7.
The Nucleus
The presence of a nucleus is
the principal feature that distinguishes eukaryotic from prokaryotic cells. By
housing the cell's genome, the nucleus serves both as the repository of genetic
information and as the cell's control center. DNA replication, transcription,
and RNA processing all take place within the nucleus, with only the final stage
of gene expression (translation) localized to the cytoplasm.
By separating the genome from
the cytoplasm, the nuclear envelope allows gene expression to be regulated by
mechanisms that are unique to eukaryotes. Whereas prokaryotic mRNAs are
translated while their transcription is still in process, eukaryotic mRNAs
undergo posttranscriptional processing (e.g., splicing) before being
transported from the nucleus to the cytoplasm. The presence of a nucleus thus
allows gene expression to be regulated by posttranscriptional mechanisms, such
as alternative splicing. By limiting the access of proteins to the genetic
material, the nuclear envelope also provides novel opportunities for the
control of gene expression at the level of transcription. For example, the
expression of some eukaryotic genes is controlled by the regulated transport of
transcription factors from the cytoplasm into the nucleus—a form of transcriptional
regulation unavailable to prokaryotes. The separation of the genome from the
site of mRNA translation thus plays a central role in eukaryotic gene
expression.
7.1 The Nuclear Envelope and Traffic between
the Nucleus and Cytoplasm
The nuclear envelope separates
the contents of the nucleus from the cytoplasm and provides the structural
framework of the nucleus. The nuclear membranes, acting as barriers that
prevent the free passage of molecules between the nucleus and the cytoplasm,
maintain the nucleus as a distinct biochemical compartment. The sole channels
through the nuclear envelope are provided by the nuclear pore complexes, which
allow the regulated exchange of molecules between the nucleus and cytoplasm.
The selective traffic of proteins and RNAs through the nuclear pore complexes
not only establishes the internal composition of the nucleus, but also plays a
critical role in regulating eukaryotic gene expression.
The nuclear envelope has a
complex structure, consisting of two nuclear membranes, an underlying nuclear
lamina, and nuclear pore complexes (Fig 7.1). The nucleus is surrounded by a
system of two concentric membranes, called the inner and outer nuclear
membranes. The outer nuclear membrane is continuous with the endoplasmic
reticulum, so the space between the inner and outer nuclear membranes is
directly connected with the lumen of the endoplasmic reticulum. In addition,
the outer nuclear membrane is functionally similar to the membranes of the
endoplasmic reticulum and has ribosomes bound to its cytoplasmic surface. In
contrast, the inner nuclear membrane carries unique proteins that are specific
to the nucleus.
Figure 7.1. Cross-section of
a typical cell nucleus.
The critical function of the
nuclear membranes is to act as a barrier that separates the contents of the
nucleus from the cytoplasm. Like other cell membranes, the nuclear membranes
are phospholipid bilayers, which are permeable only to small nonpolar
molecules. Other molecules are unable to diffuse through the phospholipid
bilayer. The inner and outer nuclear membranes are joined at nuclear pore
complexes, the sole channels through which small polar molecules and
macromolecules are able to travel through the nuclear envelope. The nuclear
pore complex is a complicated structure that is responsible for the selective
traffic of proteins and RNAs between the nucleus and the cytoplasm.
Figure 7.2. Model of lamin assembly
Underlying the inner nuclear
membrane is the nuclear lamina, a fibrous meshwork that provides structural
support to the nucleus. The nuclear lamina is composed of one or more related
proteins called lamins. Most mammalian cells, for example, contain four
different lamins, designated A, B1, B2, and C. All the
lamins are 60- to 80-kilodalton (kd) fibrous proteins that are related to the
intermediate filament proteins of the cytoskeleton. Like other intermediate
filament proteins, the lamins associate with each other to form filaments (Fig
7.2). The first stage of this association is the interaction of two lamins to
form a dimer in which the α-helical regions of two polypeptide chains
are wound around each other in a structure called a coiled coil. These lamin
dimers then associate with each other to form the filaments that make up the
nuclear lamina. The association of lamins with the inner nuclear membrane is
facilitated by the posttranslational addition of lipid—in particular,
prenylation of C-terminal cysteine residues. In addition, the lamins bind to
inner nuclear membrane proteins, which may help organize the lamin filaments
into a meshwork and mediate their attachment to the membrane.
In addition to providing
structural support to the nucleus, the nuclear lamina is thought to serve as a
site of chromatin attachment. Chromatin within the nucleus is organized into
large loops of DNA, some of which appear to be bound to the nuclear envelope.
The lamins bind chromatin and may help mediate this interaction.
The nuclear pore complexes are the only channels through which small polar molecules, ions, and macromolecules
(proteins and RNAs) are able to travel between the nucleus and the cytoplasm.
The nuclear pore complex is an extremely large structure with a diameter of
about 120 nm and an estimated molecular mass of approximately 125 million
daltons—about 30 times the size of a ribosome. In vertebrates, the nuclear pore
complex is composed of 50 to 100 different proteins. By controlling the traffic
of molecules between the nucleus and cytoplasm, the nuclear pore complex plays
a fundamental role in the physiology of all eukaryotic cells. RNAs that are
synthesized in the nucleus must be efficiently exported to the cytoplasm, where
they function in protein synthesis. Conversely, proteins required for nuclear
functions (e.g., transcription factors) must be transported into the nucleus
from their sites of synthesis in the cytoplasm. In addition, many proteins
shuttle continuously between the nucleus and the cytoplasm. The regulated
traffic of proteins and RNAs through the nuclear pore complex thus determines
the composition of the nucleus and plays a key role in gene expression.
