig. 12-26