Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function

1. Bio 525/ Spring, 2010Nuclear Architecture and Genomic Function Session 3. Nuclear Import/Export Background & Figures for Kutay et al. &Fornerod et al.

2. Mechanisms of Nuclear Import and Export Gene Expression in Prokaryotes: DNA RNA Protein Gene Expression in Eukaryotes: DNA RNAN -----RNACProtein export CYTOPLASM --------- Nuclear Proteins Shuttling Proteins NUCLEAR IMPORT/EXPORT ARE ESSENTIAL PROCESSES FOR GENE REGULATION IN EUKARYOTES AND ARE HIGHLY REGULATED PROCESSES N E NUCLEUS --------- Import Export

3. Proteins to be imported into the nucleus haveNuclear Localization Signals (NLS’s)that enable nuclear import. • NLS’sbind toimportin αsubunit of an importin α-β complex. • Transport through the NPC is mediated by interaction of degenerative sequences in the NPC proteins with theimportin β subunit. • Key to function and regulation areRAN GTP[high in nucleus byRCC1 (Ran nucleotide exchange factor)]&RAN GDP[high in cytoplasm byRAN GAP(RAN GTP activating protein)]. • The asymmetric distribution of RCC1 in the nucleus and RAN GAP in the cytoplasm drives the nuclear import process. Nuclear Import Mechanisms Kutay Paper

4. The exporting proteins have special sequences called Nuclear Export Signals (NES’s) that mediate export through binding to a class of proteins that function in export called exportins. • Exportins are typically monomeric and function in a reverse manner to importin under the control of RAN. • Thus the cargo complex requires RAN-GTP which is found only in the nucleus. • Disassociation of the ‘cargo” from the exportin requires RAN-GDP which occurs only in the cytoplasm. NUCLEAR EXPORT Fornerod paper

5. Machinery of nuclear import/export contd… • RNA Export: • In the current model, RNA export occurs by the export of multiple RNP proteins that cover the mRNA during transport. • So a more precise term is: “RNP (ribonucleoprotein) export”. • In the cell nucleus pre-mRNA is packaged into particles called pre-mRNP or hnRNP particles. protein RNA pre-mRNA RNP particle

6. Machinery of RNA Export NLS NES Shuttling protein • During RNA splicing changes occur in the proteins associated with the hnRNP particles. • Most dramatic change occurs at the moment of nuclear export where some of the RNP proteins are “nuclear restricted” and are therefore released from the RNP particles while others stay associated with the RNA. • CBC(cap binding protein complex) initiates the export of RNA from the 5’ end. • In the cytoplasm the remaining nuclear RNP proteins are removed and the RNA gets associated with cytoplasmic specific proteins which enable the mRNA to associate with ribosomes and carry out protein synthesis. • The disassociated RNP proteins are imported back into the nucleus where they associate with other pre m-RNA’s. • The RNP proteins imported back into the nucleus “shuttle” between nucleus and the cytoplasm through the NPC. • Shuttling proteins have both a NES and NLS sequences.

7. Kutay et al., “Export of Importin α from the Nucleus is Mediated by a Specific Nuclear Transport Factor” [CAS: Cellular Apoptosis Susceptibility Gene] [WHAT IS MECHANISM OF IMPORTIN α EXPORT?] 1. Re export of importin α in permeabilized cells requires another factor in addition to the reconstitution system (Figure 1) 2. Identification of importin α, CAS, importin β, Ran BP as components that bind RAN-GTP but not Ran-GDP Sepharose beads (Figure 2) 3. CAS binding to importin α requires Ran GTP (Figure 3) 4. Importin α, CAS & Ran GTP form a stable heterotrimeric complex by gel filtration (Figure 4) 5. RCC1 nucleotide exchange & GTPase activation on Ran are inhibited in the trimeric CAS/importin α/Ran GTP complex & the complex is disassembled by Ran BP and Ran GAP (Figure 5) 6. CAS can restore reexport activity of importin α in extracts (Fig 7) and is termed an “exportin”.

8. Kutay etal ., Cell 1997, Figure 1 • Reexport of importin α in permeabilized cells requires another factor in addition to the reconstitution system

9. Kutay etal ., Cell 1997, Figure 2 • Identification of importin α, CAS, importin β, Ran BP as components that bind RAN-GTP but not RAN-GDP sepharose beads BACKGROUND BASIS FOR THIS EXPERIMENT Earlier experiments showed that low levels of RAN-GTP in the nucleus (yeast mutants in RCC1 or microinjecting RAN-GAP Into Xenopus nuclei), blocks importin α export suggesting that this export is dependent on Ran-GTP

10. Kutay etal ., Cell 1997, Figure 3 • CAS binding to importin α requires Ran GTP React CAS, importin α& β together. Determine what binds to Importin α by tagging it with an IgG binding domain. Can then retrieve the importin α and anything that binds to it with IgG Sepharose and analyze what binds with SDS-PAGE

11. Kutay etal ., Cell 1997, Figure 4 • Importin α , CAS & RanGTP form a stable heterotrimeric complex by gel filtration while RanGTP & importin α do not form a complex RanGTP & importin α do not form a complex RanGTP, importin α & CAS form a complex

12. Kutay etal ., Cell 1997, Figure 5 • RCC1 nucleotide exchange & GTPase activation on Ran are inhibited in the trimeric CAS/importin α/ Ran GTP complex & the complex is disassembled by Ran BP & Ran GAP

13. Kutay et al ., Cell 1997, Figure 7(A) • CAS is a functional export factor for importin α Nuclear accumulation of fluorescein importin α and a Texas Red- labeled BSA-NLS conjugate was studied using three different extracts

