Block III Lecture 6Nucleocytoplasmic TransportFebruary 13, 2006

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Maria L. Zapp, PhDProgram in Molecular Medicine The UMass Center for AIDS ResearchBiotech II Suite 207Ext. 6-4787 Maria.Zapp@umassmed.edu

Regulation of gene expression at the level of nucleocytoplasmic transport

Compartmentalization and the need for nuclear transport

One distinct characteristic of eukaryotic cells is the existence of nuclear and cytoplasmic compartments separated by a nuclear envelope (NE). The NE is a double membrane that is continuous with the ER and is perforatedby nuclear pore complexes (NPCs).

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Prokaryotes Eukaryotes

Adapted from Lewin, 1988

Cellular mRNAs, tRNAs, and rRNAs are transcribed in the nucleus and must be exported to the cytoplasm for protein translation. Conversely, nuclear proteins such as histones, pre-mRNA splicing and transcription factors are synthesized in the cytoplasm and must be imported into the nucleus to perform their functions.

Cellular RNAs and proteins are transported bidirectionally across the NE through the NPCs. This cellular process is known as “nuclear-cytoplasmic or nucleocytoplasmic transport”.

Nucleocytoplasmic transport has two distinct components: nuclear import and nuclear export.

In part, regulation relies on the controlled exchange of molecules between two compartments.

Solution:

Problem:

Hour One:

OVERVIEW OF LECTURE 6

• Permeability of the nuclear envelope• Molecular movement into the nucleus

• Nuclear protein importSignals for nuclear localizationAssays for studying nuclear import

• Identification of nuclear import receptors• The nuclear import cycle

Hour Two:• Nuclear export of biomolecules

Assays for studying nuclear export

• Identification of nuclear export receptors • mRNP biogenesis and mRNA nuclear export

Model systems for studying nuclear export (HIV & MPMV)

• Regulation of nucleocytoplasmic transport

Nuclear Envelope (NE) : Nuclear pore complexes, nuclear lamina, and lipid membranes

Nuclear pore complexes (NPCs)

Nuclear lamina

8-fold symmetry perpendicular to the membrane

Asymmetrical with respect to the plane of the NE

1. Direct passage through the nuclear pores 2. Synthesis on outer nuclear membrane (ONM) or contiguous ER followed by passage through the inner nuclear membrane (INM) 3. Synthesis in the nucleoplasm 4. Passage by diffusion through the ONM and INM 5. Passage by active transport through the ONM and the INM 6. Passage in vesicles that form from the ONM and subsequently fuse with the INM 7. Passage in vesicles formed from both nuclear membranes 8. Passage through holes in the NM (i.e. at mitosis)

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Possible pathways for molecular movement into the nucleus:

NPC

Adapted from Maul, G. 1777

What is the permeability of the nuclear envelope?

Experiment : Inject a radiolabeled tracer * into the cytoplasm of oocytes. Incubate for various times. Quench oocytes by placing at -190oC. Prepare 100m sections (- 50oC). Determine theintracellular concentrations of tracer by ultra-low temperature autoradiography. Count the grain densities.

Microinjection assay using X. laevis oocytes

Longitudinal cross-section

NucleusCytoplasm

Micropipette filled with tracer substance

**

Tritiated ( 3H-labeled) dextrans of 3 different sizes Radii= 12.0 ( ) 23.3 ( ) 35.5 ( )

*Å Å Å

***

Tracer substances:

Oocyte

Representative 100 M sections

**Minus tracer Plus tracer

Time course of nuclear permeation, expressed as the average nuclear:cytoplasmic grain density, X n/c as a function of diffusion time after injection, td (min). Vertical bars = s. e. mean.

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are needed to see this picture.35.5Å

23.3 Å12.0Å

3H-labeled dextrans Radii= 12.0 ( ) 23.3 ( ) 35.5 ( )Å Å Å

Time (minutes)

***

**

*

** *

Schematic representation

of the dataviewed in cross-

section

Paine, et al., 1975. Nature 254: 109-114.

Results:

Summary

1. These data demonstrate that the NE is less permeable to larger dextrans (>23.3 Å) than smaller dextrans (<12.0 Å).

2. The permeability of the NE plays a major role in limiting the rate of nuclear entry.

3. These classical studies suggested that the NE is a diffusion- restrictive barrier. The data are consistent with nuclear entry kinetics expected for passage through an envelope with pores.