Figure
Depending on their size,
molecules can travel through the nuclear pore complex by one of two different
mechanisms (Fig
complex by an active process
in which appropriate proteins and RNAs are recognized and selectively
transported in only one direction (nucleus to cytoplasm or cytoplasm to
nucleus). The traffic of these molecules occurs through regulated channels in
the nuclear pore complex that, in response to appropriate signals, can open to
a diameter of more than 25 nm—a size sufficient to accommodate large
ribonucleoprotein complexes, such as ribosomal subunits. It is through these
regulated channels that nuclear proteins are selectively imported from the cytoplasm
to the nucleus while RNAs are exported from the nucleus to the cytoplasm.
Figure
Visualization of nuclear pore
complexes by electron microscopy reveals a structure with eightfold symmetry
organized around a large central channel, which is the route through which
proteins and RNAs cross the nuclear envelope. Detailed structural studies,
including computer-based image analysis, have led to the development of
three-dimensional models of the nuclear pore complex (Fig
The basis for selective
traffic across the nuclear envelope is best understood for proteins that are
imported from the cytoplasm to the nucleus. Such proteins are responsible for
all aspects of genome structure and function; they include histones, DNA
polymerases, RNA polymerases, transcription factors, splicing factors, and many
others. These proteins are targeted to the nucleus by specific amino acid
sequences, called nuclear localization signals, that direct their transport
through the nuclear pore complex.
The first nuclear localization
signal to be mapped in detail was characterized by Alan Smith and colleagues in
1984. These investigators studied simian virus 40 (SV40) T antigen, a
virus-encoded protein that initiates viral DNA replication in infected cells.
As expected for a replication protein, T antigen normally is localized to the
nucleus. The signal responsible for its nuclear localization was first
identified by the finding that mutation of a single lysine residue prevents
nuclear import, resulting instead in the accumulation of T antigen in the
cytoplasm. Subsequent studies defined the T antigen nuclear localization signal
as the seven-amino-acid sequence Pro-Lys-Lys-Lys-Arg-Lys-Val.(Fig 7.4) Not only
was this sequence necessary for the nuclear transport of T antigen, but its
addition to other, normally cytoplasmic, proteins was also sufficient to direct
their accumulation in the nucleus.
Figure 7.4. Nuclear localization signals
Nuclear localization signals
have since been identified in many other proteins. Most of these sequences,
like that of T antigen, are short stretches rich in basic amino acid residues
(lysine and arginine). In many cases, however, the amino acids that form the
nuclear localization signal are close together but not immediately adjacent to
each other. For example, the nuclear localization signal of nucleoplasmin (a
protein involved in chromatin assembly) consists of two parts: a Lys-Arg pair
followed by four lysines located ten amino acids farther downstream. Both the
Lys-Arg and Lys-Lys-Lys-Lys sequences are required for nuclear targeting, but
the ten amino acids between these sequences can be mutated without affecting
nuclear localization. Because this nuclear localization sequence is composed of
two separated elements, it is referred to as bipartite. Similar bipartite
motifs appear to function as the localization signals of many nuclear proteins;
thus they may be more common than the simpler nuclear localization signal of T
antigen. In addition, some proteins, such as ribosomal proteins, contain
distinct nuclear localization signals which are unrelated to the basic amino
acid-rich nuclear localization signals of either nucleoplasmin or T antigen.
Nucleoplasminis a large nuclear protein with distinct head and tail domains. The heads can be cleaved from the tails by limited proteolysis. When injected into the cytosol of a frog oocyte, intact nucleoplasmin molecules rapidly accumulate in the nucleus even though they are too large to diffuse passively through the pore complex. The signal for this nuclear import resides in the tail domains, since injected tails are taken up by the nucleus but heads are not. The role of nuclear pores in this signal-directed import is demonstrated by electron microscopy using nucleoplasmin tails coupled to spheres of colloidal gold, which are easily visualized because of their high electron density. The attached nucleoplasmin tails direct the entry of the gold particles into the nucleus via nuclear pores(Fig 7.5).
Figure 7.5. The uptake of nuclear proteins via nuclear pores.
Protein import through the
nuclear pore complex can be operationally divided into two steps, distinguished
by whether they require energy (Fig 7.6). In the first step, which does not
require energy, proteins that contain nuclear localization signals bind to the
nuclear pore complex but do not pass through the pore. In this initial step,
nuclear localization signals are recognized by a cytosolic receptor protein,
and the receptor-substrate complex binds to the nuclear pore. The prototype
receptor, called importin, consists of two subunits. One subunit (importin α)
binds to the basic amino acid-rich nuclear localization signals of proteins
such as T antigen and nucleoplasmin. The second subunit (importin β)
binds to the cytoplasmic filaments of the nuclear pore complex, bringing the
target protein to the nuclear pore. Other types of nuclear localization
signals, such as those of ribosomal proteins, are recognized by distinct
receptors which are related to importin β and function similarly to
importin β during the transport of their target proteins into the
nucleus.
Figure 7.6. Protein import
through the nuclear pore complex
The second step in nuclear
import, translocation through the nuclear pore complex, is an energy-dependent
process that requires GTP hydrolysis. A key player in the translocation process
is a small GTP-binding protein called Ran, which is related to the Ras proteins
(Fig 7.7). The conformation and activity of Ran is regulated by GTP binding and
hydrolysis, like Ras or several of the translation factors involved in protein
synthesis. Enzymes that stimulate GTP binding to Ran are localized to the
nuclear side of the nuclear envelope whereas enzymes that stimulate GTP
hydrolysis are localized to the cytoplasmic side. Consequently, there is a
gradient of Ran/GTP across the nuclear envelope, with a high concentration of
Ran/GTP in the nucleus and a high concentration of Ran/GDP in the cytoplasm.
This gradient of Ran/GTP is thought to determine the directionality of nuclear
transport, and GTP hydrolysis by Ran appears to account for most (if not all)
of the energy required for nuclear import. Importin β forms a complex
with importin α and its associated target protein on the cytoplasmic
side of the nuclear pore complex, in the presence of a high concentration of
Ran/ GDP. This complex is then transported through the nuclear pore to the
nucleus, where a high concentration of Ran/GTP is present. At the nuclear side
of the pore, Ran/GTP binds to importin β, displacing importin α and the target protein. As a result, the target protein is released within the
nucleus. The Ran/GTP-importin β complex is then exported to the cytosol,
where the bound GTP is hydrolyzed to GDP, releasing importin β to
participate in another cycle of nuclear import.
