Specialized Subdomains of the Nucleus

Cell nuclei contain numerous discrete subdomains or bodies with distinctive structural organizations and/or biochemical composition (Fig. 14-2 and Table 14-1). The most prominent of these is the nucleolus, discussed in the next section. Although these subdomains are often referred to as organelles, unlike cytoplasmic organelles, nuclear subdomains are not membrane bounded. In fact, many proteins that have been examined by the fluorescence recovery after photobleaching technique (see Fig. 6-3) exhibit a relatively rapid exchange between a respective body and a nucleoplasmic pool of the protein. Therefore, although these bodies are discrete at steady state, they represent highly dynamic associations of macromolecular complexes. These domains, like the nucleolus, reflect the functional compartmentalization of the nucleoplasm.

Figure 14-2 examples of major subnuclear structures. A, Components involved in RNA processing are scattered throughout the nucleus but concentrated in domains called speckles that are rich in interchromatin granules. Inhibition of RNA processing causes splicing components to accumulate in enormous concentrations of interchromatin granule clusters. Several cells were injected with a short oligonucleotide that disrupts the function of the U1 snRNP in RNA processing (see Chapter 16), and were then stained with an antibody recognizing the Sm splicing components (green). The injected cells were marked by introducing an inert fluorescent dextran marker into the cytoplasm (red). B, Nucleus with simultaneous staining of nucleoli (blue), PML nuclear bodies (red), Cajal bodies (green), and the nuclear envelope (purple). C, Nucleus with simultaneous staining of chromatin (blue), nucleoli (red), speckles (green), and Cajal bodies (white). D, Nucleus with simultaneous staining of DNA (blue) and the polymorphic interphase karyosomal association (PIKA)/OPT domain (red). Nucleoli appear as unstained areas. A number of proteins involved in the sensing and repair of DNA damage concentrate in the PIKA.

(A, Courtesy of David Spector, Cold Spring Harbor Lab, New York. B–C, Courtesy of Angus Lamond, University of Dundee, Scotland. D, Courtesy of William S. Saunders and William C. Earnshaw.)

Table 14-1 MAJOR NUCLEAR SUBDOMAINS

RNA transcription and processing occur at up to 10,000 discrete sites spread throughout the average mammalian nucleus. These sites likely correspond to structures, originally observed by electron microscopy on the surface of regions of condensed chromatin, called perichromatin fibrils. Perichromatin fibrils contain various splicing factors and RNA-packaging proteins.

When factors involved in RNA processing are detected by fluorescence microscopy, 20 to 50 bright speckles are seen against a diffuse background of nucleoplasmic staining (Fig. 14-2). The diffuse staining probably corresponds to splicing factors associated with perichromatin fibrils at the thousands of sites where RNA transcription and processing take place. Speckles are less prominent in cells that transcribe RNA at high levels, and they become strikingly prominent when RNA processing is inhibited (Fig. 14-2).

Most speckles correspond to clusters of interchromatin granules, particles 20 to 25 nm in diameter that are distributed throughout the interchromosomal domain. Proteomic analysis of isolated interchromatin granules reveals that they contain approximately 240 stably associated proteins, most involved with various aspects of RNA processing. Interchromatin granules may be sites for the assembly, modification, or storage of protein complexes involved in pre-mRNA processing. Consistent with this, a number of components involved in RNA processing were shown to be highly dynamic. When tagged with green fluorescent protein, these mRNA-processing proteins cycle between speckles and sites of transcription in less than 1 minute in vivo.

Metabolic labeling experiments indicate that speckles are not major sites of active transcription, and most messenger RNAs (mRNAs) are seldom or never seen associated with speckles. However, mRNAs for certain genes accumulate either within or immediately adjacent to speckles. These “speckles” could be highly active transcription sites that have recruited a significant population of splicing factors and are indistinguishable from interchromatin granule clusters by fluorescent microscopy.

When cells enter mitosis, speckles disperse as RNA-processing factors redistribute diffusely throughout the cytoplasm. During telophase, processing factors reaggregate in the cytoplasm into punctate structures termed mitotic interchromatin granule clusters. These factors are subsequently imported into the nucleus at the completion of mitosis.