CONCLUSION: The NE is a molecular sieve that restricts molecular movement between the nucleus and the cytoplasm.

Protein constituents of the NPC are known as Nucleoporins or “NUPs”. ≈Thirty different NUPs, each present in 8 to 56 copies/ pore. Three different classes of NUPS: pore membrane anchor proteins, FG repeat containing NUPs which span entire pore and are essential for translocation of cargo, and varied- motif structural proteins.

Schematic representation of the Nuclear Pore Complex (NPC)

N

C

B

B

N =nuclear face

C =cytoplasmic face

B = both nuclear and cytoplasmic face

Selectivity at the nuclear pore Part I: Nuclear-cytoplasmic transport of proteins

Key observations: Large proteins can enter the nucleus and remain there. Cytoplasmic proteins do not enter the nucleus, and remain localized in the cytoplasm. Some proteinsre-equilibrate between the nucleus and the cytoplasm.

Conclusion: Nuclear proteins contain nuclear-targeting signals

Approach and Results:

Nucleoplasmin is a pentameric nuclear protein that contains aprotease-resistant “core” or headdomain and a protease-sensitive“tail”domain.

Nucleoplasmin injected into the cytoplasm of frog oocytes entersthe nucleus.

When the tail domain is removed by digestion, the residual core domainremains a pentamer and is UNABLEto enter the nucleus. The detached tail domains rapidly accumulate inthe nucleus, suggesting the tail domain contains a signal for nuclear accumulation.

Nuclear protein transport occurs through the NPCs and requires ATP

Key observations: Direct visualization of intracellular migration of nucleoplasmin- coated colloidal-gold particles through oocyte NPCs using EM. Particle movement is altered dramatically by ATP depletion and low temperature. Additional EM work visualized an RNA-coated gold particle moving through the NPC to the cytoplasm.

Significance:

These oocyte-based approaches helpdemonstrate that cellular proteinsand RNA are transported bidirectionallythrough the NPC.

Conclusions:

1. The steady-state distribution of cellular proteins between the nucleus and the cytoplasm is governed by an intrinsic property of the polypeptides.2. Nuclear proteins contain specific Nuclear Localization Signals (NLS) that promote nuclear uptake.3. Nuclear protein uptake occurs via NPCs.

Bonner, et al., 1975. J.Cell Biol. 64: 431-437. Dingwall, et al., 1982. Cell 30: 449-458. Feldherr, et al., 1984. J. Cell Biol. 99:2216-2222.

The NLS of a protein selectively promotes its import into the nucleus

Approaches to identify sequences which mediate nuclear localization of proteins

i. Deletion analysis of SV40 virus large T-antigen

Construction and characterization of viral protein mutants defective in nuclear import. The first NLS was identified in SV40 large T-antigen and consists of numerous charged amino acid residues. The SV40 T-antigen sequence is the “prototype”of classical NLSs.

Lanford and Butel, 1984. Cell 37:801-813. Kalderon, et al., 1984. Cell 39: 499-509.

Immunofluorescence (IF) micrographs showing the intracellular distribution of the SV40 virus T-antigen containing or lacking a short peptide that serves as an NLS. (Left panel) The wild type T-antigen protein contains the lysine-rich sequence indicated and it is imported to its site of action in the nucleus, as shown by IF staining with an antibody against the T-antigen. (Right panel) An SV40 T- antigen protein with a mutant NLS peptide (Lys--> Thr ) remains in the cytosol.

Yeast Cell

Nucleus

Cytoplasm

Analysis of Protein Localization

-galactosidaseyMAT2 yMAT2--galactosidase

ii. Construction and analysis of chimeric fusion proteins

Generate a yeast expression vector : Sequences that encode Mat 2 were clonedin frame with sequences that encode -gal. Transform plasmid into yeast cells and analyze the intracellular distribution of the fusion protein.

Richardson, et al., 1984. Cell 44: 77-85. Hall, et al., 1984. Cell 36: 1057-1065. Goldfarb, et al., 1986. Nature 322:641-644.

Reporterprotein

852 AA

Yeast MAT2 E. coli -galactosidase1 AA

Mat2 = A yeast protein involved in mating. The protein is nuclear localized.

-galactosidase (-gal ) = A bacterial enzyme involved in metabolism. The protein is localized in the cytoplasm of yeast cells.

