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Transcript
9
The Nucleus
9 The Nucleus
Chapter Outline
• The Nuclear Envelope and Traffic
between the Nucleus and the
Cytoplasm
• Internal Organization of the Nucleus
• The Nucleolus and rRNA Processing
Introduction
The nucleus is the principal feature that
distinguishes eukaryotic from prokaryotic
cells.
It serves as the repository of genetic
information and as the cell’s control
center.
Separation of the genome from the site of
mRNA translation thus plays a central
role in eukaryotic gene expression.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The nuclear envelope separates the
contents of the nucleus from the
cytoplasm.
The selective traffic of proteins and
RNAs through nuclear pore complexes
establishes the nuclear composition
and plays a critical role in regulating
eukaryotic gene expression.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The nuclear envelope consists of two
membranes, an underlying nuclear
lamina, and nuclear pore complexes.
The outer membrane is continuous with
the endoplasmic reticulum. It is
enriched in membrane proteins that
bind the cytoskeleton.
The inner membrane has proteins that
bind the nuclear lamina.
Figure 9.1 The nuclear envelope (Part 1)
Figure 9.1 The nuclear envelope (Part 2)
Figure 9.1 The nuclear envelope (Part 3)
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Each nuclear membrane is a
phospholipid bilayer permeable only to
small nonpolar molecules.
Nuclear pore complexes are the sole
channels for small polar molecules,
ions, proteins, and RNA to pass
through the nuclear envelope.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The nuclear lamina is a fibrous mesh
that provides structural support.
It consists of fibrous proteins called
lamins, and other proteins.
Mutations in lamin genes cause several
different inherited tissue-specific
diseases.
Figure 9.3 Electron micrograph of the nuclear lamina
Molecular Medicine 9.1 Nuclear Lamina Diseases: (A) A child with Hutchinson-Gilford progeria. (B)
Intron-exon structure of the LMNA gene and lamin A protein
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Mammalian cells have three lamin genes,
(A, B, and C), which code for at least
seven proteins.
Two lamins interact to form a dimer in
which the α-helical regions of two
polypeptide chains wind around each
other to form a coiled coil.
The lamin dimers associate with each
other to form the nuclear lamina.
Figure 9.4 Model of lamin assembly
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Lamins bind to inner nuclear membrane
proteins such as emerin and lamin B
receptor.
The lamins and lamin-associated
proteins also bind to chromatin.
Lamins also extend in a loose meshwork
throughout the interior of the nucleus.
Figure 9.5 The nuclear lamina
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear pore complexes are large,
complex structures.
In vertebrates, they is composed of
about 30 different pore proteins
(nucleoporins).
The control of molecular traffic between
the nucleus and the cytoplasm plays a
fundamental role in the physiology of all
eukaryotic cells.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Molecules travel through pore
complexes by two mechanisms:
Passive transport—small molecules
pass freely in either direction through
open aqueous channels.
Macromolecules (proteins and RNAs)
are transported by a selective,
energy-dependent mechanism.
Figure 9.6 Molecular traffic through nuclear pore complexes
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Electron microscopy shows that pore
complexes have eightfold symmetry
organized around a large central
channel.
Figure 9.7 Electron micrograph of nuclear pore complexes
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
A nuclear pore complex consists of eight
spokes connected to rings at the
nuclear and cytoplasmic surfaces.
The spoke-ring assembly surrounds a
central channel.
Protein filaments extend from the rings,
forming a basketlike structure on the
nuclear side.
Figure 9.8 Model of the nuclear pore complex
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Proteins that must enter the nucleus
have amino acid sequences called
nuclear localization signals.
These are recognized by nuclear
transport receptors which direct
transport of the proteins through the
nuclear pore complex.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear localization signals were first
identified in 1984, using a viral
replication protein SV40 T antigen.
Using T antigen mutants, they determined
the amino acid sequence that was
responsible for nuclear localization.
When the same sequence was attached
to other proteins, they also were
transported to the nucleus.
Key Experiment 9.1 Identification of Nuclear Localization Signals: Determining cellular localization
of fusion proteins
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The T antigen nuclear localization signal
is a single stretch of amino acids.
Others are bipartite, consisting of two
amino acids sequences separated by
another amino acid sequence.
Figure 9.9 Nuclear localization signals
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear localization signals are
recognized by nuclear transport
receptors called importins.
Activity of nuclear transport receptors is
regulated by Ran, a GTP-binding protein.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
For Ran, enzymes for GTP hydrolysis to
GDP are on the cytoplasmic side of the
nuclear envelope; enzymes for
exchange of GDP for GTP are on the
nuclear side.