Cajal bodies (formerly known as coiled bodies) are compact structures about 0.3 to 1.0μm in diameter (Fig. 14-2) that resemble balls of tangled threads in the electron microscope. Nuclei contain 1 to 10 Cajal bodies, which accumulate many factors involved in mRNA processing, as well as a number of nucleolar components, but lack non-snRNP protein-splicing factors that are present in speckles. They also contain an 80-kD human autoantigen of unknown function, called p80-coilin. In contrast to speckles, Cajal bodies disperse when transcription and splicing are blocked, and they are particularly prominent in rapidly growing cells with high levels of gene expression. However, like speckles, some Cajal bodies disassemble during mitosis and reform during the G₁ phase after transcription is reinitiated. Cajal bodies are connected with the maturation of newly imported snRNP and snoRNP particles (see Chapter 16). They may also have other functions, including possibly regulating expression of snRNA gene clusters.

Mammalian nuclei also contain about 10 to 30 bodies, varying in size from 0.3 to 1μm, known as promyelocytic leukemia (PML) bodies (other names are listed in Table 14-1) that are often juxtaposed with Cajal bodies (Fig. 14-2). PML bodies were initially defined by the presence of a protein called PML, and other components have since been identified. PML has a RING finger amino acid sequence motif and is therefore likely to be a ligase for ubiquitin or ubiquitin-like proteins (see Fig. 23-8). Its targets are unknown, but several components of PML bodies are conjugated to the ubiquitin-like protein SUMO-1.

The PML gene was identified by analysis of a chromosome translocation between chromosomes 15 and 17 found in patients with acute promyelocytic leukemia (APL). In many patients, this translocation produces a gene fusion between PML and the retinoic acid receptor alpha (RARa). The fusion protein is termed PML-RARa. Antibodies to PML protein stained subnuclear structures that were given the name PML bodies. In APL cells, PML bodies are “shattered” into many tiny punctate foci scattered throughout the nucleus. However, when APL cells are treated with drugs that are clinically effective in the treatment of patients with APL, such as retinoic acid and arsenic trioxide, the PML bodies reform, and the PML-RARa fusion protein is degraded. This reveals a tantalizing link between these structures and the cancerous phenotype. At present, the function of PML bodies remains uncertain.

The Nucleolus: The Most Prominent Nuclear Subdomain

The nucleolus, first described only five years after the nucleus, in 1835, is the most conspicuous and best-characterized nuclear subdomain (Fig. 14-3). Most mammalian cell nuclei have one to five nucleoli, which are specialized regions 0.5 to 5.0μm in diameter surrounding transcriptionally active ribosomal RNA (rRNA) gene clusters. Within nucleoli occur the bulk of the steps for ribosome biogenesis, from the transcription and processing of rRNA to the initial assembly of ribosomal subunits. The ribosome is a complex macromolecular machine with four different structural RNA molecules and about 85 proteins that are assembled into two subunits (see Figs. 17-6 and 17-7). rRNA transcription by RNA polymerase I comprises nearly one half of total cellular RNA synthesis in some cell types. This high level of synthesis is necessary to produce about 5 million ribosomes in each cell cycle, more than 30 every second in budding yeast.

Figure 14-3 nucleolus and nucleolar organizer region. A, Electron micrograph of a thin section of a typical nucleolus. The fibrillar centers, dense fibrillar component, and granular component are indicated. B, Use of silver staining to visualize the nucleolus in interphase nuclei and the nucleolar organizer regions on mitotic chromosomes of the rat kangaroo.

(A, From Fawcett DW: The Cell. Philadelphia, WB Saunders, 1981. B, From Robert-Fortel I, Junera HR, Geraud G, et al: Three-dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy. Chromosoma 102:146–157, 1993.)

Over 690 proteins associate stably with human nucleoli. Many more may associate transiently, and this composition changes to reflect different metabolic states of the cell (Fig. 14-4). Many of these nucleolar proteins are involved with either ribosomal RNA synthesis and modification or with ribosome subunit assembly. Surprisingly, the functions of many (˜80) other nucleolar proteins remain unknown and may reflect the involvement of nucleoli in other biological processes. Other stable RNAs, including the RNA component of the signal recognition particle (SRP; see Fig. 20-5), are also processed in the nucleolus, and the nucleolus might have other as yet undiscovered functions.

Figure 14-4 dynamic analysis of chromatin and a major nucleolar component. A, Fluorescence recovery after photobleaching (FRAP) of H2B-GFP shows that chromatin is immobile within the cell nucleus. B, FRAP of fibrillarin-GFP shows that this major component of nucleoli is highly dynamic. Scale bar: 5 mm.