Figure 7.7. Role of the Ran
protein in nuclear import(right)
Figure 7.8. Nuclear export
Some proteins remain within
the nucleus following their import from the cytoplasm, but many others shuttle
back and forth between the nucleus and the cytoplasm. Some of these proteins
act as carriers in the transport of other molecules, such as RNAs; others
coordinate nuclear and cytoplasmic functions (e.g., by regulating the
activities of transcription factors). Proteins are targeted for export from the
nucleus by specific amino acid sequences, called nuclear export signals. Like
nuclear localization signals, nuclear export signals are recognized by
receptors within the nucleus that direct protein transport through the nuclear
pore complex to the cytoplasm. Interestingly, the nuclear export receptors
(called exportins) are related to importin β. Like importin β,
the exportins bind to Ran, which is required for nuclear export as well as for
nuclear import (Fig 7.8). Strikingly, however, Ran/GTP promotes the formation
of stable complexes between exportins and their target proteins, whereas it dissociates
the complexes between importins and their targets. This effect of Ran/GTP
binding on exportins dictates the movement of proteins containing nuclear
export signals from the nucleus to the cytoplasm. Thus, exportins form stable
complexes with their target proteins in association with Ran/GTP within the
nucleus. Following transport to the cytosolic side of the nuclear envelope, GTP
hydrolysis
leads to dissociation of the
target protein, which is released into the cytoplasm.
Figure 7.9 The nuclear import of the glucocorticoid
receptor.
The glucocorticoid receptor is a gene regulatory protein that, in the non-hormone-treated cell, is bound in the cytosol to the chaperone protein hsp90. When activated by the binding of the appropriate steroid hormone, it is released from hsp90 and is directed into the nucleus by a nuclear localization signal; once in the nucleus, it binds to specific DNA sequences and regulates the transcription of a discrete set of genes.(Fig 7.9)
An intriguing
aspect of the transport of proteins into the nucleus is that it is another
level at which the activities of nuclear proteins can be controlled.
Transcription factors, for example, are functional only when they are present
in the nucleus, so regulation of their import to the nucleus is a novel means
of controlling gene expression.The regulated nuclear import of both
transcription factors and protein kinases plays an important role in
controlling the behavior of cells in response to changes in the environment,
because it provides a mechanism by which signals received at the cell surface
can be transmitted to the nucleus.
In one mechanism of regulation, transcription factors (or other proteins) associate with cytoplasmic proteins that mask their nuclear localization signals; because their signals are no longer recognizable, these proteins remain in the cytoplasm. A good example is provided by the transcription factor NF-κB, which activates transcription of immunoglobulin-κ light chains in B lymphocytes (Fig 7.10). In unstimulated cells, NF-κB is found as an inactive complex with an inhibitory protein (IκB) in the cytoplasm. Binding to IκB appears to mask the NF-κB nuclear localization signal, thus preventing NF-κB from being transported into the nucleus. In stimulated cells, IκB is phosphorylated and degraded by ubiquitin-mediated proteolysis, allowing NF-κB to enter the nucleus and activate transcription of its target genes.
The nuclear import of other transcription factors is regulated directly by their phosphorylation, rather than by association with inhibitory proteins. For example, the yeast transcription factor SWI5 is imported into the nucleus only at a specific stage of the cell cycle. Otherwise, SWI5 is retained in the cytoplasm as a result of phosphorylation at serine residues adjacent to its nuclear localization signal, preventing nuclear import. Regulated dephosphorylation of these sites activates SWI5 at the appropriate stage of the cell cycle by permitting its translocation to the nucleus.
Figure 7.10 Regulation of
nuclear import of transcription factors.
Whereas many proteins are
selectively transported from the cytoplasm into the nucleus, most RNAs are
exported from the nucleus to the cytoplasm. Since proteins are synthesized in
the cytoplasm, the export of mRNAs, rRNAs, and tRNAs is a critical step in gene
expression in eukaryotic cells. Like protein import, the export of RNAs through
nuclear pore complexes is an active, energy-dependent process that requires the
Ran GTP-binding protein.
Figure 7.11. Transport of snRNAs
between nucleus and cytoplasm
RNAs are transported across
the nuclear envelope as RNA-protein complexes, which in some cases are large
enough to visualize by electron microscopy. The substrates for transport are
ribonucleoprotein complexes rather than naked RNAs, and RNAs are targeted for
transport from the nucleus by nuclear export signals on the proteins bound to them.
These proteins are recognized by exportins and transported from the nucleus to
the cytoplasm as described earlier. Pre-mRNAs and mRNAs are associated with a
set of at least 20 proteins (forming heterogeneous nuclear
ribonucleoproteins, or hnRNPs) throughout their processing in the
nucleus and eventual transport to the cytoplasm. At least two of these hnRNP
proteins contain nuclear export signals and are thought to function as carriers
of mRNAs during their export to the cytoplasm. As discussed in a later section
of this chapter, ribosomal RNAs are assembled with ribosomal proteins in the
nucleolus, and intact ribosomal subunits are then transported to the cytoplasm.
Their export from the nucleus appears to be mediated by nuclear export signals
present on ribosomal proteins. For tRNAs, the specific proteins that mediate
nuclear export remain to be identified.
In contrast to mRNAs, tRNAs,
and rRNAs, which function in the cytoplasm, the snRNAs function within the
nucleus as components of the RNA processing machinery. Perhaps surprisingly,
these RNAs are initially transported from the nucleus to the cytoplasm, where
they associate with proteins to form functional snRNPs and then return to the
nucleus (Fig 7.11). Proteins that bind to the 5′ caps of snRNAs appear to be
involved in the export of the snRNAs to the cytoplasm, whereas sequences
present on the snRNP proteins are responsible for the transport of snRNPs from
the cytoplasm to the nucleus.