This leads to higher concentration of
Ran/GTP in the nucleus, and
determines the directionality of
transport.
Figure 9.10 Distribution of Ran/GTP across the nuclear envelope
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
A specific importin binds to the nuclear
localization signal of a cargo protein in
the cytoplasm.
This complex binds to the cytoplasmic
filaments of the pore complex.
Transport proceeds by sequential binding
to specific nuclear pore proteins located
further and further toward the nuclear
side of the pore complex.
Figure 9.11 Protein import through the nuclear pore complex
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nucleoporins with multiple Phe-Gly
repeats (FG-proteins) line the central
channel.
At the nuclear side the cargo/importin
complex is disrupted by binding of
Ran/GTP.
This causes a conformation change in
the importin, which releases the cargo
protein into the nucleus.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The importin-Ran/GTP complex is then
exported back to the cytoplasm where
the GTP is hydrolyzed to GDP.
The importin is released and can
participate in another round of transport.
Ran/GDP is transported back to the
nucleus by its own import receptor
(NTF2), where Ran/GTP is regenerated.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Proteins are targeted for export from the
nucleus by specific amino acid
sequences, called nuclear export
signals.
These signals are recognized by
receptors in the nucleus (exportins),
which direct protein transport to the
cytoplasm.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Many importins and exportins are
members of a family of nuclear
transport receptors known as
karyopherins.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Ran is required for nuclear export as
well as import.
Ran/GTP promotes binding of exportins
and their cargo proteins, but
dissociates complexes between
importins and their cargos.
Figure 9.12 Nuclear export
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Protein transport is another point at which
nuclear protein activity can be controlled;
for example regulation of import and
export of transcription factors.
Changes in receptor affinity of only two
pore complex proteins contributed to the
evolutionary split between Drosophila
melanogaster and Drosophila simulans.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
In one mechanism, transcription factors
(or other proteins) associate with
cytoplasmic proteins that mask their
nuclear localization signals, and so
they remain in the cytoplasm.
Figure 9.13 Regulation of nuclear import of transcription factors
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Other transcription factors are regulated
by phosphorylation.
Example: the yeast transcription factor
Pho4 is phosphorylated at a site
adjacent to its nuclear localization
signal, which interferers with its import.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Most RNAs are exported from the
nucleus to the cytoplasm for use in
protein synthesis.
It is an active, energy-dependent
process requiring the transport
receptors to interact with the nuclear
pore complex.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
RNAs are transported as
ribonucleoprotein complexes (RNPs).
rRNAs are associated with ribosomal
proteins and specific RNA processing
proteins in the nucleolus.
mRNAs are associated with at least 20
proteins during processing and
eventual transport to the cytoplasm.
Figure 9.14 Transport of a ribonucleoprotein complex
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Many small RNAs (snRNAs and
snoRNAs) function within the nucleus.
snRNAs are transported to the
cytoplasm by an exportin (Crm1),
where they associate with proteins to
form snRNPs and return to the nucleus.
Figure 9.15 Transport of snRNAs between nucleus and cytoplasm
Internal Organization of the Nucleus
The nucleus has an internal structure
that organizes the genetic material and
localizes nuclear functions.
In animal cells, lamins serve as sites of
chromatin attachment and organize
other proteins into functional nuclear
bodies.
Internal Organization of the Nucleus
During interphase, heterochromatin
remains highly condensed and is
transcriptionally inactive; the
euchromatin is decondensed and
distributed throughout the nucleus.
Figure 9.16 Heterochromatin in interphase nuclei
Internal Organization of the Nucleus
Two types of heterochromatin:
• Constitutive heterochromatin contains
DNA that is not transcribed, such as
satellite sequences at centromeres.
• Facultative heterochromatin contains
sequences that are not transcribed in
the cell being examined but are
transcribed in other cell types.
Internal Organization of the Nucleus
During interphase, chromosomes are
organized and divided into discrete
functional domains.
This organization was first suggested in
1885 and confirmed in 1984 by studies
of polytene chromosomes in Drosophila
salivary glands.
Figure 9.17 Chromosome organization
Internal Organization of the Nucleus
Each chromosome occupies a discrete
region of the nucleus.
They are closely associated with the
nuclear envelope at many sites.
Many of these associations result in
repression of gene expression but
some promote transcription of specific
genes.
Figure 9.18 Organization of Drosophila chromosomes
Internal Organization of the Nucleus
In mammalian cell nuclei, actively
transcribed genes are localized to the
periphery of the territories.
RNA processing and transport are
thought to occur in channels
(interchromosomal domains) that
separate the chromosomes.
