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Block III Lecture 7 Nucleocytoplasmic transport February 16, 2005. Maria L. Zapp, Ph.D. Program in Molecular Medicine and The UMass Center for AIDS Research. Regulation of gene expression at the level of nucleocytoplasmic transport. Prokaryotes. Eukaryotes.

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slide1
Block III Lecture 7

Nucleocytoplasmic transport

February 16, 2005

Maria L. Zapp, Ph.D.

Program in Molecular Medicine and

The UMass Center for AIDS Research

slide2
Regulation of gene expression at the level of

nucleocytoplasmic transport

Prokaryotes

Eukaryotes

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 perforated

by nuclear pore complexes (NPCs).

Adapted from Lewin, 1988

slide3
Problem:

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.

Solution:

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

slide4
Nuclear Envelope (NE) : Nuclear pore complexes, nuclear lamina, and lipid membranes

Nuclear pore complexes (NPCs)

Nuclear lamina

slide5
Possible pathways for molecular movement into the nucleus:

NPC

1. Direct passage through the nuclear pores

2. Synthesis on the 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)

Adapted from Maul, G. 1777

slide6
Micropipette filled with tracer substance

*

*

*

*

*

*

Tracer substances:

Nucleus

Tritiated ( 3H-labeled) dextrans of 3 different sizes

Radii= 12.0 ( ) 23.3 ( ) 35.5 ( )

Cytoplasm

Å

Å

Å

*

*

Longitudinal cross-section

What is the permeability of the nuclear envelope?

Microinjection assay

using X. laevis oocytes

Representative 100 mM sections

Minus tracer

Plus tracer

Oocyte

Experiment : Inject a radiolabeled tracer * into the cytoplasm of oocytes. Incubate for various

times. Quench oocytes by placing at -190oC. Prepare 100mm sections (- 50oC). Determine the

intracellular concentrations of tracer by ultra-low temperature autoradiography. Count the

grain densities.

slide7
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.

3H dextrans

Radii= 12.0 ( ) 23.3 ( ) 35.5 ( )

Å

Å

Å

*

*

*

Å

35.5

*

*

*

Å

12.0

Å

23.3

*

*

*

Schematic

representation

of the data

Time (minutes)

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

slide8
Conclusion:

The NE is a molecular sieve that

restricts molecular movement

between the nucleus and the

cytoplasm.

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.

slide9
Schematic representation of the Nuclear Pore Complex (NPC)

Protein constituents of the NPC are known as nucleoporins or “NUPs”.

slide10
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 proteins

re-equilibrate between the nucleus and the cytoplasm.

Approach and Results:

Nucleoplasmin is a pentameric

nuclear protein that contains a

protease-resistant “core” domain

and a protease-sensitive “tail”

domain.

Nucleoplasmin injected into the

cytoplasm of frog oocytes enters

the nucleus.

When the tail domain is removed by

digestion, the residual core domain

remains a pentamer and is UNABLE

to enter the nucleus. The detached

tail domains rapidly accumulate in

the nucleus, suggesting the tail

domain contains a signal for nuclear

accumulation.

Conclusion: Nuclear proteins contain nuclear-targeting signals

slide11
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 help

demonstrate that cellular proteins

and RNA are transported bidirectionally

through 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.

slide12
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.

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.

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

slide13
1 AA

852 AA

Yeast MATa2

E. colib-galactosidase

Reporter

protein

Yeast Cell

Nucleus

Cytoplasm

ii. Construction and analysis of chimeric fusion proteins

Mata2 = A yeast protein involved in mating. The protein is nuclear localized.

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

Generate a yeast expression vector : Sequences that encode Mat a2 were cloned

in frame with sequences that encode b-gal. Transform plasmid into yeast cells

and analyze the intracellular distribution of the fusion protein.

Analysis of Protein Localization

yMATa2

yMATa2-b-galactosidase

b-galactosidase

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

slide14
Summary:

1. The addition of an NLS can facilitate nuclear entry of a protein that is too large to

enter by diffusion.

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.

slide15
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.

Cytoplasm

Nucleus

+ NLS Protein

MT-NLS protein

DNLS Protein

Injection substrates:

D NLS Protein (lacks an NLS)

+ NLS Protein (contains an NLS)

MT-NLS protein (contains a mutant NLS)

slide16
GST-NLS-EGFP

hRIP

hRIP

GST-DNLS-EGFP

ii. Mammalian cell transient transfection assay

Glutathione-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-DNLS-EGFP) in mammalian tissue culture cells.

