Nuclear Function and Trafficking
Lecture Outline:
1. The Nucleus in Context
A. Compartmentalizing DNA into the nucleus
B. Origin of the nucleus
C. ER is continuous with nuclear membrane
2. Organization and Function of the Nucleus
A. Comparison of complexity of transcription/translation in eukaryotes & prokaryotes
B. DNA synthesis
C. RNA synthesis and processing
D. Non-membraneous compartments in the nucleus
E. Splicing
F. Ribosome assembly
G. Processing of eukaryotic mRNA for export
3. The Nuclear Membrane
A. Function
B. Structure
4. The Nuclear Pore Complex
A. Structure of the NPC
B. Molecular components of the NPC
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Lecture Outline, cont.:
5. Trafficking through the Nuclear Pore
A. Functional relevance
B. Mechanism of nuclear trafficking
C. Mechanism of nuclear import
D. Mechanism of nuclear export
E. Shuttling proteins
F. Specific nuclear export proteins
G. Specific nuclear export pathways
6. Experimental Approaches used to Study Nuclear Trafficking
7. Examples from Pathobiology
A. Regulation of NF-kB transport
B. NFAT, a shuttling protein
C. How HIV-1 exploits cellular trafficking machinery for genomic RNA export
D. How other retroviruses exploit cellular trafficking machinery for RNA export
E. How nuclear import machinery is exploited by adenovirus for viral entry
F. Herpesvirus egress
8. References
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1. The Nucleus in Context:
Prokaryotic cell
A. Compartmentalization of the nucleus:
A key feature of eukaryotic cells
1. Eukaryotic but not prokaryotic cells
contain a nucleus that sequesters DNA
2. Other differences between eukaryotes and
prokaryotes:
Larger cell volumes in eukaryotes
Presence of cytoskeleton in eukaryotes
Elaborate set of internal membranes and
organelles in eukaryotes
Larger amounts of DNA in eukaryotes,
and more regulatory non-coding DNA
Metabolism of eukaryotes is dependent
on oxidation occurring in
mitochondria
Eukaryotic cell
3. Note: single cell eukaryotes are called
protists & include yeast and protozoa
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1. The Nucleus in Context, cont.:
B. Origin of the Nucleus: Most likely, from invagination of PM of an ancient
bacterium. Note: Topology of a compartment can be determined from its
evolutionary origins. Thus the interior of the nucleus is the topological equivalent
of the cytoplasm.
C. The ER is continuous with the nuclear membrane. ER lumen is continuous
with the space between the inner and outer membrane, and topologically equivalent
to the extracellular space.
Evolution of the Eukaryotic Nucleus
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2. Organization and Function of the Nucleus
A. Comparison of Complexity of Transcription/Translation in Eukaryotes and Prokaryotes
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2. Organization and Function of the Nucleus
B. DNA replication: in mammalian cells occurs in clustered sites throughout nucleus
C. RNA synthesis and processing:
Types of RNA:
Ribosomal RNAs (rRNA )= made/processed/assembled in nucleolus; made by RNA
Pol I and III; constitutes 80% of RNA in some cells; methylated using snoRNAs
Small nucleolar RNAs (snoRNA)= guide RNAs used for processing rRNAs, snRNAs
Messenger RNA (mRNA) = made by Pol II; are processed in nucleus (see below), then
exported and undergo translation in cytoplasm
Transfer RNA (tRNA) = ~80 nt; synthesized by Pol III; different ones for different aa’s,
aminoacylated then export to cytoplasm; provide aa’s for translation
Small nuclear RNAs (snRNA) = < 200 nt; components of the spliceosome; U1-U6
SnoRNAs are used as guides
in the processing of rRNA
precursors
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tRNA structure
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2. Organization and Function of the Nucleus, cont.
D. Non-membraneous compartments in the nucleus:
Nucleolus: Site where rRNAs, tRNAs and some snRNPs are synthesized,
processed, and assembled into RNPs. Also contains snoRNAs that act as
guides for RNA modifications. Site of assembly of ribosomes.
