Transcript Chapter 10

10
The Nucleus
10 The Nucleus
• The Nuclear Envelope and Traffic
between the Nucleus and the
Cytoplasm
• The Organization of Chromosomes
• Nuclear Bodies
Introduction
The nucleus is the main feature that
distinguishes eukaryotic from prokaryotic
cells.
It houses the genome, and thus is the
repository of genetic information and the
cell’s control center.
Separation of the genome from the site of
mRNA translation plays a central role in
eukaryotic gene expression.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The nuclear envelope separates the
nuclear contents from the cytoplasm.
It controls traffic of proteins and RNAs
through nuclear pore complexes, and
plays a critical role in regulating gene
expression.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The nuclear envelope consists of:
• Two nuclear membranes
• An underlying nuclear lamina
• Nuclear pore complexes
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The outer membrane is continuous with
the endoplasmic reticulum (ER).
The space between inner and outer
membranes is directly connected with
the lumen of the ER.
The inner membrane has integral
proteins, including ones that bind the
nuclear lamina.
Figure 10.1 The nuclear envelope (Part 1)
Figure 10.1 The nuclear envelope (Part 2)
Figure 10.1 The nuclear envelope (Part 3)
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear membranes are phospholipid
bilayers permeable only to small
nonpolar molecules.
Nuclear pore complexes are the only
channels for small polar molecules,
ions, and macromolecules.
Figure 10.2 Electron micrograph showing nuclear pores
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear lamina: a fibrous mesh that
provides structural support.
Consists of fibrous proteins (lamins) and
other proteins.
Figure 10.3 Electron micrograph of the nuclear lamina
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Lamins are a class of intermediate
filament proteins that associate to form
higher order structures.
Two lamins interact to form a dimer: the
α-helical regions wind around each
other to form a coiled coil.
The lamin dimers associate with each
other to form the lamina.
Figure 10.4 Lamin assembly
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Lamins bind to inner membrane proteins
such as emerin and lamin B receptor
(LBR).
They are connected to the cytoskeleton
by LINC protein complexes.
Lamins also bind to chromatin.
Figure 10.5 The nuclear lamina
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Mutations in lamin genes cause several
inherited tissue-specific diseases.
The bases of the pathologies in each of
these diseases is still unclear.
Molecular Medicine, Ch. 10, p. 371 (Part 1)
Molecular Medicine, Ch. 10, p. 371 (Part 2)
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear pore complexes are large;
about 30 proteins (nucleoporins).
RNAs synthesized in the nucleus must
be exported to the cytoplasm for
protein synthesis.
Proteins needed for nuclear functions
must be imported from synthesis sites
in the cytoplasm.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Molecules pass through pore complexes
by two mechanisms:
• Passive diffusion—small molecules
pass freely in either direction.
• Proteins and RNAs are selectively
transported; requires energy.
Figure 10.6 Molecular traffic through nuclear pore complexes
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Electron microscopy shows pore
complexes have eight subunits
organized around a large central
channel.
Figure 10.7 Electron micrograph of nuclear pore complexes
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Eight spokes are 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 10.8 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 signals are recognized by
nuclear transport receptors.
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.
The amino acid sequence responsible for
nuclear localization was determined
using T antigen mutants.
When the same sequence was attached
to other proteins, they were also
transported to the nucleus.
Key Experiment, Ch. 10, p. 374 (2)
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The T antigen nuclear localization signal
is a single stretch of amino acids.
Other signals are bipartite: two amino
acids sequences are separated by
another amino acid sequence.
Figure 10.9 Nuclear localization signals
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Nuclear localization signals (NLS) are
recognized by receptors called
importins, which carry proteins through
the nuclear pore complex.
Importins work in conjunction with the
GTP-binding protein Ran, which controls
directionality of movement.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Importins bind to the NLS of a protein,
then to nuclear pore proteins and the
complex is transported across the
membrane.
Ran/GTP binds to the importin, and this
complex is transported back.
In the cytoplasm, Ran GAP hydrolyzes
the GTP on Ran to GDP, releasing the
importin.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
The Ran/GDP formed in the cytoplasm
is then transported back to the nucleus
by its own import receptor, where
Ran/GTP is regenerated.
Figure 10.10 Protein import through the nuclear pore complex
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Protein export from the nucleus:
Proteins are targeted for export by
amino acid sequences called nuclear
export signals (NES).
NES 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.
Table 10.1 Examples of Karyopherins
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Exportins form stable complexes with
cargo proteins in association with
Ran/GTP in the nucleus.
In the cytoplasm, GTP hydrolysis and
release of Ran/GDP leads to
dissociation of the cargo protein.
Figure 10.11 Nuclear export
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
RNAs are transported to the cytoplasm
as ribonucleoprotein complexes
(RNPs).
Karyopherin exportins transport tRNAs,
rRNAs, miRNAs.