7.2 Internal Organization of the Nucleus
The nucleus is more than a
container in which chromatin, RNAs, and nuclear proteins move freely in aqueous
solution. Instead, the nucleus appears to have an internal structure that
organizes the genetic material and localizes some nuclear functions to discrete
sites. The most obvious aspect of the internal organization of the nucleus is
the nucleolus, which, as discussed in the following section, is the site at
which the rRNA genes are transcribed and ribosomal subunits are assembled.
Additional elements of internal nuclear structure are suggested by the
organization of chromosomes and by the potential localization of functions such
as DNA replication and pre-mRNA processing to distinct nuclear domains.
Figure
7.12 Heterochromatin and
Euchromatin
Chromatin becomes highly
condensed during mitosis to form the compact metaphase chromosomes that are
distributed to daughter nuclei. During interphase, some of the chromatin
(heterochromatin) remains highly condensed and is transcriptionally inactive;
the remainder of the chromatin (euchromatin) is decondensed and distributed
throughout the nucleus (Fig 7.12). Cells contain two types of heterochromatin.
Constitutive heterochromatin contains DNA sequences that are never transcribed,
such as the satellite sequences present at centromeres. Facultative
heterochromatin contains sequences that are not transcribed in the cell being
examined, but are transcribed in other cell types. Consequently, the amount of
facultative heterochromatin varies depending on the transcriptional activity of
the cell. Much of the heterochromatin is localized to the periphery of the
nucleus, possibly because one of the principal proteins associated with
heterochromatin binds to a protein of the inner nuclear membrane.
The phenomenon of X
chromosome inactivation provides an example of the role of heterochromatin
in gene expression. In many animals, including humans, females have two X
chromosomes, and males have one X and one Y chromosome. The X chromosome
contains thousands of genes that are not present on the much smaller Y
chromosome. Thus, females have twice as many X chromosome genes as males have.
Despite this difference, female and male cells contain equal amounts of the
proteins encoded by X chromosome genes. This results from a dosage compensation
mechanism in which one of the two X chromosomes in female cells is inactivated
by being converted to heterochromatin early in development. Consequently, only
one copy of the X chromosome is available for transcription in either female or
male cells. The mechanism of X chromosome inactivation is fascinating though
not yet fully understood; it appears to involve the action of a regulatory RNA
that coats the inactive X chromosome and induces its conversion to
heterochromatin.
Although interphase chromatin
appears to be uniformly distributed, the chromosomes are actually arranged in
an organized fashion and divided into discrete functional domains that play an
important role in regulating gene expression. The nonrandom distribution of
chromatin within the interphase nucleus was first suggested in 1885 by C. Rabl,
who proposed that each chromosome occupies a distinct territory, with
centromeres and telomeres attached to opposite sides of the nuclear envelope.
This basic model of chromosome organization was confirmed nearly a hundred
years later (in 1984) by detailed studies of polytene chromosomes in Drosophila salivary glands. Rather than randomly winding around one another, each
chromosome was found to occupy a discrete region of the nucleus. The
chromosomes are closely associated with the nuclear envelope at many sites,
with their centromeres and telomeres clustered at opposite poles.
Individual chromosomes also
occupy distinct territories within the nuclei of mammalian cells (Fig7.13).
Actively transcribed genes appear to be localized to the periphery of these
territories, adjacent to channels separating the chromosomes. Newly transcribed
RNAs are thought to be released into these channels between chromosomes, where
RNA processing takes place.
Figure 7.13. Organization of chromosomes in the
mammalian nucleus
Like the DNA in metaphase
chromosomes, the chromatin in interphase nuclei appears to be organized into
looped domains containing approximately 50 to 100 kb of DNA. A good example of
this looped-domain organization is provided by the highly transcribed
chromosomes of amphibian oocytes, in which actively transcribed regions of DNA
can be visualized as extended loops of decondensed chromatin. These chromatin
domains appear to represent discrete functional units, which independently
regulate gene expression.
The effects of chromosome
organization on gene expression have been demonstrated by a variety of
experiments showing that the position of a gene in chromosomal DNA affects the
level at which the gene is expressed. For example, the transcriptional activity
of genes introduced into transgenic mice depends on their sites of integration
in the mouse genome. This effect of chromosomal position on gene expression can
be alleviated by sequences known as locus control regions, which result
in a high level of expression of the introduced genes irrespective of their
site of integration. In contrast to transcriptional enhancers, locus control
regions stimulate only transfected genes that are integrated into chromosomal
DNA; they do not affect the expression of unintegrated plasmid DNAs in
transient assays. In addition, rather than affecting individual promoters,
locus control regions appear to activate large chromosome domains, presumably
by inducing long-range alterations in chromatin structure.
The separation between
chromosomal domains is maintained by boundary sequences or insulator
elements, which prevent the chromatin structure of one domain from
spreading to its neighbors. In addition, insulators act as barriers that
prevent enhancers in one domain from acting on promoters located in an adjacent
domain. Like locus control regions, insulators function only in the context of
chromosomal DNA, suggesting that they regulate higher-order chromatin
structure. Although the mechanisms of action of locus control regions and
insulators remain to be elucidated, their functions clearly indicate the
importance of higher-order chromatin organization in the control of eukaryotic
gene expression.
An internal organization of
the nucleus is indicated also by the localization of some nuclear processes to
distinct regions of the nucleus. Rather than taking place throughout the
nucleus, activities such as DNA replication and pre-mRNA processing may be
localized to discrete subnuclear structures or domains. The nature and function
of these nuclear substructures are not yet clear, however, and understanding
the organization of functional domains within the nucleus is an incompletely
explored area of cell biology.