Summary:

1. The addition of an NLS can facilitate nuclear entry of a protein that is too large to enter by diffusion (> 40KDa).

2. Nuclear proteins contain specific amino acid sequences that selectively promote nuclear localization.

3. Additional NLS peptide competition studies in frog oocytes indicated that nuclear protein localization or “nuclear import” is a saturable process. The saturation kinetics and competition effects observed suggested nuclear protein import is a carrier-mediated process.

4. Nuclear import of proteins is a receptor-mediated process. The NLS may interact with a component of the nuclear transport machinery.

5. Large proteins may interact with cellular “receptors” for nuclear import. Specific interactions would result in a selective distribution of proteins between the nucleus and the cytoplasm.

Development of novel assays for nuclear protein import

To determine whether the protein of interest contained an NLS.To identify the molecular steps required for nuclear protein import.To identify cellular factors that mediate nuclear protein import.

i. Mammalian cell microinjection assay Inject a fluorescently-labeled protein into the cytoplasm of a mammalian cell, then determine its intracellular localization using fluorescence microscopy.

Nucleus

Cytoplasm

NLS Protein + NLS Protein MT- NLS protein

NLS Protein (lacks an NLS)

+ NLS Protein (contains an NLS)

MT- NLS protein (contains a mutant NLS)

Injection substrates:

MFP= Control or “Marker” protein.

MFP is a cellular protein localized at

the nuclear periphery.

ii. Mammalian cell transient transfection assayGlutathione-S-Transferase (GST) is an enzyme from S. japonicum. GST = 26 kDa.Green Fluorescent Protein (EGFP) is a light-converting protein from A. victoria. GST= 27kDa.Enhanced GFP (EGFP) is a variant of wild type GFP protein, which has been optimized for brighter fluorescence and high expression in mammalian cells.

•Construct plasmids for transient expression of a GST- EGFP fusion protein that contains an NLS (GFP-NLS-EGFP) or lacks an NLS (GST-NLS-EGFP) in mammalian tissue culture cells.• Introduce DNA into cells using standard methods•Analyze the intracellular distribution of the protein using indirect fluorescence microscopy.

MFPGST-NLS-EGFP

GST-NLS-EGFP MFP

iii. in vitro reconstituted nuclei.

Assemble an assay mix containing isolated intact nuclei from mammalian cells, frog egg extract, and a fluorescently labeled protein.

Results: Isolated mammalian cell nuclei import nuclear proteins efficiently when incubated in this mix, but exclude non-nuclear proteins. Nuclear import of the protein substrate displays the same characteristics for an active protein import system: a requirement for an NLS, ATP, an intact NE, and temperature dependence.

Summary

1. These three assay systems provided evidence that nuclear protein import occurs in two distinct steps: rapid binding or “docking” at the NE, followed by trans- location through the NPC.2. The binding and translocation steps can be uncoupled by incubating cells at low temperature or by treating them with inhibitors of ATP production. Translocation through the NPC is energy-dependent.3. The NPC contains multiple docking sites that guide the movement of NLS- containing proteins from the cytoplasm to the nucleoplasmic face of the NPC.4. Docking of the NLS-containing protein to the NPC, as well as its subsequent movement through the NPC requires cellular transport factors.

Newmeyer, et al., 1986. EMBO J. 5:501-510 ; J. Cell Biol. 103: 2091-2103. Richardson, et al., 1988. Cell 52: 655-664. Adams, et al., 1990. J. Cell Biol.111: 807-816. Adams and Gerace, 1991. Cell 66: 837-847. Moore and Blobel, 1993. Nature 365: 661-663; PNAS 91: 10212-10216. Melchior, et al., 1993. J. Cell Biol. 123:1649-1659. Rexach and Blobel, 1995. Cell 83: 638-692.

Fluorescently labeled (FITC) or epitope-tagged import substrate can be introduced into cells and nuclear uptake monitored microscopically. Cells are depleted of their soluble cytoplasmic components; thus re-import requires re-addition of a cytosolic fraction(s).

RESULTS:Cytosolic fractions were added to digitonin-permeabilized cells to restore nuclear importof an FITC-labeled or epitope-tagged NLS-containing protein. Fractions demonstrated to support protein import into nuclei were subfractionated to identify components of the protein import machinery. Ultimately, cytosolic fractions were replaced with purified recombinant factors for functional analysis.