Figure 10.12 Transport of a ribonucleoprotein complex (Part 1)
Figure 10.12 Transport of a ribonucleoprotein complex (Part 2)
Figure 10.12 Transport of a ribonucleoprotein complex (Part 3)
Figure 10.12 Transport of a ribonucleoprotein complex (Part 4)
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
mRNA transport does not involve
karyopherins and is independent of Ran.
A distinct transporter complex moves the
mRNA through the nuclear pore.
Helicase on the cytoplasm side releases
the mRNA and ensures unidirectional
transport.
Figure 10.13 mRNA export
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Many noncoding RNAs (snRNAs and
snoRNAs) function within the nucleus.
snRNAs are initially exported from the
nucleus by an exportin (Crm1).
In the cytoplasm, the snRNAs associate
with proteins to form snRNPs, which
are recognized by an importin and
transported back to the nucleus.
Figure 10.14 Transport of snRNAs between nucleus and cytoplasm
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Regulation of protein transport is a
mechanism for controlling protein activity
in the nucleus.
Example: Regulation of import and export
of transcription factors is a way of
controlling gene expression.
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
NLS, and so they remain in the
cytoplasm.
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Transcription factor NF-kB is complexed
with IkB in the cytoplasm.
If IkB is phosphorylated and degraded
by ubiquitin-mediated proteolysis, NFkB can enter the nucleus and activate
transcription of its target genes.
Figure 10.15 Regulation of nuclear import of transcription factors
The Nuclear Envelope and Traffic between the Nucleus and the
Cytoplasm
Other transcription factors are regulated
directly by phosphorylation.
Example: Yeast transcription factor Pho4
is phosphorylated at a site adjacent to
its NLS, which interferes with its import.
The Organization of Chromosomes
Chromatin becomes highly condensed
during mitosis to form the compact
metaphase chromosomes.
During interphase, most of the chromatin
decondenses and is distributed
throughout the nucleus.
The Organization of Chromosomes
But even in interphase, the
chromosomes occupy distinct regions
and are organized such that
transcriptional activity of a gene is
correlated with its position.
DNA replication and transcription take
place in clustered regions within the
nucleus.
The Organization of Chromosomes
This organization was first suggested in
1885 and confirmed in 1984 by studies
of polytene chromosomes in Drosophila
salivary glands.
Each chromosome was found to occupy
a discrete region of the nucleus, called
a chromosome territory.
Figure 10.16 Chromosome organization (Part 1)
Figure 10.16 Chromosome organization (Part 2)
Figure 10.17 Organization of Drosophila chromosomes (Part 1)
Figure 10.17 Organization of Drosophila chromosomes (Part 2)
The Organization of Chromosomes
In situ hybridization with fluorescent
probes specific for repeated sequences
on individual chromosomes has been
used to visualize the location of
chromosomes within a nucleus.
Figure 10.18 Organization of chromosomes in the mammalian nucleus
The Organization of Chromosomes
In living cells, chromosome conformation
capture (3C) techniques reveal sites of
interactions between chromosomal
regions.
They are identified by cross-linking
interacting DNA sequences, which are
then amplified and identified by highthroughput sequencing.
The Organization of Chromosomes
In interphase cells, the euchromatin is
decondensed and transcriptionallyactive, and is distributed throughout the
nucleus.
Heterochromatin is highly condensed
and not transcribed, and is often
associated with the nuclear envelope or
periphery of the nucleolus.
Figure 10.19 Heterochromatin in interphase nuclei
The Organization of Chromosomes
Some human chromosomes are rich in
transcribed genes, whereas others
contain relatively few genes.
Fluorescent in situ hybridization shows
that gene-rich chromosomes are
located in the center of the nucleus;
gene-poor chromosomes are at the
periphery.
Figure 10.20 Fluorescent in situ hybridization to transcriptionally-active and-inactive chromosomes
(Part 1)
Figure 10.20 Fluorescent in situ hybridization to transcriptionally-active and-inactive chromosomes
(Part 2)
The Organization of Chromosomes
Genomes are divided into topologically
associating domains (TADs).
Regions within a domain interact
frequently with one another, but only
rarely with regions in other domains.
Figure 8.20 Chromosomal domains and CTCF
The Organization of Chromosomes
Some domains are associated with the
nuclear lamina (called laminaassociated domains or LADs).
The genes within LADs are generally
transcriptionally repressed.
LADS correspond to heterochromatin.
Figure 10.21 Distribution of transcriptionally-active and -inactive chromatin
The Organization of Chromosomes
The nucleolus is also surrounded by
heterochromatin (called nucleolusassociated domains or NADs).
DNA sequences found in NADs
substantially overlap with those in
LADs.
The Organization of Chromosomes
Most nuclear processes occur in distinct
regions.
DNA replication takes place in large
complexes called replication
factories, where replication of multiple
DNA molecules takes place.
The Organization of Chromosomes
These can be seen by labeling cells with
bromodeoxyuridine, (an analog of
thymidine), then staining with
fluorescent antibodies.