Figure 9.19 Organization of chromosomes in the mammalian nucleus
Internal Organization of the Nucleus
Different cell types express different
genes, so their facultative
heterochromatin is different, and
varying regions of the chromosomes
interact with the nuclear lamina in
different cells and tissues.
The Hilbert curve is a one-dimensional
fractal trajectory that densely fills
higher-dimensional space without
crossing itself.
Leiberman-Aiden et al. (2009)
Science 326:289-293
3-D structure of human genome: fractal globule architecture
Internal Organization of the Nucleus
In interphase, the chromatin is organized
into looped domains containing about
50–100 kb of DNA.
In amphibian oocytes, actively
transcribed regions of DNA can be
seen as large loops of extended
chromatin.
Figure 9.20 Looped chromatin domains
Internal Organization of the Nucleus
Most nuclear processes are localized to
distinct regions of the nucleus.
Many proteins are localized in lowdensity, sponge-like subnuclear bodies
that allow macromolecules to move in
and out.
The nature and function of these nuclear
substructures are not yet clear.
Internal Organization of the Nucleus
Mammalian cell nuclei contain clustered
sites of DNA replication.
They are identified by labeling newly
synthesized DNA with
bromodeoxyuridine, which is
incorporated in place of thymidine.
Immunofluorescence shows the newly
replicated DNA is present in discrete
clusters.
Figure 9.21 Clustered sites of DNA replication
Internal Organization of the Nucleus
DNA replication appears to take place in
distinct functional bodies with multiple
replication complexes.
These have been called replication
factories.
Internal Organization of the Nucleus
The mRNA splicing machinery is
concentrated in discrete nuclear bodies,
called nuclear speckles.
They can be seen with
immunofluorescent staining with
antibodies against snRNPs and splicing
factors.
Figure 9.22 Localization of splicing components
Internal Organization of the Nucleus
PML bodies were first identified as
localization sites of a transcriptional
regulatory protein involved in acute
promyelocytic leukemia (PML).
They are sites of accumulation of
transcription factors and chromatinmodifying proteins, but their function
remains unknown.
Figure 9.23 A PML body
Internal Organization of the Nucleus
Cajal bodies contain the characteristic
protein coilin and are enriched in small
RNPs.
They may function as sites of RNP
assembly and processing.
Figure 9.24 Cajal bodies in the nucleus
The Nucleolus and rRNA Processing
The nucleolus is the site of rRNA
transcription and processing, and some
aspects of ribosome assembly.
Cells need large numbers of ribosomes
at specific times for protein synthesis.
Actively growing mammalian cells have
5 to 10 million ribosomes that must be
synthesized each time the cell divides.
The Nucleolus and rRNA Processing
The nucleolus is not surrounded by a
membrane.
The 5.8S, 18S, and 28S rRNAs are
transcribed as a single unit in the
nucleolus by RNA polymerase I,
yielding a 45S ribosomal precursor
RNA.
Figure 9.25 Ribosomal RNA genes
The Nucleolus and rRNA Processing
All cells contain multiple copies of the
rRNA genes.
In oocytes, the rRNA genes are amplified
to support synthesis of ribosomes needed
for early development.
rRNA genes are amplified two-thousandfold in Xenopus oocytes, in thousands of
nucleoli, resulting in 1012 ribosomes per
oocyte.
Figure 9.26 Nucleoli in amphibian oocytes
The Nucleolus and rRNA Processing
Nucleoli have three regions: the fibrillar
center, dense fibrillar component, and
granular component.
They are thought to represent sites of
progressive stages of rRNA
transcription, processing, and ribosome
assembly.
Figure 9.27 Structure of the nucleolus
The Nucleolus and rRNA Processing
Following each cell division, nucleoli
become associated with the nucleolar
organizing regions that contain the
5.8S, 18S, and 28S rRNA genes.
Transcription of 45S pre-rRNA leads to
fusion of small prenucleolar bodies. In
most cells, the initially separate nucleoli
then fuse to form a single nucleolus.
The Nucleolus and rRNA Processing
Each nucleolar organizing region
contains a cluster of tandemly repeated
rRNA genes separated by
nontranscribed spacer DNA.
The genes and their growing RNA
chains can be seen in electron
micrographs.
Figure 9.28 Transcription of rRNA genes
The Nucleolus and rRNA Processing
In higher eukaryotes the primary
transcript of rRNA genes is the large
45S pre-rRNA.
The pre-rRNA is processed via a series
of cleavages, which is similar in all
eukaryotes.
Figure 9.29 Processing of pre-rRNA
The Nucleolus and rRNA Processing
Processing of pre-rRNA also includes
addition of methyl groups to bases and
ribose residues, and conversion of
uridine to pseudouridine.