Cellular factors which selectively interact with the NLS:Identification of nuclear protein import receptors

i. Development of an in vitro reconstitution assay for protein import using digitonin-permeabilized mammalian cell nuclei. This unique assay system offers several technical advantages for identifying mediators of protein import :

Adams, et al., 1990. J. Cell Biol. 111:807-816.

Plus Lysate

PlusBuffer

+ATP -ATP

ii. Chemical crosslinking of cellular proteins that bind to an NLS-containing protein.

Molecular events in nucleocytoplasmic transport

Overview

Nucleocytoplasmic transport is largely mediated by a superfamily of transport“receptors” that interact directly with the NPC. These transport receptors are related, albeit often distantly, to the cellular protein importin- (Imp ), and sharean N-terminal GTPase binding motif. Based on the direction these transport receptors carry their cargo, they are called “importins” or “exportins.” These transport receptors are sometimes referred to as “karyopherins”, a more historical nomenclature.

Transport receptors bind their cargo on one side of the NE, translocate to the otherside, release the cargo, and return to their original cellular compartment to mediatethe next round of transport. Cargo contain targeting motifs (for import or export).Specifically, importins bind cargo in the cytoplasm and release it in the nucleus; conversely, exportins bind their cargo in the nucleus and release it in the cytoplasm.

In the simplest case, the cargo is recognized directly by its cognate transport receptor. In others, cargo recognition is more complicated and requires additional “adapter”molecules. In the most complex cases, the same receptor binds one cargo for nuclearimport and a different cargo for nuclear export.

The nuclear protein import cycle

Key adapter molecules : 1. Importin- (Imp ) or the “NLS receptor” mediates NLS recognition. 2. Importin-(Imp ) mediates interactions with the NPC to drive translocation of cargo. 3. A nuclear GTPase system- Ran, RCC1, Ran GAP, Ran binding proteins 1 and 2, NTF-2

cytoplasm

nucleus

RanGTP

RanGTP

K

K

NE

K Cargo bearing an NLS

Importin Importin

1. Imp directly binds to the NLS of the cargo, then interacts with Imp .2. Imp docks the trimeric complex to the NPC and mediates translocation.3. Translocation is terminated by direct binding of Ran-GTP to Imp which releases the complex from the NPC, and dissociates Imp from Imp .4. Imp and are recycled to the cytoplasm separately. Imp Ran-GTP complexes leave the nucleus directly. Imp requires a specialized exportin (CAS 1), thus helping to explain how NLS-containing proteins remain in the nucleus.5. Proteins with an M9-like NLS bind directly to Transportin, and do not require an adapter or -like protein. Ran also regulates these interactions.

Ran GTPase system: Regulation of cargo loading onto transport receptors

RanGTP

NE

RanGDP

RanGDP

RanGTP

nucleus

cytoplasm

GEF/Rcc1

GTP GDP

GAP/Rna1

RanBP1

The intrinsic GTPase activity of Ranis activated by the concerted action of the GAP and RanBP1. Because both proteins are in the cytoplasm, Ran is in the GDP-bound form in this compartment. Conversion of Ran-GDP to Ran-GTP requires the GEF. Because the GEF is bound to chromatin, nuclear Ran is in the GTP-bound form.

The overall result of this nuclear GTPase cycle is a Ran-GTP gradient across the NE with a high concentration of Ran-GTP in the nucleus, and a low concentration in the cytoplasm.

Ran is a small nuclear GTPase that switches between a GDP- and a GTP-bound form. This switch can only be accomplished by the aid of regulators of Ran’s nucleotidebound state. These regulatory proteins are localized on opposite sides of the NE: the Ran GTPase-nucleotide Exchange Factor (GEF) is nuclear, whereas the Ran GTPaseActivating Protein (GAP) is cytoplasmic. Ran binding proteins are also cytoplasmic.

The nucleotide state of Ran determines compartment identity

Summary

Importins bind their cargo in the cytoplasm, and release them upon binding Ran-GTP in the nucleus. Importins then return to the cytoplasm as Ran-GTP complexes minus cargo. Ran-GTP must then be removed from the importins to allow binding of another cargo molecule.

Exportins bind their cargo in the nucleus forming a trimeric complex with Ran-GTP.This cargo-exportin-Ran-GTP complex is then transferred to the cytoplasm, whereit disassembles following GTP hydrolysis. The cargo free, Ran-GTP free exportincan then re-enter the nucleus and bind another cargo molecule.