Cajal bodies/ GEMS: sites where snRNAs and snoRNAs undergo modification and
final assembly events.
Speckles or interchromatin granular clusters: storage site for mature snRNPs
Compartments in the Nucleus:
April 4, 2006
Bright pink = Cajal bodies
Dark pink = nucleoli
Green = speckles
Blue = bulk chromatin
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2. Organization and Function of the
Nucleus, cont.
E. Splicing:
1. UsnRNAs:
Transcribed in nucleolus, exported via
Crm1 pathway to cytoplasm where
they associate with snRNP
proteins (see below).
Import back into nucleus for final
modification and assembly in
Cajal bodies and Gems.
Storage in nuclear speckles.
2. Machinery consists of:
5 small nuclear RNAs (snRNAs),
U1, 2, 4, 5, 6
Each snRNA is complexed with
proteins to form an snRNP
3. Critical snRNP proteins include
SMN, mutations in which cause
spinal muscular atrophy
Gemins
Sm proteins
4. snRNPs form the spliceosome core
Biogenesis of UsnRNPs
From Dreyfus et al., Current Opinion in Cell Bio 14: 305 (2002)
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2. Organization and Function of
the Nucleus, cont.
F. Ribosome assembly:
1. Nucleolus is not surrounded by a
membrane.
2. Three rRNAs (5.8S, 18S, and 28S) are
transcribed as a unit and then
processed within nucleolus by RNA
pol I.
3. One rRNA (5S) is transcribed outside
nucleolus by RNA pol III & is brought
into nucleolus for assembly.
4. Ribosomal proteins translated in
cytoplasm & transported to
nucleolus.
5. Assembly of ribosomal proteins with
rRNA occurs in nucleolus.
6. Export of fully assembled large and
small ribosomal subunits to the
cytoplasm
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2. Organization and Function of the Nucleus, cont.
G. Processing of Eukaryotic mRNA for Export. Only a small percentage of pre-mRNAs are
exported. Events that mature mRNAs must undergo in order to be exported include:
1. Marking of exon boundaries with SR proteins
2. Proper splicing of introns
3. Addition of 5’ methyl-guanosine CAP by 3 enzymes
4. Addition of 3’ poly A tail by cleavage and polyadenylation specificity factors
5. hnRNPs that help distinguish between mature and immature mRNAs: some
stay in nucleus, some exit with mRNA into cytoplasm
6. Exit via interaction with specific nuclear transport receptors (i.e. TAP)
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3. The Nuclear Membrane
A. Function of the Nuclear Membrane:
•
•
•
1. Separation of transcription from
translation allows for posttranscriptional processing.
2. Limiting access of genome by proteins
allows regulation of transcription.
3. Acts as a barrier for the passage of
molecules between nucleus and
cytoplasm.
B. Structure of the Nuclear Membrane:
•
•
•
•
•
•
1. TWO lipid bilayers: outer nuclear
membrane (OM) and inner nuclear
membrane (IM).
2. Outer nuclear membrane is connected to
the ER.
3. Perinuclear space is continuous with the
ER lumen.
4. Different composition of OM and IM, with
OM similar to ER membrane.
5. IM and OM are joined at the nuclear pore
complex.
6. Nuclear lamina underlying IM provides
structure & is site of chromatin
attachment.
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4. The Nuclear Pore Complex - Except during mitosis, essentially all traffic
between nucleus and cytoplasm in higher eukaryotes occurs via nuclear
pore complexes.
A. Structure of the Nuclear Pore Complex (NPC):
1. Definition: channels for trafficking of small polar molecules, ions, &
macromolecules.
2. Size: diameter = 120 nm, 8-fold rotational symmetry; molecular mass =
125 million daltons; composed of 50 -100 different proteins called
nucleoporins.
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4. The Nuclear Pore Complex, cont.