Introduce DNA into cells using standard methods (i. e. CaPO4-mediated DNAprecipitation, cationic liposomes,

DEAE-dextran or Electroporation).

Analyze the intracellular distribution of the protein using indirect fluorescence microscopy.

hRIP= Control or “Marker” protein.

hRIP is an endogenous protein that is localized at the nuclear periphery.

slide17
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.

slide18
-ATP

+ATP

Plus

Buffer

Plus

Lysate

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 :

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 import

of 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.

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

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

slide19
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-b(Imp b), and share

an 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 other

side, release the cargo, and return to their original cellular compartment to mediate

the next round of transport. 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 nuclear

import and a different cargo for nuclear export.

slide20
Ran

GTP

Ran

GTP

a

NE

b

b

a

a

K

b

K

Cargo bearing an NLS

K

Importin a

b

a

Importin b

The nuclear protein import cycle

Key adapter molecules :

1. Importin-a (Imp a) or the “NLS receptor” mediates NLS recognition.

2. Importin-b (Imp b) mediates interactions with the NPC to drive translocation of cargo.

3. A nuclear GTPase system- Ran, RCC, a Ran GAP, binding proteins 1 and 2, NTF-2

1. Imp a directly binds to the NLS of the

cargo, then interacts with Imp b.

2. Imp b docks the trimeric complex

to the NPC and mediates translocation.

3. Translocation is terminated by direct

binding of Ran-GTP to Imp b, which

releases the complex from the NPC,

and dissociates Imp a from Imp b.

4. Imp a and b are recycled to the

cytoplasm separately. Imp b / Ran-GTP

complexes leave the nucleus directly.

Imp a 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 a-like protein.

Ran also regulates these interactions.

nucleus

cytoplasm

slide21
GAP/

Rna1

Ran

GDP

Ran

GTP

Ran GTPase system: Regulation of cargo loading onto transport receptors

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 nucleotide

bound state. These regulatory proteins are localized on opposite sides of the NE: the

Ran GTPase-nucleotide Exchange Factor (GEF) is nuclear, whereas the Ran GTPase

Activating Protein (GAP) is cytoplasmic. Ran binding proteins are also cytoplasmic.

The intrinsic GTPase activity of Ran

is 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.

GTP

GDP

Ran

GTP

Ran

GDP

GEF/Rcc1

nucleus

NE

cytoplasm

RanBP1

The nucleotide state of Ran determines compartment identity

slide22
Summary

The existence of a Ran-GTP gradient provides a plausible explanation as to how

functional asymmetry can be imposed on the transport cycle.

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, where

it disassembles following GTP hydrolysis. The cargo free, Ran-GTP free exportin

can then re-enter the nucleus and bind another cargo molecule.

The release of the one cargo molecule requires energy in the form of one molecule

of GTP hydrolyzed per transport cycle.

slide23
Selectivity across the nuclear pore

Part 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.

slide24
Microinjection assay for RNA export in Xenopus oocytes

32P-labeled RNA transcript injected

into the nucleus

Nucleus

Incubate at 16oC

Longitudinal cross-sectional

view of nuclear-specific

microinjection

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

C

N

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

T

N

C

N

C

RNA of interest

Control RNA

t0

t0

t0

t2hr

t2hr

Time (t):

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

slide25
Microinjection / RNA titration assay in Xenopus oocytes.

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

Cold rRNA competitor

(pmol)

No RNA Competitor

0. 5

2. 5

5.0

N C

N C

TN C

N C

N C

32P-rRNA

T= total input RNA

C=cytoplasmic RNA

N= nuclear RNA

t = time (min)

TN C

N C

N C

N C

N C

32P-U1snRNA

TN C

N C

N C

N C

N C

32P-mRNA

t0

t45

t45

t45

t45

Time (minutes):

Summary

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.

slide26
Yeast cell

WT strain at 37oC

WT strain at 25oC

nucleus

cytoplasm

Mutant strain at 25oC

Mutant strain at 37oC

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.

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).