The nuclei of mammalian cells
appear to contain clustered sites of DNA replication within which the
replication of multiple DNA molecules takes place. These discrete sites of DNA replication
have been defined by experiments in which newly synthesized DNA was visualized
within cell nuclei. This was accomplished by labeling cells with
bromodeoxyuridine—an analog of thymidine that can be incorporated into DNA and
then detected by staining with fluorescent antibodies. In such experiments, the
newly replicated DNA was detected in approximately 200 discrete clusters
distributed throughout the nucleus. Since approximately 4000 origins of
replication are active in a diploid mammalian cell at any given time, each of
these clustered sites of DNA replication must contain approximately 40
replication forks. Thus, DNA replication appears to take place in large
structures that contain multiple replication complexes organized into distinct
functional domains, which have been called replication factories.
Actively transcribed genes
appear to be distributed throughout the nucleus, but components of the splicing
machinery are concentrated in discrete subnuclear structural domains. The
localization of splicing components to discrete domains within the nucleus has
been demonstrated by immunofluorescent staining with antibodies against snRNPs
and splicing factors. Rather than being distributed uniformly throughout the
nucleus, these components of the splicing apparatus are concentrated in 20 to
50 discrete structures termed nuclear speckles. It is thought that speckles are
storage sites of splicing components, which are then recruited from the
speckles to actively transcribed genes where pre-mRNA processing occurs.
In addition to speckles,
nuclei contain several other types of morphologically distinct structures,
collectively called nuclear bodies. The two major types of these nuclear
structures are coiled bodies and PML bodies. Coiled bodies are enriched in
snRNPs and are believed to function as sites of snRNP assembly. The function of
PML bodies is unknown; they are not enriched in snRNPs and do not appear to be
major sites of transcription or DNA replication. Thus, although nuclear bodies
indicate the presence of substructural domains within the nucleus, their
functions remain to be fully elucidated.
Some scientists believe that a
central component of the internal organization of the nucleus is the nuclear
matrix, which is defined as the structural skeleton of the nucleus. The concept
of the nuclear matrix was proposed in 1975 on the basis of experiments in which
nuclei were treated with DNase to digest most of the DNA and extracted with
high salt buffers to remove histones and other nuclear proteins. Such treatments
left a residual network of fibers (the nuclear matrix), which was suggested to
provide an internal structural framework for the nucleus, analogous to the role
of the cytoskeleton as the structural framework of the cell. In addition, it
has been proposed that the nuclear matrix serves to organize and anchor
functional domains of the nucleus, including chromatin loops, DNA replication
factories, splicing domains, and structures involved in mRNA transport.
Many scientists, however, are
not convinced that the nuclear matrix exists in the living cell. They believe
that the matrix observed after extraction of nuclei may be the result of
artificial aggregation of proteins and nucleic acids during preparation.
Although the nuclear matrix is visualized after extraction of nuclei by several
different methods, its molecular composition has not been clearly defined. In
the absence of definitive characterization of its structural components, the
nuclear matrix remains an area of dispute among cell biologists.
7.3 The Nucleolus
The most prominent
substructure within the nucleus is the nucleolus, which is the site of rRNA
transcription and processing, and of ribosome assembly. Cells require large
numbers of ribosomes to meet their needs for protein synthesis. Actively
growing mammalian cells, for example, contain 5 million to 10 million ribosomes
that must be synthesized each time the cell divides. The nucleolus is a
ribosome production factory, designed to fulfill the need for large-scale
production of rRNAs and assembly of the ribosomal subunits.
Figure 7.14. Ribosomal RNA genes.
The nucleolus, which is not
surrounded by a membrane, is organized around the chromosomal regions that
contain the genes for the 5.8S, 18S, and 28S rRNAs. Eukaryotic ribosomes
contain four types of RNA, designated the 5S, 5.8S, 18S, and 28S rRNAs. The
5.8S, 18S, and 28S rRNAs are transcribed as a single unit within the nucleolus
by RNA polymerase I, yielding a 45S ribosomal precursor RNA (Fig 7.14). The 45S
pre-rRNA is processed to the 18S rRNA of the 40S (small) ribosomal subunit and
to the 5.8S and 28S rRNAs of the 60S (large) ribosomal subunit. Transcription
of the 5S rRNA, which is also found in the 60S ribosomal subunit, takes place
outside of the nucleolus and is catalyzed by RNA polymerase III.
To meet the need for
transcription of large numbers of rRNA molecules, all cells contain multiple
copies of the rRNA genes. The human genome, for example, contains about 200 copies
of the gene that encodes the 5.8S, 18S, and 28S rRNAs and approximately 2000
copies of the gene that encodes 5S rRNA. The genes for 5.8S, 18S, and 28S rRNAs
are clustered in tandem arrays on five different human chromosomes (chromosomes
13, 14, 15, 21, and 22); the 5S rRNA genes are present in a single tandem array
on chromosome 1.
The importance of ribosome
production is particularly evident in oocytes, in which the rRNA genes are
amplified to support the synthesis of the large numbers of ribosomes required
for early embryonic development. In Xenopus oocytes, the rRNA genes are
amplified approximately 2000-fold, resulting in about 1 million copies per
cell. These amplified rRNA genes are distributed to thousands of nucleoli,
which support the accumulation of nearly 1012 ribosomes per oocyte.
Morphologically, nucleoli
consist of three distinguishable regions: the fibrillar center, dense fibrillar
component, and granular component.(Fig 7.15) These different regions are
thought to represent the sites of progressive stages of rRNA transcription,
processing, and ribosome assembly. The rRNA genes are located in the fibrillar
centers, with transcription occurring primarily at the boundary of the
fibrillar centers and dense fibrillar component. Processing of the pre-rRNA is
initiated in the dense fibrillar component and continues in the granular
component, where the rRNA is assembled with ribosomal proteins to form nearly
completed preribosomal subunits, ready for export to the cytoplasm.