A. Structure of the Nuclear Pore Complex (NPC), cont.:
3. Number: Average cell has over 4000 NPCs.
4. Channel: Pathway for free diffusion is 9 nm x 15 nm. Can open to a diameter of more
than 25 nm allowing passage of large complexes.
5. Passage across the NPC: Proteins < 40 kD pass through the NPC by diffusion, but
this is often inefficient. Larger proteins and RNA pass thru NPC only selectively
(Gated Transport).
6. Microscopic structure:
Eight spokes around a central
channel connected to a ring
anchored into nuclear
membrane (on cytoplasmic and
nuclear sides).
Filaments extend from both
sides forming basket on
nuclear side.
NPC extensions may be
initial cargo-docking sites
during nucleo-cytoplasmic
transport. A subset of NPC
components contain
phenylalanine glycine (FG)
repeats that act as docking
sites for transport factors.
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From Alberts,Molecular Biology of the Cell, Fig. 12-10
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Trafficking In and Out of The Nucleus
4. The Nuclear Pore Complex, cont.
B. Molecular components of the NPC:
1. Nucleoporins (NPs):
Make up the cytoplasmic filaments, nuclear
basket, and line the pore
Contain different types of FG (Phe/Gly)
repeats
Nuclear transport factors make multiple
sequential contacts with distinct NPs,
resulting in docking and translocation of
nuclear transport complexes
Exact mechanism by which NPs mediate
translocation of complexes unclearbut may
involve sequential association with of
receptor plus cargo with FG repeats lining
pore
From Bayliss, R. et al. Traffic 1:448 (2000)
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4. The Nuclear Pore Complex, cont.
B. Molecular components of the NPC, cont.:
2. Soluble nuclear transport receptors in cytosol and nucleus:
Nuclear transport receptors include karyopherins
Bind to nucleoporins (NPs)
Different factors for export and import, i.e. Exportin & Importin, see below
Nuclear transport factors bind to cargo (or adaptors that bind cargo) that
have localization or export signals (NLS, NES)
Cargo can be protein (with NLS, NES) or RNA
Nuclear transport factors bind to and are regulated by RanGTP
J. Lingappa, 2003
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4. The Nuclear Pore Complex, cont.
B. Molecular components of the NPC, cont.:
3. Ran-GTP:
A small GTPase that gives directionality to nuclear transport, see below
Members of the Ras oncogene family (like Rab, which regulates vesicle fusion)
Binds to nuclear transport receptors
4. Lamins make up the nuclear lamina:
Lamins are 80 kD proteins related to intermediate filaments of cytoskeleton
Form dimers that polymerize to form filaments and a lattice
Associate with the inner nuclear membrane via prenylation
Give structural support to the nucleus and may be involved in recruitment of nuclear
envelope components
Structure of the nuclear lamina
From Hutchison, Nature Rev. Mol. Cell Bio. 3: 849 (2002)
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5. Trafficking through the Nuclear Pore:
A. Functional relevance of trafficking:
1. Bring into nucleus transcription factors, proteins for ribosome and
spliceosome assembly, and other proteins needed for nuclear
functions.
2. Export RNAs and ribosomes out of nucleus in a regulated manner. Each
is exported via a specific pathway.
3. Shuttling of cellular proteins that go back and forth between nucleus and
cytoplasm (nuclear transport receptors, HnRNPs, etc.).