14. Kutay etal ., Cell 1997, Figure 7(B) • CAS is a functional export factor for importin α Nuclear import of BSA-NLS and importin α export in the depleted egg extract supplemented with importin β as described

15. Kutay etal ., Cell 1997, Figure 7(C) • CAS is a functional export factor for importin α Nuclear import of 2 μM Texas Red nucleoplasmin and 2μM FITC importin α was allowed for 15 min in the presence of recombinant importin factors, namely importin β, Ran, RanBP1 and NTF2

16. Kutay etal ., Cell 1997, Figure 8 Scheme of the importin α transport cycle involving a heterotrimeric complex of Ran-GTP, Importin α& CAS

17. Fornerod et al., “CRM1 is an Export Receptor for Leucine-Rich Nuclear Export Signals” BACKGROUND • CRM1 was first identified in S. pombe & is a target for the cytotoxin leptomycin B {LMB} • In mammalian cells LMB blocks HIV Rev nuclear export • Human CRM1 is part of a protein family that are similar in sequence to importin β at the N-term where Ran binding occurs. Moreover, CRM1 binds the nucleoporin protein NUP 214 • This article, therefore, attempts to determine the possible role of CRM1 as an export factor or “exportin”

18. Fornerod et al., “CRM1 is an Export Receptor for Leucine-Rich Nuclear Export Signals” 1. Leptomycin B inhibits rev and U-snRNA export (Figure 1) 2. Leptomycin B binds to CRM1 but has no effect on import/export of CRM1 or CBP 80 (Figure 2) 3. hCRM1 accelerates U-snRNA export (Figure 3) 4. hCRM1 accelerates Rev export & reverses LMB inhibition (Fig 4) 5. Interaction of hCRM1 with Ran GTP requires NES peptide & this interaction is inhibited by leptomycin B (Figure 5) 6. Chemical crosslinking produces a complex(es) of hCRM1, Ran GTP & NES. Formation of this complex is inhibited by leptomycin B (Fig 6) 7. hCRM1 is “exportin 1” and a model for directional transport (Fig 7)

19. Fornerod., Cell 1997, Figure 1(A) • Leptomycin B inhibits Rev but not importin α export Microinjection of 35S labeled HIV-1 Rev and importin α proteins into the nucleusof Xenopus laevis oocytes

20. Fornerod., Cell 1997, Figure 1(B) • LMB has no effect on CB 80 or hnRNPA1 import Microinjection of 35S labeled CBP 80 and hnRNP A1 proteins into cytoplasm

21. Fornerod., Cell 1997, Figure 1(C) • Leptomycin B has no effect on DHFR mRNA or tRNA but inhibits U1 and U5 snRNA export Microinjection of 32P RNAs into the nucleus

22. Fornerod., Cell 1997, Figure 2(A) • Leptomycin B binds to CRM1 but has no effect on import/export of CRM1 35S–labeled CRM1 proteins are incubated with and without LMB and run on non-denaturing gels

23. Fornerod., Cell 1997, Figure 2(B) • Leptomycin B binds to CRM1 but has no effect on import/export of CRM1 or CBP 80 Xenopus oocytes incubated + LMB, then microinjection of 35S-hCRM1 and CBP80 into the cytoplasm

24. Fornerod., Cell 1997, Figure 3(A) • hCRM1 accelerates U-snRNA export DHFR mRNA (lanes 1-6) or hCRM1 mRNA (7-9) injected into the cytoplasm of Xenopus oocytes. After 24 hr, 32P-label-RNA injected into the nucleus and RNA extracted at 0 or 150 min

25. Forenrod., Cell 1997, Figure 3(B) • hCRM1 accelerates U-snRNA export

26. Forenrod., Cell 1997, Figure 4 hCRM1 accelerates Rev export & reverses inhibition by LMB Water (lanes 1-6, 10-12) or hCRM1 mRNA was microinjected into cytoplasm of Xenopus oocytes. After 23 hr oocytes were placed in control media (1-9) or LMB (10-15). After 2 hr 35S- Rev and transportin (TRN) were injected into the nucleus. Proteins were extracted at 0 (1-3) and 65 min (4-15)

27. Forenrod., Cell 1997, Figure 5 (A) • Interaction of hCRM1 with Ran GTP requires NES peptide & this interaction is inhibited by leptomycin B 35S-CRM1 (1-14) or importin-β (15-16) incubated with RAN-GTP beads in - (1-2, 15-16) or + (3-14) Rev NES peptide from 1-1000 µM. At 1mM, LMB (11, 12) or SDS (13,14) were added. Samples were separated into super- natant (not Ran-GTP bound) or pellet (bound) and analyzed by SDS-PAGE

28. Forenrod., Cell 1997, Figure 5 (B) • Interaction of hCRM1 with Ran GTP requires NES peptide: comparing (100μM) wt or mutant NESes that are inactive

29. Fornerod., Cell 1997, Figure 5 (C) • Interaction of hCRM1 with Ran GTP requires NES peptide & this interaction is inhibited by leptomycin B Labeled CRM1 produced with E.coli lysates instead of rabbit reticulocyte in vitro translation

30. Fornerod., Cell 1997, Figure 6 Chemical crosslinking produces complex(es) of hCRM1, Ran GTP &NES. Formation of this complex is inhibited by LMB 35S- CRM1 is incubated with combinations of Ran-GTP and NESes, X-linked with glutarald- ehyde and run on SDS- PAGE

31. Fornerod., Cell 1997, Figure 7 • hCRM1 is “exportin 1” and a model for directional transport