Translocation of receptor-cargo via NPCs is independent of energy (i.e. facilitated diffusion). The energy requirement (NTP hydrolysis) reflects the necessity to dissociate and recycle import and export receptors. The release of one cargo molecule requires energy in the form of one molecule of GTP hydrolyzed per transport cycle.

The existence of a Ran-GTP gradient provides a plausible explanation as to how functional asymmetry can be imposed on the transport cycle.

Selectivity across the nuclear porePart II. Nucleocytoplasmic transport of RNA

RNA Cargo: 1. Messenger RNA (mRNA) transcripts must exit the nucleus to engage the protein translation machinery. 2. Ribosomal (rRNA) and transfer (tRNA) RNAs must exit the nucleus to participate in protein translation. 3. Small nuclear RNAs required for pre-mRNA splicing must exit the nucleus to undergo maturation to small ribonucleoprotein particles (snRNPs) within the cytoplasm. 4. Certain viral RNAs must exit the nucleus for viral replication.

Advances in the nuclear protein import field contributed significantly to our current understanding of nucleocytoplasmic RNA transport.

Identification of cellular factors that mediate nuclear protein import (soluble importins, insoluble NPC components).

Establishment of novel assay systems to directly analyze the movement of biomolecules between the nucleus and the cytoplasm.

Microinjection assay for RNA export in Xenopus oocytes

Isolate RNA in fractions and analyze RNA species using PAGE and autoradiography.

32P-labeled RNA transcript injected into the nucleus

Incubate at 16oC

Manually dissect into nuclear (N ) and cytoplasmic (C ) fractions.

Nucleus

Time (t): t0 t0 t0 t2hr t2hr

RNA of interest

N CT N C

Control RNA

T= total RNA injected N= nuclear RNA fraction C = cytoplasmic RNA fraction

Longitudinal cross-sectional view of nuclear-specific

microinjection

N C

1. Similar to nuclear protein import, cellular RNA export is a saturable, carrier-mediated, energy dependent process. 2. Competition studies using this assay system indicate that specific factors are required for export of an individual class of cellular RNAs, and that such factors may be limiting. Conversely, nuclear export of the different classes of cellular RNAs may require common or shared factors which are not limiting.

Summary

Microinjection / RNA titration assay in Xenopus oocytes.

T= total input RNAC= cytoplasmic RNAN= nuclear RNA t= time (min)

32P-rRNA

Cold rRNA competitor

32P-U1snRNA

N C

0. 5

t0 t45

32P-mRNA

t45

T N C N C

Time (minutes):

T N C N C

T N C N C

2. 5 5.0

N C N C

N C

N C

N C N C

(pmol)

N C N C

t45 t45

No RNA Competitor

Purpose: To determine whether different classes of RNAs use the same or different export pathways.

Approach: Test whether export of a specific class of RNA is affected by the presence of increasing amounts of an RNA competitor.

Results

Genetic analysis of nuclear RNA export in budding yeast

Yeast genetic approaches facilitated the identification and functional characterization of cellular factors that mediate nuclear RNA export.

Approaches: i. Development of temperature sensitive (ts ) mutant strains ii. Synthetic lethality screens for transport-defective strains.

Cole, et al., 2002. Methods Enzymol. 351:568-587.

Yeast cell

nucleus

cytoplasm

FISH analysis of poly A(+) RNA localization in wild-type or temperature sensitive (ts) yeast cells

WT strain at 25oC WT strain at 37oC

Mutant strain at 25oC Mutant strain at 37oC

Example approach i.Incubate yeast cells with a chemical mutagen, and screen for mutants defective in mRNA export at the non-permissive temperature (37oC) using fluorescent RNA in situ hybridization (FISH).

Strains defective in mRNA export accumulate poly A(+) RNA in the nucleus at 37oC, but not at 25oC.

poly A (+) RNA visualized using a FITC-conjugated oligo probe complementary to the poly A tail (i.e. FITC-oligo dT (52))

25oC permissive temperature 37oC non-permissive temperature

Result

Retrovirus Lifecycle

Nuclear export of

viral RNAsis essential

for viral replication

HIV-1 Rev-mediated nuclear export as a model system to study RNA export

The Rev protein facilitates the cytoplasmic accumulation of unspliced (US) or incompletely spliced (IS) HIV RNAs, which encode the viral structural proteins. Completely spliced (CS) HIV RNAs are not targets for Rev function. In the absence of Rev, these RNAs are retained in the nucleus. Thus, Rev function is essential for viral replication.