The Nucleolus and rRNA Processing
Nucleoli contain over 300 proteins and
many small nucleolar RNAs
(snoRNAs) that function in pre-rRNA
processing.
snoRNAs are complexed with proteins,
forming snoRNPs. They assemble on
pre-rRNA to form processing
complexes similar to the formation of
spliceosomes on pre-mRNA.
The Nucleolus and rRNA Processing
Most snoRNAs guide RNAs to direct
specific base modifications of pre-rRNA.
They contain short sequences of
nucleotides that are complementary to
18S or 28S rRNA and include the sites
of base modification in the rRNA.
Figure 9.30 Role of snoRNAs in base modification of pre-rRNA
The Nucleolus and rRNA Processing
Formation of ribosomes requires
assembly of pre-rRNA with ribosomal
proteins and 5S rRNA.
Ribosomal proteins are imported to the
nucleolus from the cytoplasm, where
they assemble with the pre-rRNA prior
to cleavage.
The Nucleolus and rRNA Processing
5S rRNAs are similarly assembled into
preribosomal particles elsewhere in the
nucleolus.
Additional ribosomal proteins and the 5S
rRNA are incorporated as cleavage and
processing proceeds.
The two nascent ribosomal subunits
separate and are exported to the
cytoplasm.
Figure 9.31 Ribosome assembly
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
• NE is essential in defining higher-order nuclear structure
by providing anchoring sites for chromatin domains at
the nuclear periphery.
• “Laminopathies” include cardiac and skeletal
myopathies, partial lipodystrophy (loss of adipocytes),
peripheral neuropathy.
• NE not only has a ubiquitous role in the maintenance of
nuclear architecture, but also might provide cell-specific
functions.
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
X-linked Emery-Dreifuss muscular dystrophy
• Laminopathies: defects in genes that encode nuclear-lamina and
lamina-associated proteins – specifically A-type lamins and emerins
• A mutation in the emerin gene leading to a loss of emerin from the
nuclear periphery
• Childhood onset of progressive muscle wasting and weakening
• Early contractures of the Achilles tendons, tendons of the elbows
and necks….
• Late-onset symptoms: abnormal cardiac rhythms, conduction block,
and cardiomyopathy…
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Autosomal-dominant muscular dystrophy
• Mutations in Lamin A gene (LMNA) mostly missense mutations
affecting both Lamin A and Lamin C proteins
• “Autosomal-dominant” means that it is haploinsufficient.
• Mouse model with Lmna deletion: a human EDMD model
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Lmna knockout mouse model
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Lmna knockout mouse model
Anti-LaminB
Anti-LaminA
Anti-Emerin
LaminA를 발현시켜 주니 Emerin 이
제대로 localize된다.
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Skeletal and cardiomyopathy
• Mutations in Lamin A gene (LMNA)
• One mutation is a frameshift at codon 320.
• This mutation is predicted to produce lamin proteins without NLS
and are defective in assembly.
• Skeletal myopathy and cardiac conduction defects
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Neuropathy
•
•
•
•
Mutations in Lamin A gene (LMNA)
Charcot-Marie-Tooth type 2 (CMT2) syndrome
R298C
This mutation is likely to perturb the lateral interactions between Atype lamins.
• Symptoms: muscle weakness accompanied by loss of large
myelinated muscle fibers and axonal degeneration
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Lipodystrophy
•
•
•
•
•
Mutations in Lamin A gene (LMNA), mainly in exon 11
Dunnigan-type familial partial lipodystrophy (FPLD)
Changes in fat distribution (more in upper body, face, and neck)
High TG, high insulin, insulin-resistant diabetes
Lmna mutant mice do not show such phenotype. Thus, FPLD is not
caused simply by loss of some lamin function.
• R482W, maybe a gain-of-function mutation?
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
The Nuclear Envelope and Human Diseases
Burke & Stewart (2002) Nat Rev Mol Cell Biol 3:575
Disease Mechanisms
• Nuclear fragility
• Weak nuclear envelope leading to nuclear damage and cell
death
• Muscle contraction – mechanical stress on weak NE
• Lipodystrophy는 mechanical stress와 무관
• Gene-expression effects
• Heterochromatin의 구성을 바꾸어 gene expression pattern에
변화가 온다?
Hutchinson-Gilford Progeria Syndrome
Untreated
Nuclear LMNA blebbing
•
•
•
•
•
treated
Premature aging
LMNA mutation (Nature, 2003)
Processing of Prelamin A to Lamin A is affected.
Farnesylated prelamin A accumulates in the nuclear envelope.
Farnesyltransferase inhibitor (FTI) to release prelamin A from NE
partially reverses the nuclear damage.
• Phase II clinical trial ongoing