4. Pathogens (mainly viruses) usurp nuclear trafficking machinery:
Viral genome import into and export out of the nucleus
Virus entry into nucleus
Virus exit from nucleus
Shuttling proteins encoded by viruses
5. Pathogens can also destroy cellular nuclear trafficking machinery.
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5. Trafficking through the Nuclear Pore, cont.:
B. Mechanism of Nuclear Trafficking:
1. Ran GTPase
a. Little hydrolysis or exchange activity on its own.
b. Regulated by GTPase-activating protein (RanGAP) that promotes hydrolysis that is
predominantly in cytoplasm and guanine exchange factor (RanGEF) that promotes
GTP exchange that is predominantly in the nucleus
c. So, in the cytoplasm Ran exists in inactive GDP-bound form
d. In contrast, in the nucleus, Ran is largely bound to GTP
e. Ran gradient is critical for most nuclear transport, except bulk mRNA transport which
is mediated by factors that are not karyopherins
f. Ran gradient is created by having RanGap (which promotes GTP hydrolysis) only in
cytoplasm and RanGEF (which promotes loading of Ran with GTP) only in nucleus
from Nalkieny and Dreyfus, Cell 99:677 (1999)
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Trafficking In and Out of The Nucleus
5. Trafficking through the Nuclear Pore,
cont.:
C. Mechanism of Nuclear Import:
Nuclear Import
a. Cargo and/or adaptors encode nuclear
localization signals (NLS)
b. NLS can be composed of short stretches
of basic amino acids, not always
contiguous (can be bipartite). Other
types of NLS exist.
c. NLS containing proteins (NLS proteins)
bind directly or via an adaptor to nuclear
import receptors (i.e. Importin)
d. Cargo/adaptors bind to import receptors,
some of which are karyopherins
(importins), in the absence of RanGTP.
Note that protein cargo is completely
folded (unlike in mitochondrial import).
e. Importin composed of an a subunit that
binds NLS and a b subunit that binds
nucleoporins in cytoplasmic filaments.
f. After translocation thru NPC, import
receptors release cargo upon binding to
Ran-GTP in nucleus.
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Trafficking In and Out of The Nucleus
5. Trafficking through the Nuclear Pore, cont.:
D. Mechanism of Nuclear Export:
a. Cargo and/or adaptors encode nuclear export signals (NES)
b. NLS often composed of leucine rich regions.
c. NES-containing proteins bind nuclear export receptors, some but not all of which are
karyopherins (i.e. exportin).
d. Nuclear export factors require RanGTP to bind cargo in nucleus.
e. Nuclear export factors release cargo and Ran-GTP upon translocation to cytoplasm
where hydrolysis of RanGTP is induced by Ran-GAP.
Nuclear Import
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Nuclear Export
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5. Trafficking through the Nuclear Pore, cont.:
E.
1.
2.
3.
4.
Shuttling Proteins (includes HIV-1 Rev, Vpr, and many other viral proteins):
Proteins contain both nuclear localization and export signals (NLS + NES).
These proteins shuttle back and forth between nucleus and cytosol.
Rate of export and import determines in which compartment it resides.
Export/ import of shuttling proteins can be regulated, i.e. by
phosphorylation-dephosphorylation of residues adjacent to
NLS or NES signals resulting in blockade/exposure of signals.
from Nalkieny and Dreyfus, Cell 99:677 (1999)
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5. Trafficking through the Nuclear Pore, cont.:
F. Specific Nuclear Export Proteins:
1. Crm1 (Exportin), a karyopherin, mediates export of most cellular proteins that
shuttle, several U snRNAs, all rRNAs, and a subset of mRNAs.
Crm1 discovered because it is required for the nuclear export of late HIV-1
mRNAs (see below), other lentiviruses, and HTLV
Retroviral Rev and HTLV Rex proteins are adaptor proteins that recruit Crm1 to
retroviral mRNA
Crm1 pathway specifically inhibited by leptomycin B (LMB) which modifies a
cysteine residue present on the Crm1 protein
2. NXF proteins (include Tap) are not karyopherins; these mediate export of most
mRNAs. MPMV and other simple retroviruses use Tap/Nxt as export factors.