Figure 10.22 Replication factories (Part 1)
Figure 10.22 Replication factories (Part 2)
The Organization of Chromosomes
Transcription occurs at clustered sites
(transcription factories) that contain
newly synthesized RNA.
Coregulated genes, such as
immunoglobulin genes from different
chromosomes, may be transcribed in
the same factory.
Figure 10.23 Transcription factories
Nuclear Bodies
Nuclear bodies are organelles within
the nucleus that concentrate proteins
and RNAs for specific processes.
They are not enclosed by membranes;
they are dynamic structures maintained
by protein-protein and protein-RNA
interactions.
Table 10.2 Examples of Nuclear Bodies
Nuclear Bodies
The nucleolus functions in rRNA
synthesis and ribosome production.
Cells need large numbers of ribosomes
at specific times for protein synthesis.
Actively growing mammal cells have 5 to
10 million ribosomes that must be
synthesized each time the cell divides.
Nuclear Bodies
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.
Transcription of the 5S rRNA takes place
outside the nucleolus and is catalyzed
by RNA polymerase III.
Nuclear Bodies
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.
Nuclear Bodies
Nucleoli have three regions: fibrillar
center, dense fibrillar component, and
granular component.
They represent sites of progressive
stages of rRNA transcription,
processing, and ribosome assembly.
Figure 10.24 Structure of the nucleolus
Nuclear Bodies
Each nucleolar organizing region
contains a cluster of tandemly repeated
rRNA genes separated by spacer DNA.
The genes are actively transcribed by
RNA polymerase I, and their growing
RNA chains can be seen in electron
micrographs.
Figure 10.25 Ribosomal RNA genes (Part 1)
Figure 10.25 Ribosomal RNA genes (Part 2)
Nuclear Bodies
In higher eukaryotes, the primary
transcript of rRNA genes is the 45S
pre-rRNA.
The pre-rRNA is processed via a series
of cleavages, which is similar in all
eukaryotes.
Figure 10.26 Processing of pre-rRNA
Nuclear Bodies
Processing of pre-rRNA also includes
substantial base modification:
Addition of methyl groups to bases and
ribose residues, and conversion of
uridine to pseudouridine.
Nuclear Bodies
Nucleoli have over 300 proteins and 200
small nucleolar RNAs (snoRNAs)
that function in pre-rRNA processing.
snoRNAs are complexed with proteins,
forming snoRNPs. They form
processing complexes similar to
spliceosomes on pre-mRNA.
Nuclear Bodies
Most snoRNAs guide RNAs to direct
specific base modifications of pre-rRNA.
Two families of snoRNAs associate with
different proteins, which catalyze ribose
methylation or pseudouridine formation.
snoRNAs have sequences
complementary to 18S or 28S rRNA,
which include the sites of base
modification.
Figure 10.27 Role of snoRNAs in base modification of pre-rRNA
Nuclear Bodies
Formation of ribosomes requires
assembly of pre-rRNA with ribosomal
proteins and 5S rRNA.
Ribosomal proteins are produced in the
cytoplasm and imported to the
nucleolus, where they assemble with
the pre-rRNA prior to cleavage.
Nuclear Bodies
5S rRNAs are produced elsewhere in
the nucleolus.
Additional ribosomal proteins and the 5S
rRNA assemble to form pre-ribosomal
particles.
Pre-ribosomal particles are then
exported to the cytoplasm, yielding the
40S and 60S ribosomal subunits.
Figure 10.28 Ribosome assembly
Nuclear Bodies
Polycomb proteins repress transcription
of genes via methylation of histone H3
lysine 27 residues.
Immunofluorescence shows these
proteins are concentrated in domains
called Polycomb bodies.
Some Polycomb bodies contain clusters
of repressed domains from different
chromosomal regions.
Figure 8.36 Polychrome proteins
Figure 10.29 Polycomb bodies (Part 1)
Figure 10.29 Polycomb bodies (Part 2)
Nuclear Bodies
Cajal bodies are involved in assembly of
snRNPs and other RNA-protein
complexes.
snRNAs are modified by ribose
methylation and pseudouridylation.
The enzyme for RNA methylation
(fibrillarin) is concentrated in Cajal
bodies.
Figure 10.30 Cajal bodies in the nucleus (Part 1)
Figure 10.30 Cajal bodies in the nucleus (Part 2)
Nuclear Bodies
Cajal bodies also have small Cajal bodyspecific RNAs (scaRNAs).
scaRNAs are related to snoRNAs and
similarly serve as guides to direct
ribose methylation and
pseudouridylation of snRNAs.
Nuclear Bodies
Cajal bodies appear to play a role in
assembly of telomerase, which
replicates the ends of chromosomal
DNA.
Cajal bodies may promote assembly of
the RNA-protein telomerase complex
and facilitate its delivery to telomeres.
Nuclear Bodies
Following assembly and maturation in
Cajal bodies, snRNPs are transferred
to speckles, which also contain
splicing factors.
Speckles are recruited to actively
transcribed genes where pre-mRNA
processing occurs.
Figure 10.31 Nuclear speckles