(A–B, Courtesy of Tom Misteli. B, From Phair RD, Misteli T: High mobility of proteins in the mammalian cell nucleus. Nature 404:604–609, 2000.)

Ribosomal Biogenesis in Functionally Distinct Regions of the Nucleolus

The nucleolus contains three morphologically distinct regions in thin sections viewed by transmission electron microscopy (Fig. 14-3). Fibrillar centers contain concentrations of rRNA genes, together with significant amounts of RNA polymerase I and its associated transcription factors. Actively transcribed ribosomal genes are found near the border between the fibrillar centers and a dense fibrillar component that surrounds them. The granular component is the site for many steps in ribosome subunit assembly and is made up of densely packed clusters of preribosomal particles 15 to 20 nm in diameter.

Ribosomal RNA loci have a modular organization, with genes alternating with spacer regions in large tandemly arranged clusters (see Fig. 16-9). The repeat unit in this array (gene plus spacer) is approximately 40,000 base pairs in humans. Humans have approximately 300 to 400 copies of the ribosomal DNA (rDNA) repeat unit located in clusters on chromosomes 13, 14, 15, 21, and 22. Usually, only a fraction of these genes is actively transcribed. An additional rRNA, 5S, is encoded by distinct genes and transcribed by RNA polymerase III (see Fig. 15-10).

A simple yet efficient mechanism guarantees a balance between the RNA components of the two ribosomal subunits. The major rRNA components are encoded by a single precursor RNA molecule. In humans, this 13,000-base precursor is commonly described by its sedimentation coefficient in sucrose gradients as 45S. Following its transcription, the RNA precursor is processed in a series of cleavages to yield the 18S, 5.8S, and 28S rRNA molecules (see Fig. 16-9). In addition to the cleavages, rRNA processing also involves extensive base and sugar modifications, including approximately 100 2′-O-methyl ribose and approximately 90 pseudouridine residues per molecule. The earliest stages of rRNA processing probably occur in the dense fibrillar component of the nucleolus. Later stages take place in the granular component. Ribosomal protein synthesis occurs in the cytoplasm on free ribosomes, and the newly synthesized proteins are transported into the nucleus for assembly into ribosomes, predominantly in the granular component.

Disassembly of the Nucleolus during Mitosis

The nucleolus disassembles during each mitotic cycle, starting with the dispersal of the dense fibrillar and granular components during prophase. This disassembly is apparently driven by specific phosphorylation of nucleolar proteins. Ultimately, the fibrillar centers alone remain associated with the mitotic chromosomes, forming what are termed nucleolus-organizing regions (NORs [Fig. 14-3B]). NORs are often the sites of a prominent secondary constriction of the chromosome. (The primary constriction is the centromere.) The nucleolar proteins nucleolin and RNA polymerase I remain bound at NORs as cells enter and exit mitosis.

Nucleolar reformation begins in telophase as processing factors and unprocessed pre-RNA remaining from the previous cell cycle associate with NORs (10 in human), which then cluster into one to five foci. Next, a wide variety of nucleolar components assemble into particles termed prenucleolar bodies that associate with the NORs in a process requiring transcription of the rRNA genes. Normally, nascent transcripts, rather than ribosomal genes, nucleate assembly of the nucleolus in each cell cycle. If antibodies to RNA polymerase I are microinjected into mitotic cells, rRNA transcription is blocked, and nucleoli do not reform in the next G₁ phase.

Structure and Assembly of the Nuclear Lamina

The nuclear lamina is a protein meshwork, typically 20 to 40 nm thick, composed of type V intermediate filament proteins called nuclear lamins (Fig. 14-6). Mammalian lamins are generally divided into two families. Lamin A is encoded by a gene that gives rise to four polypeptides (including lamin C) by alternative splicing (see Fig. 16-6). Members of the lamin B family are the products of two distinct genes.

Figure 14-6 the nuclear lamina. A, Thin-section electron micrograph of a nuclear envelope with a prominent nuclear lamina and nuclear pores. B, Field emission scanning electron micrograph of the inner surface of an amphibian oocyte nuclear envelope. The nuclear pores are prominent, protruding above the underlying nuclear lamina.

(A, Reference: Fawcett DW: The Cell. Philadelphia, WB Saunders, 1981, Fig. 156 [upper]. B, From Zhang C, Jenkins H, Goldberg MW, et al: Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract. J Cell Sci 109:2275–2286, 1996.)