However, unlike HIV-1 and lentiviruses that encode adaptor proteins like Rev
that recruit Crm 1, simple retroviruses encode a cis-acting RNA sequence
(consititutive transport element; CTE) that recruits NXF protein transport
factors
3. Exp-t (Exportin t), a karyopherin, mediates export of tRNA by binding directly to
tRNA unlike other karyopherins that bind RNA via adaptors; only binds mature
/ aminoacylated tRNAs
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5. Trafficking through the Nuclear Pore, cont.:
G. Specific Nuclear Export Pathways:
from Cullen
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Trafficking In and Out of The Nucleus
6. Experimental approaches used to study nuclear trafficking:
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•
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•
•
A. Permeabilized cells/ Cell-free assay:
Digitonin perforates PM (because of high cholestrol conc.) releasing cytosol
Nuclear membrane, nucleus, and other organelles remain intact
Allows add back of cytosolic fractions, biochemical manipulations, & antibody
blockade
B. Immunofluorescent Tags:
Transfect cells with proteins tagged with GFP, RFP, YFP, etc. Assess nuclear vs.
cytoplasmic location by IF.
C. Use of chimeric proteins:
Import/Export signals can be engineered into heterologous proteins (usually
encoding fluorescent tags) to demonstrate effect of the signal
Or, fuse an adaptor onto a cargo that doesn’t bind transport receptor
D. Heterokaryon assay:
For proteins that are predominantly nuclear and shuttle in and out of nucleus.
Nuclei transfected/injected to express tagged protein (+ marker protein that stays
in "donor" nucleus)
Cells are then fused to another set of cells using PEG.
Examine whether tagged protein shows up in cytoplasm alone (export), or in
recipient nucleus as well (export followed by import, implying
shuttling).
E. Biochemical Fractionation:
Ultracentrifugation to separate nuclei from cytoplasm
F. Inhibitors: Leptomycin B
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7. Examples from Pathobiology
A. Regulation of NF-kB Nuclear
Transport:
1. Masking NLS by binding with an
inhibitory protein: e.g. transcription
factor NF-kB.
•
I-kB is an inhibitory protein that
forms an inactive complex with NFkB in cytosol.
•
I-kB binding masks the NLS of NFkB, preventing its translocation into
the nucleus.
•
Upon stimulation of lymphocytes,
I-kB is phosphorylated and
ubiquitinated resulting in
proteasome-mediated degradation
of I-kB.
•
Once released from I-kB, NF-kB
can translocate into nucleus &
activate transcription.
2. Note that for other proteins, NLS
can be masked by direct
phosphorylation of residues
adjacent to the NLS.
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7. Examples from Pathobiology, cont.:
B. Regulation of NF-AT, a shuttling protein:
Phosphorylated NF-AT is found in the cytosol of resting T cells. T cell activation
leads to increased intracellular Ca+2 and dephosphorylation of NF-AT (via calcineurin,
a phosphatase). This exposes an NLS and possibly masks an NES, resulting in
nuclear import and activation of transcription by NF-AT.
Decreased Ca+2 leads to re-phosphorylation of NF-AT which inactivates the NLS
and re-exposes the NES, causing NF-AT to relocate to the cytosol.
Some immunosuppresive drugs act by blocking the ability of calcineurin to
dephosphorylate NF-AT and thereby block nuclear transport of NF-AT.
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7. Examples from Pathobiology, cont.:
C. How HIV-1 exploits cellular machinery for genomic RNA trafficking:
•
•
•
1. Cellular RNAs: almost always exported from the nucleus in a fully
spliced form (albeit sometimes spliced in alternate ways), with retention of
incompletely spliced RNA in nucleus. Question: why is this important for
the cell?
2. But HIV-1 exports incompletely spliced RNA out of nucleus. How?
a. HIV-1 produces multiply-spliced mRNAs encoding Tat, Rev, Nef
(early proteins); singly-spliced mRNAs encoding Vif, Vpr, Vpu, and Env
(regulatory proteins); and an unspliced mRNA encoding Gag & GagPol (late
proteins).
b. Initially, only multiply-spliced mRNAs are exported out of the
nucleus, resulting in translation of Tat, Rev, and Nef.
c. Rev is transported back into nucleus, and binds to RRE stem loop
present in HIV-1 singly and multiply-spliced genomic RNA retained in
nucleus. Rev has an NES that recruits the nuclear export factor Crm1 to
the Rev/RRE complex. Thus, Rev acts as a virally-encoded adaptor to
mediate nuclear export of genomic RNA via Crm1 pathway, resulting in later
expression of regulatory and late proteins.