9kb

4kbclass

2kbclass

mock WT HIV Rev mutant HIV

Northern blot of cytoplasmic HIV RNAs

US

IS

CS

US

IS

CS

Functional domains of the HIV-1 Rev protein

i. In vitro binding assays demonstrated that Rev contains an arginine-rich motif (ARM) which binds, in a sequence-specific manner, to a cis-acting RNA sequence known as the Rev Responsive Element (RRE). The RRE is located in the second intron of unspliced (i.e. gag-pol) or incompletely spliced (i.e. env) viral RNAs.

ii. Genetic analysis in mammalian cells identified a second functional domain, a leucine-rich “Effector” domain. Point mutations within its coding sequences abolish Rev function (L78, 79Eto D78, A79). This particular Rev mutant, Rev M10, is a trans-dominant negative inhibitorof Rev function. These key observations suggested the Rev Effector domain interacts with acellular cofactor (s).

RRRRWR LPPLERLTLD1aa 116 aa

Nuclear Export Signal (NES)

Effector DomainARM Domain

NLS / RNA bindingOligomerization

LE = Amino acids 78 and 79 of Rev. Note: The mutant Rev M10 protein contains amino acid substitutions in these residues

Rev’s Mechanism of Action:

Model of HIV-1 Rev-Mediated RNA Export

Rev binds directly to the RRE within incompletely spliced viral RNAs (i.e gag-pol and env ).The Rev effector domain interacts with cellular factors which mediate RNA export.

Rev M10 does not support viral replication and does not promote the cytoplasmic accumulation of RRE-containing viral RNAs. The inability of Rev M10 to exit the nucleus was shown to correlate with its inability to support Rev function. Thus, the Rev effector domain contains a “Nuclear Export Signal” (NES).

AAAAARRE

RRE

AAAAA

Nucleus

Rev AAAAA

Rev

Rev

RRE

RevRev

RRE = Rev Responsive Element

HIV Rev protein

Putative host factor

RNA in situ hybridization assay for studying Rev-mediated RNA export

Additional experimental approaches that have been developed for analyzing Rev function: 1. HIV-1 or chimeric HIV-based genetic analysis. 2. Transfection assays using an Rev-dependent reporter construct. 3. Oocyte microinjection using recombinant Rev protein or peptides. 4. Yeast-based colorimetric assays using a Rev-dependent reporter construct.

Sanchez-Velar et al., 2004. Genes & Devel. 18: 23-34; Meyers and Malim, 1994. Gene s & Devel. 8:1538-1547; Hope, et al., 1990. J. Virol. 91 :1231-1238.

HIV gag-polRev

HIV gag-polRevM10

No DNA HIV gag-pol

Approach: Mammalian cells are transiently transfected with a plasmid that expresses an RRE-containing HIV RNA (gag-pol) in the absence or presence of a Rev expression plasmid (Rev). The intracellular

distribution of these RNAs is analyzed by fluorescent RNA in situ hybridization(FISH) using a Cy3-conjugated oligo probe that is complementary to the RRE RNA.

Note: Cy3 is an orange fluorescing cyanine dye that produces an intense red signal easy detected using a rhodamine filter (660nm).

+ probe + probe + probe + probe

Summary

RNA export can be viewed as a protein process associated with an RNA cargo.

HIV-1 Rev-mediated and certain classes of cellular RNAs require NES-containing proteins as RNA transport cofactors.

HIV-1 Rev-mediated and cellular RNA pathways share one or more dedicated components.

Several cellular proteins contain leucine-rich NESs: TFIIIA, IB, PKI

Unique NES in the hnRNP A1 protein, the M9 domain, acts as an NLSand an NES.

Evidence :1. Leptomycin B (LMB), a lipophilic antibiotic, was shown to block Rev or Rev- dependent RNA export in HeLa cells.2. LMB had been previously shown to be toxic to fission yeast. The molecular target of LMB is the CRM1 gene; mutants resistant to LMB map to that gene.3. Immunoprecipitation studies revealed that human CRM1, a member of the importin- protein family, interacts directly with NUP 214/CAN.

Collective data from mammalian cell-based assays, oocyte microinjection studies, and genetic screens in yeast demonstrated CRM1 /Xpo1 is the nuclear export receptor (NER) for Rev. Additional studies showed CRM1 is the NER for cellular and viral proteins that contain a leucine-rich NES; nuclear export of these proteins is inhibited by LMB.

