1. Fig. 12-17

2. Fig. 2-15 Deamination

3. U1snRNP U2snRNP Phosphorylated CTD Pol II { Cross-exon recognition complex

4. hnRNP proteins Splicing Cleavage/ polyadenylation AAAAAAAAAAA hnRNP proteins CBP PABPII nascent pre-mRNA mRNP

5. Fig. 5-19

6. Fig. 5-22

7. Nuclear pore complexes are made of multiple copies of ~100 different proteins. The general term for one of the proteins that make up the nuclear pore complex is “nucleoporin.” The specific name for a nucleoporin is generally based on its molecular weight, such as “Nup 150.” NPCs are roughly octagonal, membrane-embedded structures from which eight ~100 nm fibers made of specific Nups extend into the cytoplasm. Similarly, eight ~100 nm fibers extend into the nucleoplasm where there ends are connected, forming a “nuclear basket.”

8. Fig. 12-18

10. The nucleoplasmic side of an NPC is attached to an orthagonal network of lamin intermediate filaments called the “nuclear lamina.” This nuclear lamina supports the inner nuclear membrane, giving strength and shape to the nuclear envelope.

11. Fig. 21-16

12. Ions, small metabolites, and small proteins (< ~60 kDa) can diffuse through a water-filled channel in the NPC. However, large proteins and mRNPs cannot diffuse freely in and out of the nucleus. They must be selectively transported out of or into the nucleus with the aid of soluble transporter proteins called “importins” or “exportins” or “karyopharins.” These karyopharins bind to short peptide sequences in the proteins to be transported and to nucleoporins to transport their “cargo” proteins through the central transporter of the NPC.

13. A class of nucleoporins called FG-nucleoporins line the channel in the NPC central transporter and are also found in the cytoplasmic and nucleoplasmic filaments. FG-nucleoporins contain domains with repeats of multiple phenylalanine (F) and glycine (G) amino acid residues separated by stretches of hydrophilic amino acids.

14. The domains of FG nucleoporins containing the FG repeats are postulated to form extended polypeptide strands with hydrophobic repeats punctuating the extended strands. FG-repeat Hydrophilic region

15. These domains are postulated to extend into the channel of the central transporter where the hydrophobic FG repeats from separate FG-nucleoporins interact forming a molecular meshwork. K. Ribbeck and D. Gorlich (2001) EMBO J. 20:1320

16. This molecular meshwork in the central transporter of the NPC is postulated to allow small proteins to diffuse through the meshwork, but to prevent large proteins and mRNPs from diffusing through.

17. Fig. 12-19 SV40 T-antigen nuclear localization signal -- NLS: PKKKRKV Pyruvate kinase Pyruvate kinase-PKKKRKV

18. All large nuclear proteins studied contain a peptides sequence of < 50 aa that cause most soluble cytoplasmic proteins to be transported into the nucleus when fused to that protein. Many of these NLSs are rich in lysine (K) like the SV40 NLS. But other NLSs have a variety of sequences that are not K-rich.

19. A digitonin-permeabilized cell system provided an assay for cytoplasmic proteins required for the transport into nuclei of proteins with an NLS. This non-ionic detergent makes wholes in the plasmamembrane that allows cytoplasmic proteins to leak out of the cell without disrupting the nuclear membrane. Proteins added to these digitonin-treated cells can come in contact with the intact nuclear envelope. If a fluorescently labeled protein with an NLS was added to these digitonin treated cells, it did not accumulate in the nucleus. However, if a cytoplasmic protein extract was added to the cells along with the fluorescently labeled protein, it did.

20. Fig. 12-20

21. Using the transport of a fluorescently labeled protein with an NLS into digitonin permeabilized cells as an assay for the soluble cytoplasmic proteins required for nuclear transport, only four proteins were found to be required: Ran a small G protein similar to Ras. Importin  Importin  Nuclear transport factor 2 -- NTF2

22. These domains are postulated to extend into the channel of the central transporter where the hydrophobic FG repeats from separate FG-nucleoporins interact forming a molecular meshwork. K. Ribbeck and D. Gorlich (2001) EMBO J. 20:1320

23. This molecular meshwork in the central transporter of the NPC is postulated to allow small proteins to diffuse through the meshwork, but to prevent large proteins and mRNPs from diffusing through.

24. Fig. 12-19 SV40 T-antigen nuclear localization signal -- NLS: PKKKRKV Pyruvate kinase Pyruvate kinase-PKKKRKV

25. All large nuclear proteins studied contain a peptides sequence of < 50 aa that cause most soluble cytoplasmic proteins to be transported into the nucleus when fused to that protein. Many of these NLSs are rich in lysine (K) like the SV40 NLS. But other NLSs have a variety of sequences that are not K-rich.

26. A digitonin-permeabilized cell system provided an assay for cytoplasmic proteins required for the transport into nuclei of proteins with an NLS. This non-ionic detergent makes wholes in the plasmamembrane that allows cytoplasmic proteins to leak out of the cell without disrupting the nuclear membrane. Proteins added to these digitonin-treated cells can come in contact with the intact nuclear envelope. If a fluorescently labeled protein with an NLS was added to these digitonin treated cells, it did not accumulate in the nucleus. However, if a cytoplasmic protein extract was added to the cells along with the fluorescently labeled protein, it did.

27. Fig. 12-20

28. Using the transport of a fluorescently labeled protein with an NLS into digitonin permeabilized cells as an assay for the soluble cytoplasmic proteins required for nuclear transport, only four proteins were found to be required: Ran a small G protein similar to Ras. Importin  Importin  Nuclear transport factor 2 -- NTF2

29. K. Ribbeck and D. Gorlich (2001) EMBO J. 20:1320 = FG repeat Importin

30. NTF2 binds RanGDP and interacts with FG-repeats. NTF2 does not bind RanGTP.

31. + NTF2 +

32. Fig. 12-21

33. 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.

34. Fig. 12-22

35. Nucleus restricted hnRNPs Nuclear pores Nuclear mRNP hnRNP Protein shuttle Nucleus Cytoplasm Exchange of nuclear mRNPs proteins for cytoplasmic mRNP proteins Cytoplasmic mRNP Cytoplasmic mRNP proteinsPABPI PABPII

36. Fig. 12-23