Figure
7.15 Organization of nucleolus
Figure
7.16 The formation of nucleoli
Figure
7.17 The cycle of nucleoli(right)
Following each cell division,
nucleoli form around the chromosomal regions that contain the 5.8S, 18S, and
28S rRNA genes, which are therefore called nucleolar organizing regions.
The formation of nucleoli requires the transcription of 45S pre-rRNA, which
appears to lead to the fusion of small prenucleolar bodies that contain
processing factors and other components of the nucleolus.(Fig 7.16) In most
cells, the initially separate nucleoli then fuse to form a single nucleolus.
The size of the nucleolus depends on the metabolic activity of the cell, with
large nucleoli found in cells that are actively engaged in protein synthesis.
This variation is due primarily to differences in the size of the granular
component, reflecting the levels of ribosome synthesis(Fig 7.17)..
Each nucleolar organizing
region contains a cluster of tandemly repeated rRNA genes that are separated
from each other by nontranscribed spacer DNA. These genes are very actively
transcribed by RNA polymerase I, allowing their transcription to be readily
visualized by electron microscopy. In such electron micrographs, each of the
tandemly arrayed rRNA genes is surrounded by densely packed growing RNA chains,
forming a structure that looks like a Christmas tree. The high density of
growing RNA chains reflects that of RNA polymerase molecules, which are present
at a maximal density of approximately one polymerase per hundred base pairs of
template DNA.
Figure 7.18. Processing of pre-rRNA
The primary transcript of the
rRNA genes is the large 45S pre-rRNA, which contains the 18S, 5.8S, and 28S
rRNAs as well as transcribed spacer regions (Fig 7.18). External transcribed
spacers are present at both the 5′ and 3′ ends of the pre-rRNAs, and two
internal transcribed spacers lie between the 18S, 5.8S, and 28S rRNA sequences.
The initial processing step is a cleavage within the external transcribed
spacer near the 5′ end of the pre-rRNA, which takes place during the early
stages of transcription. This cleavage requires the U3 small nucleolar RNP that
remains attached to the 5′ end of the pre-rRNA, forming the characteristic
knobs. Once transcription is complete, the external transcribed spacer at the
3′ end of the molecule is removed. In human cells, this step is followed by a
cleavage at the 5′ end of the 5.8S region, yielding separate precursors to the
18S and 5.8S + 28S rRNAs. Additional cleavages then result in formation of the
mature rRNAs. Processing follows a similar pattern in other species, although
there are differences in the order of some of the cleavages.
In addition to cleavage, the
processing of pre-rRNA involves a substantial amount of base modification resulting
both from the addition of methyl groups to specific bases and ribose residues
and from the conversion of uridine to pseudouridine. In animal cells, pre-rRNA
processing involves the methylation of approximately a hundred ribose residues
and ten bases, in addition to the formation of about a hundred pseudouridines.
Most of these modifications occur during or shortly after synthesis of the
pre-rRNA, although a few take place at later stages of pre-rRNA processing.
The processing of pre-rRNA
requires the action of both proteins and RNAs that are localized to the
nucleolus. The small nuclear RNAs (snRNAs) are involved in pre-mRNA splicing.
Nucleoli contain a large number (about 200) of small nucleolar RNAs
(snoRNAs) that function in pre-rRNA processing. Like the spliceosomal
snRNAs, the snoRNAs are complexed with proteins, forming snoRNPs. Individual
snoRNPs consist of single snoRNAs associated with eight to ten proteins. The
snoRNPs then assemble on the pre-rRNA to form processing complexes in a manner
analogous to the formation of spliceosomes on pre-mRNA.
Some snoRNAs are responsible
for the cleavages of pre-rRNA into 18S, 5.8S, and 28S products. For example,
the most abundant nucleolar snoRNA is U3, which is present in about 200,000
copies per cell. As already noted, U3 is required for the initial cleavage of
pre-rRNA within the 5′ external transcribed spacer sequences. Similarly, U8
snoRNA is responsible for cleavage of pre-rRNA to 5.8S and 28S rRNAs, and U22
snoRNA is responsible for cleavage
of pre-rRNA to 18S rRNA.
Figure 7.19. Role of snoRNAs in base modification of pre-rRNA
The majority of snoRNAs,
however, function to direct the specific base modifications of pre-rRNA,
including the methylation of specific ribose residues and the formation of pseudouridines
(Fig 7.19). Most of the snoRNAs contain short sequences of approximately 15
nucleotides that are complementary to 18S or 28S rRNA. Importantly, these
regions of complementarity include the sites of base modification in the rRNA.
By base pairing with specific regions of the pre-rRNA, the snoRNAs act as guide
RNAs that target the enzymes responsible for ribose methylation or
pseudouridylation to the correct site on the pre-rRNA molecule.
The formation of ribosomes
involves the assembly of the ribosomal precursor RNA with both ribosomal
proteins and 5S rRNA (Fige 7.20). The genes that encode ribosomal proteins are
transcribed outside of the nucleolus by RNA polymerase II, yielding mRNAs that
are translated on cytoplasmic ribosomes. The ribosomal proteins are then
transported from the cytoplasm to the nucleolus, where they are assembled with
rRNAs to form preribosomal particles. Although the genes for 5S rRNA are also
transcribed outside of the nucleolus, in this case by RNA polymerase III, 5S
rRNAs similarly are assembled into preribosomal particles within the nucleolus.
The association of ribosomal
proteins with rRNA begins while the pre-rRNA is still being synthesized, and
more than half of the ribosomal proteins are complexed with the pre-rRNA prior
to its cleavage. The remaining ribosomal proteins and the 5S rRNA are
incorporated into preribosomal particles as cleavage of the pre-rRNA proceeds.
The smaller ribosomal subunit, which contains only the 18S rRNA, matures more rapidly
than the larger subunit, which contains 28S, 5.8S, and 5S rRNAs. Consequently,
most of the preribosomal particles in the nucleolus represent precursors to the
large subunit. The final stages of ribosome maturation follow the export of
preribosomal particles to the cytoplasm, forming the active 40S and 60S
subunits of eukaryotic ribosomes.