Identification of a cellular factor that interacts with the NES: Discovery of the nuclear export receptor

CRM 1

Rev-mediated export

Rev NES

Cellular RNA export

in Xenopus oocytes:

injection/ titration experiments

Leptomycin B (LMB)

Member of the importin-family of transport

receptors

Constitutive Transport Element (CTE)-mediated nuclear RNA export

Hammarskjold, M.L. (2001). Curr. Top. Microbiol. Immunol. 259: 77-93.

Mechanism of Action:TAP: p15 binds directly to the CTE to promote nuclear export of MPMV RNAs. TAP: p15 function requires interaction with a subset of FG-containing NUPs and other components of the cellular export machinery. The TAP:p15 complex was subsequently shown to mediate the nuclear export of cellular poly (A) + mRNA.

CTE Constitutive Transport Element

TAP : p15 heterodimer

CTE = cis-acting RNA element located in the 3’UTR of Mason-

Pfizer Monkey Virus RNA (MPMV)

CTE

AAAAA

CTE

AAAAA

CTE

Nu

cleu

s

TAP p15

p15

AAAAA

AAAAATAPp15

TAPp15

Cis-acting Elements and Trans-acting Factors Mediating Nuclear Export

Pemberton &Paschal. (2005). Traffic 6:187-198; Rodriguez, et al. ( 2004). Bio Cell 96: 639-655

Pre-mRNA splicing coupled export model

Adapted from Reed , R. and Magni, K. (2001). Nat Cell Biol. 3 :E201-4.

mRNA transport factors are recruited to the mRNA during splicingNascent pre-mRNAs are packaged into hnRNPs. During spliceosome assembly, exons are packaged by non-hnRNP spliceosome components such as SR proteins. After splicing, hnRNP particles remain associated with the introns, which are retained in the nucleus. Partial or mutant pre-mRNAs unable to enter the splicing pathway are also retained in packaged hnRNPs. In contrast, the spliced mRNP is targeted for export by factors recruited during splicing, in particular the export factor Aly/REF. The spliced mRNA is exported by a conserved machinery composed of non-hnRNP factors such as TAP/p15, hGle1, hGle 2, and hDbp5.

TAP/ p15 heterodimer

can be recruited to

poly (A) + mRNA

by different adapter proteins

Cellular Factors Implicated in mRNA Export

Adapted from Conti, E. and Izaurralde, E. (2001). Curr. Opin. Cell. Biol. 13: 310-320.

•No members of the importin-

family of proteins

involved in mRNA export

•TAP/p15 heterodimer

does not bind Ran GTPase

Deposition of the Exon-Junction Complex (EJC)

Formation of an export competent mRNPcomplex requires:

• Highly dynamic rearrangements of

protein: proteinand protein:RNA

interactions

•Dissociation of retention factors and association

of signals for interaction with the export

machinery

•Involves RNA binding proteins, NUP

interactions, and ATPase/ RNA helicases

Recent studies indicate that the formation of an export competent mRNA is coupled to

mRNA biogenesis (i.e. transcription, 5’ capping, 3’ end formation, pre-mRNA splicing , and mRNA surveillance.

1. To regulate a given response

2. To communicate cytoplasmic and nuclear events allowing cells to respond to environmental changes or cell cycle position

3. To generate a more robust molecular switch or affect its nature (i.e on / off)

Nucleocytoplasmic Transport : Regulation

Two important issues concerning regulated nuclear translocation

1. Steady-State Localization of a Cellular Protein. The steady-state distribution of a protein is determined by its relative rate of nuclear import and export. Changes in the rate of import or export can lead to a shift in the steady-state localization of the protein. Since both import and export can be regulated, it is essential to experimentally observe import in the absence of export (or vice versa ) to determine which rate is subject to regulation.

Eukaryotic cells control many biological processes by regulating the movement of macromolecules in and out of the nucleus. Similar to other steps in gene expression, nucleocytoplasmic transport may be subject to positive or negative regulation.

2. Protein ShuttlingShuttling proteins move continuously between the nucleus and the cytoplasm.The steady-state localization of a shuttling protein reflects a dynamic process of nuclear entry and exit.

To date, two classes of shuttling proteins have been identified:

“Carrier proteins” - Proteins associated with hnRNP particles, presumablyare exported to the cytoplasm bound to RNA and then re-imported into thenucleus for another round of transport. HIV-1 Rev is an example.