FISH analysis of poly A(+) RNA localization in wild

type or temperature sensitive (ts) yeast cells

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

25oC permissivetemperature

37oC non-permissive temperature

Result

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

Cole, et al., 2002.MethodsEnzymol. 351:568-587.

slide28
9kb

4kb

class

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

The Rev protein facilitates the

cytoplasmic accumulation of unspliced or incompletely spliced

HIV RNAs, which encode the viral structural proteins. In the absence

of Rev, these RNAs are retained in

the nucleus. Thus, Rev function is essential for viral replication.

Northern blot of cytoplasmic HIV RNAs

mock

WT HIV

Rev mutant

HIV

2kb

class

slide29
ARM Domain

Effector Domain

RRRRWR

LE

LPP

RLTLD

1aa

116 aa

NLS / RNA binding

Nuclear Export Signal

(NES)

Oligomerization

Functional domains of the HIV-1 Rev protein

LE = Amino acids 78 and 79 of Rev.

Note: The mutant Rev M10 protein

contains amino acid substitutions in

these residues

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, 79E

to D78, A79). This particular Rev mutant, Rev M10, is a trans-dominant negative inhibitor

of Rev function. These key observations suggested the Rev Effector domain interacts with a

cellular cofactor (s).

slide30
Rev

Model of HIV-1 Rev-Mediated RNA Export

Nucleus

Rev

RRE

AAAAA

cytoplasm

AAAAA

Rev

RRE

AAAAA

Rev

RRE Rev Responsive Element

Putative host factor

Rev’s Mechanism of Action:

Rev binds directly to the RRE within incompletely spliced viral RNAs (i.egag-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).

slide31
RNA in situ hybridization assay for studying Rev-mediated RNA export

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).

Rev

RevM10

No DNA

HIV gag-pol

HIV gag-pol

HIV gag-pol

+ probe

+ probe

+ probe

+ probe

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.

slide32
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, IkB, PKI

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

and an NES.

slide33
Rev-mediated export

Cellular RNA export

in Xenopus oocytes:

Rev NES

injection/ titration

experiments

Leptomycin B

(LMB)

Member of the importin-b

family of transport

receptors

Identification of a cellular factor that interacts with the NES:

Discovery of the nuclear export receptor

CRM 1

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-b 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 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.

slide34
Cis-Acting Export Signals on Proteins and RNA

Dreyfuss, et al., 2002. Nat. Rev. Mol. Cell. Biol. 3:195-205 Maniatis and Reed, 2002. Nature 416: 499-506.

slide35
TAP / p15 heterodimer

CTE Constitutive Transport

Element

Constitutive Transport Element (CTE)-mediated nuclear RNA export

CTE

The CTE is a cis-acting RNA element located in the 3’UTR of Mason-Pfizer Monkey Virus RNA (MPMV)

TAP

p15

AAAAA

Nucleus

cytoplasm

CTE

p15

AAAAA

CTE

TAP

p15

AAAAA

Mechanism of Action:

TAP/ p15 binds directly to the CTE to promote nuclear export of MPMV RNAs. TAP /p15 function requires an interaction with components of the cellular export machinery.

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

slide36
mRNA transport factors are recruited to the mRNA during splicing

Nascent 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.

Pre-mRNA splicing coupled export model

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

slide38
Nucleocytoplasmic Transport : 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.

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)

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.

slide39
2. Protein Shuttling

Shuttling 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, presumably

are exported to the cytoplasm bound to RNA and then re-imported into the

nucleus for another round of transport. HIV-1 Rev is an example.

“Non-Carrier proteins”- Proteins that use shuttling as a way of regulating

their activity. These proteins would be localized in the cytoplasm at

steady-state because their nuclear export is more efficient than nuclear

import. Their nuclear export is blocked under conditions in which their

activities 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.

slide40
How do you determine whether a protein shuttles between the

nucleus 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.

slide41
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 they

can be used sequentially to regulate nuclear localization. These mechanisms

can enhance or decrease the affinity of a cargo for its receptor (i.e. have a

positive or negative effect).

slide42
P

P

NLS

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-cellular

localization by modulating its rate of nuclear import and export.

NF-AT

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.

P

N

C

NLS

NES

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 genes

required 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 export

receptors, leading to nuclear accumulation of NF-AT. This model provides a simple explanation for

the 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.

slide43
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, they

associate 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.

slide44
Regulation of nuclear import of transcription factors

A

B

A. The transcription factor NF-B is maintained as an inactive complex with IB, which masks its NLS in the cytoplasm. In response to appropriate extracellular signals, IB 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.

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