[Frontiers in Bioscience 3, d376-398, March 26, 98]
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TRANSCRIPTION BY RNA POLYMERASE I

Katherine M. Hannan, Ross D. Hannan and Lawrence I. Rothblum

Henry Hood Research Program, Department of Molecular and Cellular Physiology, Penn State College of Medicine, Weis Centre for Research, 100 N. Academy Avenue, Danville, PA 17822-2618

Received 3/18/98 Accepted 3/23/98

3. GENERAL BACKGROUND

3.1. The Nucleolus

The interphase nucleus contains varying numbers of nucleoli. In metazoans, the nucleolus is the site of 45S rRNA synthesis, i.e. transcription of the ribosomal genes (rDNA), rRNA processing and ribonucleoprotein (RNP) assembly (6,18). The only active genes in the metazoan nucleolus are the rRNA genes, and the only RNA polymerase is RNA polymerase I.

Indeed the ribosomal genes are the central elements of the nucleolus and are localized at special chromosomal sites referred to as nucleolar organizer regions (NOR) (19). Surrounding the NOR is a fine network of filaments which forms a scaffolding distinguishable in both organization and composition from that of the "nuclear matrix". The scaffolding is thought to provide some "structural support" or organization to the arrangement of transcriptionally active rDNA and/or the assembly and transport of ribosomal subunits. This is supported by the observation that the nucleolar scaffolding is absent from cells which are inactive in rRNA synthesis such as nucleated erthyrocytes and spermatocytes (6,18).

Typically, mammalian nucleoli consist of three substructures which were named according to their appearance in transmission electron microscopy; i) Fibrillar Centers (FC); ii) Dense Fibrillar Component (DFC); and iii) Granular Component (GC) (6,20). The FC are pale staining regions in the center of the nucleoli consisting of a fine fibril (4-8 nm thick) network which is relatively opaque in the electron microscope (20). The rDNA, RNA polymerase I, and other components of the rDNA transcription system such as UBF, SL-1 and topoisomerase I have been localized to the periphery of this region (19,20,21,22,23). Thus, it is likely that the FC are the site where the primary rRNA transcript is generated. The DFC surrounds the FC and is characterized by densely packed fine fibrillae (3-5 nm thick), a high electron microscope contrast and a high content of a 34 kDa protein, fibrillarin (6,20). Fibrillarin is known to associate with proteins required in the early stages of rRNA processing, such as the U3, U8 and U13 snoRNP (small nucleolar RNP) (24). The GC is localized to the periphery of the nucleoli and consists of granular structures ranging in diameter from 10 to 15 nm, which are sometimes organized in short strings (20). The later stages of maturing ribosome precursor particles, before they are exported to the cytoplasm, have been localized to this region (20).

The boundaries of these substructures are not always discreet, in fact three different patterns of compartmentalization have been described and these are used to classify nucleoli. The type of nucleoli identified depends on the rate of ribosome production. Typically cells with a high rate of ribosome production, such as nerve and Leydig cells, have large and complex nucleoli described as compact or reticulate. Alternatively, cells with a lower rate of ribosome biogenesis, such as monocytes and lymphocytes, exhibit ring-shaped small nucleoli and a single FC (20).

A diploid human cell contains 10 NORs, thus it would be expected to have 10 nucleoli. However this is seldom the case. This discrepancy could be explained by two situations: i) not all NOR are active; or ii) more than one NOR can be included in a nucleolus. Both situations have been identified. For example, in some cells not all NORís are active (19,20,21,22). In human Hep-2 cells the transcription factor UBF associates with only six-to-eight of the possible ten NOR and in PtK1 cells UBF is found in 50% of the NORís (22). In each case, upon cell division there is an equal apportionment of UBF to the daughter cells. Alternatively, it has been shown that when lymphocytes are activated the previously inactive NOR fuse with the existing functional nucleoli (20).

During mitosis, as the cells enter prophase, the nuclei and nucleoli undergo rapid changes. For example the nuclear envelope disintegrates, chromosomes condense and subsequently the spindle apparatus form. In addition, the nucleoli disperse and disappear (20). At least a portion of some nucleolar components, such as RNA polymerase I, SL-1, UBF, and topoisomerase I, remain associated with the NOR (6,19,21,22,23), while others are released, such as NO38 (25) and the snoRNP (24,26). Nucleolar reformation usually begins during telophase with the daughter nucleoli forming at the NOR. Complete restoration of nucleolar morphology requires both ribosomal chromatin and active rDNA transcription (19). Thus, those NOR containing the RNA polymerase I transcription apparatus are more quickly able to initiate rDNA transcription and contribute to nucleolar regeneration (19,21,22,23).

In general, chromosomal DNA is organized in nucleosome structures. However, from electron microscopy, it has been suggested that the rRNA chromatin does not form a typical compact nucleosome structures. In fact, some reports suggest there are no nucleosomes on the transcribed rDNA (27,28). As one might expect, the nuclease digestion and psoralen cross-linking properties of the rDNA are atypical. This has also been examined using topoisomerase I digestion to examine the nucleoprotein structure of the rDNA. Topoisomerase I digestion sites were found to be spaced with a periodicity of 200 bp and concentrated in the regions encoding the 18S, 5.8S and 28S rRNA (28). This pattern was due to binding of nuclear proteins to the rDNA and not dependent on the DNA sequence itself (27,29). UV laser-induced histone-DNA cross-links studies demonstrated that the rDNA coding sequence, spacer enhancer and spacer promoter were associated with histones in both transcriptionally active and inactive cells (30,31). Interestingly, a recent study (32) suggested that the nucleosome structure may play a role in the regulation of initiation complex formation on the rDNA. That study demonstrated that histone octamers could compete with the transcription factors for the rDNA promoter, but only if the DNA was not first bound with an initiation complex (32). However, to date, a complete picture of the rDNA nucleosome structure and its function(s) is unclear.