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7. Examples from Pathobiology, cont.:
C. How HIV-1 exploits cellular machinery for genomic RNA trafficking, cont.:
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Trafficking In and Out of The Nucleus
7. Examples from Pathobiology, cont.:
D. Other retroviruses use a different mechanism to export unspliced genomic RNA:
Lentiviruses as well as HTLV-1 use a Rev-like mechanism for
Direct binding of CTE in
nuclear export of genomic RNA.
MPMV genomic RNA to
Type D retroviruses and avian type C retroviruses have
export receptors
structured sequences in their genomic RNAs
(the constitutive transport element, or CTE) that
binds directly to transport receptors TAP and Nxt, which
mediate most mRNA export.
Thus, these simple retroviruses bypass the need for a
virally-encoded adaptor protein like Rev.
Rev interacts with a specific loop in the RRE in the HIV-1
genome via an arginine-rich RNA-binding motif
NES
Crm1
REV
p
Ri
ab
R
/
NPC
incompletely spliced RNA
nucleus
PM
J. Lingappa, 2003
April 4, 2006
Cullen, J. Cell Sci. 116: 587 (2003)
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7. Examples from Pathobiology, cont.:
E. How nuclear import machinery is exploited by Adenovirus (Ad) for viral
entry into the nucleus:
J. Lingappa, 2003
April 4, 2006
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7. Examples from Pathobiology:
F. Herpesvirus Egress:
Herpesvirus Egress:
Herpesvirus capsids assemble in
nucleus.
Envelopment occurs at inner
nuclear membrane.
One model gaining favor proposes
that capsids then get deenveloped at the outer nuclear
membrane resulting in release
of unenveloped capsids in the
cytoplasm.
These capsids acquire a new
envelope at the Golgi.
Study of mechanism involved in deenvelopment may reveal nature
and function of proteins that
reside in space between the two
nuclear membranes.
Currently, mechanism of deenvelopment is not understood.
April 4, 2006
From Mettenleiter, J. Virol. 76: 1537 (2002)
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8. Additional Reading:
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•
•
•
•
•
•
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Cullen, B. R. Nuclear RNA export. J. Cell Science 116: 587 (2002).
Bayliss, R. The molecular mechanism of transport of macromolecules through
nuclear pore complexes. Traffic 2000 1:448 (2000).
Nakielny, S. and G. Dreyfuss. Transport of proteins and RNAs in and out of the
nucleus. Cell 99: 677 (1999).
Paushkin, et al. The SMN ocmplex, an assemblyosome of ribonucleoproteins.
Current Opinion in Cell Bio. 14: 305 (2002).
Cullen, B. R. Retroviruses as model systems for the study of nuclear RNA export
pathways. Virology 249: 203 (1998).
Whittaker, G. and A. Helenius. Nuclear import and export of viruses and virus
genomes. Virology 246: 1-23 (1998).
Trotman, L. Import of adenovirus DNA involves the nuclear pore complex receptor
CAN/Nup214 and histone H1. Nat Cell Biol. 3:1092-100 (2001).
Mettenleiter, T. C. Herpesvirus assembly and egress. J. Virol. 76: 1537 (2002).
April 4, 2006
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•
Digitonin: cell membranes that are rich in cholesterol, like PM, are permeabilized, but
internal membranes that are low in cholesterol, like nuclear envelope, remain in tact.
•
•
Point out that substrates that traffic in and out of nucleus are FOLDED
Nuclear transport is gated: selective transport across the NPC as well as diffusion
•
Actinomycin D binds to DNA and blocks movement of Pol along DNA
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