“Non-Carrier proteins”- Proteins that use shuttling as a way of regulatingtheir activity. These proteins would be localized in the cytoplasm at steady-state because their nuclear export is more efficient than nuclearimport. Their nuclear export is blocked under conditions in which theiractivities are required in the nucleus.

Thus, protein shuttling as a mode of regulation may be important for coordinating nuclear and cytoplasmic events. Additionally, it offers a simple, reversible, and rapid mechanism for regulating nuclear activity.

How do you determine whether a protein shuttles between thenucleus and the cytoplasm: A heterokaryon assay

Schematic representation of approaches for detecting nucleoplasmic shuttling of proteins.

(A) Migration of fluorescently labeled (FITC) or epitope-tagged nuclear proteins in interspecies heterokaryons.

(B) Antigen-mediated nuclear accumulation of antibodies injected into the cytosol.

In both types of experiments, cyclohexamide (CX) was used to distinguish the migration of pre-existing proteins from the contribution of newly synthesized proteins. Nuclear protein export in this assay is sensitive to LMB treatment.

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

Possible steps in nuclear translocation that could be targets for regulation

1. The binding of the cargo to an import or export receptor.2. The activity of the soluble transport machinery.3. The NPC can be modified to affect its transport properties.4. The cargo-receptor complex can be tethered to an insoluble component, thereby preventing it from binding to the NPC.

Regulation of Cargo-Receptor Complex Formation

i. Phosphorylation: Regulate the affinity of a cargo for its transport receptor, thus regulating the sub-cellular localization of the cargo.

ii. Intermolecular Association: Regulate cargo interactions with accessory adapter proteins.

Note: These modes of regulation are not mutually exclusive because theycan be used sequentially to regulate nuclear localization. These mechanismscan enhance or decrease the affinity of a cargo for its receptor (i.e. have apositive or negative effect).

Nuclear Factor of Activated T-Cells (NF-AT): A Cellular Factor Whose Function is Regulated at the Level of Nucleocytoplasmic Transport

Mode of Regulation: Phosphorylation and molecular associations affect its sub-cellularlocalization by modulating its rate of nuclear import and export.

Dephosphorylation of NF-AT results in formation of a dephosphorylated NF-AT/ calcineurin complex. Once formed, the complex translocates into the nucleus and facilitates transcription of genesrequired for T-cell specific activation.Phosphorylation of NF-AT inhibits its nuclear import rate by inducing an intra-molecular conformational change that makes the NLS inaccessible for receptor binding. Calcineurin maintains NF-AT in its unphosphorylated form, leading to a decrease in its rate of nuclear export.

Direct binding and masking of the NF-AT NES by calcineurin inhibits its association with exportreceptors, leading to nuclear accumulation of NF-AT. This model provides a simple explanation forthe observation that NF-AT/calcineurin is imported to the nucleus as a complex.

Kaffman and O’Shea (1999.Annu. Rev.Cell.Dev. Biol. 15: 291-339.

Stimulation of T-cell receptors leads to activation of signal transduction pathways which induce cytokines and cell surface molecule gene express-ion. T-cell receptor stimulation also causes an elevation in cytosolic Ca2+ levels, which activates the phosphatase Calcineurin . Active calcineurin leads to dephosphorylation of NF-AT.

PP P

NLS NES NLSN C

NF-AT

Transport of small nuclear RNAs (snRNAs) between the nucleus and the cytoplasm

Regulation by localization

snRNAs (U1, U2, etc.) are transcribed in the nucleus and exported to the cytoplasm in a CRM1-dependent fashion. In the cytoplasm, theyassociate with SM proteins to form small nuclear ribonucleoprotein

particles (snRNPs). The assembled snRNPs are then imported back into the nucleus, the site of their function.

Regulation of nuclear import of transcription factors

A B

A. The transcription factor NF-B is maintained as an inactive complex with IB, which masks its NLS in the cytoplasm. In response to appropriate extracellular signals, IB is phosphorylated and degraded by proteolysis, allowing the import of NF-B to the nucleus.

B. In contrast, the yeast transcription factor SW15 is maintained in the cytoplasm by phosphorylation in the vicinity of its NLS. Regulated dephosphorylation exposes

the NLS and allows SW15 to be transported into the nucleus at the appropriate stage of the cell cycle.