3.2. Synthesis and Assembly of Ribosomes

The synthesis of ribosomes requires the coordinate effort of all three DNA-dependent RNA polymerases (6,18). RNA polymerase I, in the nucleolus, transcribes the rRNA gene that encodes the 45S precursor of the 18S, 5.8S and 28S rRNAs (figure 1). The 45S precursor rRNA is neither capped nor polyadenylated and can account for 1/3 to 1/2 of all nuclear RNA synthesis. To a lesser extent RNA polymerase I transcribes another transcript which originates from the spacer promoter located in the intergenic spacer (figure 1). However, this second transcript is unstable and its function is yet to be established (4). RNA polymerase III, in the nucleus, transcribes the 5S RNA gene (6). RNA polymerase II, in the nucleoplasm, transcribes numerous genes encoding ribosome associated proteins (r-proteins). These mRNAs are transported to the cytoplasm, translated and the mature r-proteins returned to the nucleolus for assembly of the ribosome components (6).

Figure 1. Schematic depiction of a mammalian ribosomal DNA repeat: The top portion of the cartoon depicts one and one-half ribosomal repeats in tandem, including the terminator Sal box, intergenic spacer, repetitive elements, enhancer region and the region transcribed to yield 45S rRNA. A section of the repeat is enlarged in the bottom portion of the cartoon. This section illustrates the placement of the spacer and 45S rDNA promoters, the proximal (To) and downstream promoter terminator elements (T1-7), the transcription initiation site (+6) and the external transcribed spacers (ETS).

Mammalian ribosomal subunits are assembled in discrete stages within the GC of the nucleolus. Initially the 45S precursor rRNA is processed via a complex series of specific exo- and endo-nucleolytic cleavages. The rRNA exons are not spliced together thus the 45S precursor generates the 18S, 5.8S and 28S rRNAs. rRNA processing is directed by snoRNPs such as nucleoli U3 snRNP. U3 snRNP has been implicated in several steps, including the earliest step in rRNA processing, the cleavage at -650 in the 5í external transcribed spacer (ETS) (figure 1) (6).

The 18S, 28S and 5.8S rRNAs associate with the 5S rRNA and r-proteins to form a complex referred to as the 80S preribosome. The 80S is further processed to generate the 40S and 60S ribosomal subunits. Studies have shown that the order of r-protein addition in this process is essential for successful assembly of the ribosomal subunits. For example, a decrease in the cellular content of the r-proteins L13 or L16 can result in a deficiency of the 60S ribosomal subunit (6). The 40S and 60S ribosomal subunits are transported to the cytoplasm, where they the final stages of maturation occur. This involves the association with additional proteins, such as initiation factors. They are then able to participate in translation (6).

The accumulation of mature ribosomes in the cell depends on the balance between the rate of subunit synthesis and the rate of degradation. Since mature ribosomes are fairly stable complexes, with half lives ranging from 4.5 days in rat liver to more than 10 days in cultured L cells, ribosome degradation is not considered to contribute significantly to the regulation of ribosome content (2). To date little is known about the signaling mechanism(s) involved in ribosomal degradation, although a recent publication implicated a role for ubiquitin in this process (33). Ubiquitin may function by binding to the ribosome, thereby stabilizing or protecting it from degradation. Subsequent removal of ubiquitin would signal the cell to degrade that ribosome (6,33). Interestingly, in the majority of cells, it is the rate of ribosome synthesis, i.e. rDNA transcription, that is the primary determinant of ribosome content (2).

3.3. Proteins Associated with Ribosomes

Numerous proteins associate with ribosomes, some of which have obvious catalytic or structural functions, while for others it is unclear. For example, nucleolin (C23) is a 92-100 kDa phosphoprotein localized in the FC and DFC of the nucleolus, i.e. the sites for all stages of rDNA processing. Interestingly, nucleolin binds to the intergenic spacer region between the repeated rRNA genes. Moreover, one laboratory has demonstrated a correlation between nucleolin cellular content or activity, and the rate of rDNA transcription (34,35,36). However, the significance of this correlation is unclear. In addition, nucleolin has been implicated in the packaging and shuttling of the ribosome between the nucleus and cytoplasm. Also it has been suggested that nucleolins N-terminal HMG domain plays a role in the structure of the nucleus (20). While the extended conformation of the glycine rich C-terminal domain is suggestive of involvement in protein-protein interactions (6,18,20).

Like nucleolin, NO38 (B23, numatrin, or nucleophosmin) is an abundant 38 kDa nucleolar phosphoprotein (37) localized in the FC, which possibly plays a role in ribosome packaging and transportation. NO38 cooperatively binds, with high affinity, to single-stranded nuclei acids and exhibits an RNA helix destabilizing activity (6,18,20). Thus, NO38 may coordinate the attachment of r-proteins and other RNA-binding proteins to the rRNA (reviewed in 6,18,20).