Figure 7.20. Ribosome
assembly
7.4
The Nucleus during Mitosis
A unique feature of the
nucleus is that it disassembles and re-forms each time most cells divide. At
the beginning of mitosis, the chromosomes condense, the nucleolus disappears,
and the nuclear envelope breaks down, resulting in the release of most of the
contents of the nucleus into the cytoplasm. At the end of mitosis, the process
is reversed: The chromosomes decondense, and nuclear envelopes re-form around
the separated sets of daughter chromosomes. In this section we will consider
the mechanisms involved in the disassembly and re-formation of the nucleus. The
process is controlled largely by reversible phosphorylation and
dephosphorylation of nuclear proteins resulting from the action of the Cdc2
protein kinase, which is a critical regulator of mitosis in all eukaryotic
cells.
Figure 8.30. Closed and open
mitosis
In most cells, the
disassembly of the nuclear envelope marks the end of the prophase of mitosis.
However, this disassembly of the nucleus is not a universal feature of mitosis
and does not occur in all cells. Some unicellular eukaryotes (e.g., yeasts)
undergo so-called closed mitosis, in which the nuclear envelope remains intact
(Fig 7.21). In closed mitosis, the daughter chromosomes migrate to opposite
poles of the nucleus, which then divides in two. The cells of higher
eukaryotes, however, usually undergo open mitosis, which is characterized by
breakdown of the nuclear envelope. The daughter chromosomes then migrate to
opposite poles of the mitotic spindle, and new nuclei reassemble around them.
Figure 7.22. Dissolution of the
nuclear lamina
Disassembly of the nuclear
envelope, which parallels a similar breakdown of the endoplasmic reticulum,
involves changes in all three of its components: The nuclear membranes are
fragmented into vesicles, the nuclear pore complexes dissociate, and the
nuclear lamina depolymerizes. The best understood of these events is
depolymerization of the nuclear lamina—the meshwork of filaments underlying the
nuclear membrane. The nuclear lamina is composed of fibrous proteins, lamins,
which associate with each other to form filaments. Disassembly of the nuclear
lamina results from phosphorylation of the lamins, which causes the filaments
to break down into individual lamin dimers (Fig 7.22). Phosphorylation of the
lamins is catalyzed by the Cdc2 protein kinase. Cdc2 (as well as other protein
kinases activated in mitotic cells) phosphorylates all the different types of
lamins, and treatment of isolated nuclei with Cdc2 has been shown to be
sufficient to induce depolymerization of the nuclear lamina. Moreover, the
requirement for lamin phosphorylation in the breakdown of the nuclear lamina
has been demonstrated directly by the construction of mutant lamins that can no
longer be phosphorylated. When genes encoding these mutant lamins were
introduced into cells, their expression was found to block normal breakdown of
the nuclear lamina as the cells entered mitosis.
Figure
7.23 The function of lamin in the reconstruction of nuclear envelope
In concert with dissolution of
the nuclear lamina, the nuclear membrane fragments into vesicles (Fig 7.23).
The B-type lamins remain associated with these vesicles, but lamins A and C
dissociate from the nuclear membrane and are released as free dimers in the
cytosol. This difference arises because the B-type lamins are permanently
modified by the addition of lipid (prenyl groups), whereas the C-terminal
prenyl groups of A- and C-type lamins are removed by proteolysis following
their incorporation into the lamina. The nuclear pore complexes also dissociate
into subunits as a result of phosphorylation of several nuclear pore proteins.
Integral nuclear membrane proteins are also phosphorylated at mitosis, and
phosphorylation of these proteins may be important in vesicle formation as well
as in dissociation of the nuclear membrane from both chromosomes and the
nuclear lamina.
The other major change in
nuclear structure during mitosis is chromosome condensation. The interphase
chromatin, which is already packaged into nucleosomes, condenses approximately
a thousandfold further to form the compact chromosomes seen in mitotic cells.
This condensation is needed to allow the chromosomes to move along the mitotic
spindle without becoming tangled or broken during their distribution to daughter
cells. DNA in this highly condensed state can no longer be transcribed, so all
RNA synthesis stops during mitosis. As the chromosomes condense and
transcription ceases, the nucleolus also disappears.
The condensed DNA in metaphase
chromosomes appears to be organized into large loops, each encompassing about a
hundred kilobases of DNA, which are attached to a protein scaffold. Despite its
fundamental importance, the mechanism of chromosome condensation during mitosis
is not understood. The basic unit of chromatin structure is the nucleosome,
which consists of 146 base pairs of DNA wrapped around a histone core
containing two molecules each of histones H
Figure 7.24. The organization of chromatin in nucleosomes
Recent studies have also
identified protein complexes called condensins that play a major role in
chromosome condensation. Condensins are required for chromosome condensation in
extracts of mitotic cells and appear to function by wrapping DNA around itself,
thereby compacting chromosomes into the condensed mitotic structure. Condensins
are phosphorylated and activated by the Cdc2 protein kinase, providing a direct
link between activation of Cdc2 and mitotic chromosome condensation.
During the completion of
mitosis (telophase), two new nuclei form around the separated sets of daughter
chromosomes. Chromosome decondensation and reassembly of the nuclear envelope
appear to be signaled by inactivation of Cdc2, which was responsible for
initiating mitosis by phosphorylating cellular target proteins, including the
lamins, histone H3, and condensins. The progression from metaphase to anaphase
involves the activation of a ubiquitin-mediated proteolysis system that
inactivates Cdc2 by degrading its regulatory subunit, cyclin B. Inactivation of
Cdc2 leads to the dephosphorylation of the proteins that were phosphorylated at
the initiation of mitosis, resulting in exit from mitosis and the re-formation
of interphase nuclei.(Fig 7.25)
Figure 7.25. Cyclin degradation
during the cell cycle
The initial step in
re-formation of the nuclear envelope is the binding of the vesicles formed
during nuclear membrane breakdown to the surface of chromosomes (Fig 7.26).
