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.
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.
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.
Molecular traffic through nuclear pore complexes
Depending on their size,
molecules can travel through the nuclear pore complex by one of two different
Model of the nuclear pore complex
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.
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).
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.
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.
Role of the Ran protein in nuclear import(right)
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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)
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.
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
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.
ORGANIZATION OF THE NUCLEUS
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.
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.
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
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?