This interaction of membrane vesicles with chromosomes may be mediated by both
lamins and integral membrane proteins of the inner nuclear membrane. The
vesicles then fuse to form a double membrane around the chromosomes. This is
followed by reassembly of the nuclear pore complexes, re-formation of the
nuclear lamina, and chromosome decondensation. The vesicles first fuse to form
membranes around individual chromosomes, which then fuse with each other to
form a complete single nucleus.
Figure 7.26. Re-formation of the
nuclear envelope
The initial re-formation of
the nuclear envelope around condensed chromosomes excludes cytoplasmic
molecules from the newly assembled nucleus. The new nucleus is then able to
expand via the selective import of nuclear proteins from the cytoplasm. Because
nuclear localization signals are not cleaved from proteins that are imported to
the nucleus, the same nuclear proteins that were released into the cytoplasm
following disassembly of the nuclear envelope at the beginning of mitosis can
be reimported into the new nuclei formed after mitosis. The nucleolus, too,
re-forms as the chromosomes decondense and transcription of the rRNA genes
begins, completing the return from mitosis to an interphase nucleus
Summary
THE NUCLEAR ENVELOPE AND TRAFFIC BETWEEN THE NUCLEUS
AND CYTOPLASM
Structure of the Nuclear Envelope: The nuclear envelope separates the contents of the nucleus from the
cytoplasm, maintaining the nucleus as a distinct biochemical compartment that
houses the genetic material and serves as the site of transcription and RNA
processing in eukaryotic cells. The nuclear envelope consists of the inner and
outer nuclear membranes, which are joined at nuclear pore complexes, and an
underlying nuclear lamina.
The Nuclear Pore Complex: Nuclear pore complexes are large structures that provide the only routes through which molecules can travel between the nucleus and cytoplasm. Small molecules are able to diffuse freely through open channels in the nuclear pore complex. Macromolecules are selectively transported in an energy-dependent process.
Selective Transport of Proteins to and from the Nucleus: Proteins destined for import to the nucleus contain nuclear localization signals that are recognized by receptors that direct transport through the nuclear pore complex. Proteins that shuttle back and forth between the nucleus and the cytoplasm contain nuclear export signals that target them for transport from the nucleus to the cytoplasm. The small GTP-binding protein Ran is required for translocation through the nuclear pore complex and determines the directionality of transport.
Regulation of Nuclear Protein Import: The activity of some proteins, such as transcription factors, is controlled by regulation of their import to the nucleus.
Transport of RNAs: RNAs are transported through the nuclear pore complex as ribonucleoprotein complexes. Messenger RNAs, ribosomal RNAs, and transfer RNAs are exported from the nucleus to function in protein synthesis. Small nuclear RNAs are initially transported from the nucleus to the cytoplasm, where they associate with proteins to form snRNPs; then they return to the nucleus.
INTERNAL
ORGANIZATION OF THE NUCLEUS
Chromosomes and Higher-Order Chromatin Structure: The interphase
nucleus contains transcriptionally inactive, highly condensed heterochromatin
as well as decondensed euchromatin. Interphase chromosomes are organized within
the nucleus and divided into large looped domains that function as independent
units.
Functional Domains within the Nucleus: Some nuclear processes, such as DNA replication and pre-mRNA metabolism, may be localized to discrete subnuclear structures or domains.
THE NUCLEOLUS
Ribosomal RNA Genes and the
Organization of the Nucleolus: The nucleolus is organized around the genes
for ribosomal RNAs. It is the site of rRNA transcription and processing, and of
ribosome assembly.
Transcription and Processing of rRNA: The primary transcript of the rRNA genes is 45S pre-rRNA, which is processed to yield 18S, 5.8S, and 28S rRNAs. Processing of pre-rRNA is mediated by small nucleolar RNAs (snoRNAs).
Ribosome Assembly: Ribosomal subunits are assembled within the nucleolus from rRNAs and ribosomal proteins.
THE NUCLEUS
DURING MITOSIS
Dissolution of the Nuclear
Envelope: Entry into mitosis is signaled by activation of the Cdc2 protein
kinase. In most cells, the nuclear envelope breaks down at the end of prophase.
Depolymerization of the nuclear lamina results from phosphorylation of the
lamins by Cdc2 and other protein kinases.
Chromosome Condensation: Phosphorylation of histones H1 and H3 is correlated with the condensation of mitotic chromosomes, and H3 phosphorylation is required for proper chromosome condensation. A complex of proteins called condensin is activated by Cdc2 phosphorylation and functions in chromosome condensation by wrapping the DNA into a compact structure.
Re-formation of the Interphase Nucleus: Inactivation of Cdc2 at the end of mitosis leads to re-formation of the nuclear envelope and chromosome decondensation. Nuclear proteins are then selectively imported through nuclear pore complexes
Questions
1. You are studying a transcription factor that is regulated by phosphorylation of serine residues that inactivate its nuclear localization signal. How would mutating these serines to alanines affect subcellular localization of the transcription factor and expression of its target gene?
2.
How would mutational
inactivation of the nuclear export signal of a protein that normally shuttles
back and forth between the nucleus and cytoplasm affect its subcellular
distribution?
3.
You have constructed a mutant
importin β that no longer binds to Ran. What effect would expression of
this mutant importin have on the traffic of proteins between the nucleus and
the cytoplasm?
4.
Consider the construct
(diagrammed below) in which a single enhancer (E) acts on two separate
promoters (P1 and P2). How would insertion of an insulator between E and P1
affect transcription from P1 and P2? What about insertion of an insulator
between P1 and P2?
5.
Treatment of cells with
actinomycin D (a general inhibitor of transcription) prevents the formation of
nucleoli following mitosis. Treatment with α-amanitin (a specific
inhibitor of RNA polymerase II) does not have this effect. Why?