The nucleolus is the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus assembles around clusters of ribosomal gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. The initial step in nucleolar formation is ribosomal gene transcription, which is mediated by Pol I (RNA polymerase I) and its associated transcription factors: UBF (upstream-binding factor), SL1 (selectivity factor) and TIF-IA (transcription initiation factor IA)/Rrn3. Ribosomal gene clusters, termed NORs (nucleolar organizer regions), are found on each of the five human acrocentric chromosomes. Though transcription is repressed during metaphase, NORs that were active in the previous interphase form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under-condensation in comparison with the rest of the chromosome. Extensive binding of UBF over the ribosomal gene repeat is responsible for the formation of this chromosomal feature. During interphase, the majority of the Pol I transcription machinery, though present in nucleoli, is not actively engaged in transcription. Interaction with UBF bound across the gene repeat provides an explanation for how this non-engaged Pol I machinery is sequestered by nucleoli.
The nucleolus is the site of ribosome biogenesis
The eukaryotic nucleus is highly compartmentalized, with respect to both genome and the nuclear proteome . This compartmentalization facilitates the sequential events required for gene expression including transcription, RNA processing and RNA modification. The nucleolus represents a paradigm for this level of organization. The nucleolus is not surrounded by a membrane nor is there any convincing evidence for an underlying scaffold. In mammals, nucleoli comprise three morphologically distinct structures that reflect the steps of ribosome biogenesis . In the FCs (fibrillar centres), ribosomal genes are transcribed by the dedicated Pol I (RNA polymerase I) transcription machinery to yield the precursor rRNA. Within the dense fibrillar component pre-RNA is processed to yield mature 18, 28 and 5.8 S rRNAs. Finally, in the granular component, ribosome assembly that involves the association of rRNAs with ribosomal proteins occurs.
Nucleolar structure is ultimately dependent on active ribosomal gene transcription. Nucleolar disassembly occurs at the onset of mitosis, when transcription is inhibited . The mitotic-dependent phosphorylation of Pol I transcription factors and processing components results in the loss of nucleolar structure. As cells exit mitosis, transcriptional repression is relieved and nucleoli reform. However, if the onset of transcription is inhibited by an injection of inactivating anti-Pol I antibodies, nucleolar reformation can be prevented . Inhibition of ribosomal gene transcription during interphase results in the nucleolus becoming disordered and ultimately disappearing . Thus we can consider that both initial formation of the nucleolus and maintenance of its structure is ultimately dependent on recruitment of the Pol I transcription machinery to ribosomal genes, the focus of this review.
Ribosomal gene organization and the Pol I transcription machinery
Approximately 400 copies of the 43 kb human ribosomal gene repeat are distributed among the short arms of the human acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22). Head-to-tail tandemly repeated gene clusters representing on average 3 Mb of DNA are termed NORs (nucleolar organizer regions). During metaphase, NORs that were transcriptionally active in the previous interphase form prominent chromosomal features termed secondary constrictions in which the chromatin is differently condensed from that of the remainder of the chromosome . Association of the Pol I machinery with rDNA during mitosis correlates with the presence of a secondary constriction [7,8].
Efficient transcription of rDNA by Pol I requires the formation of a PIC (pre-initiation complex) on the promoter, including UBF (upstream-binding factor) and promoter SL1 (selectivity factor) [9–12]. SL1 comprises TBP (TATA box binding protein) and TAFs [TBP-associated factors I (TAFI110, TAFI63 and TAFI48)] . SL1 interacts with promoter DNA in a highly sequence-specific manner. PICs recruit an initiation-competent subfraction of Pol I, defined by the presence of TIF-IA (transcription initiation factor IA)/Rrn3 [14,15].
Targeting of the Pol I transcription machinery to nucleoli
Immunolocalization experiments clearly demonstrate that the major fraction of each component of the Pol I transcription machinery is localized to the FCs of nucleoli (Figure 1). What are the mechanisms underlying this localization? Two models can be proposed. First, components of the Pol I machinery could be recruited solely through interaction with the promoter and formation of productive transcription complexes. Alternatively, factors could be recruited to FCs prior to incorporation into productive transcription complexes.
The Pol I transcription machinery is highly enriched in nucleoli
Although clearly involved in promoter function, mediated through its ability to interact with other components of the Pol I machinery, highly abundant UBF (∼5×105 molecules/cell) has additional roles. It is a so-called ‘architectural’ transcription factor and is a prime candidate for maintaining the specialized chromatin state of secondary constrictions. Evidence in support of this was initially provided by the finding that UBF binds extensively across the rDNA repeat in vivo . Further support was provided by the finding that large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes recruit UBF even to sites outside the nucleolus . During metaphase these arrays form novel secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. The conclusions from this work were that targeting of UBF to the nucleolus is entirely a consequence of its DNA-binding specificity and not due to the presence of nucleolar targeting signal. Secondly, extensive UBF binding over the ribosomal gene repeat specifies a specialized chromatin structure.
It has been known for some time that the majority of Pol I within cells is not engaged in transcription, yet appears to be targeted to FCs. Recent live cell imaging experiments with GFP (green fluorescent protein)-tagged Pol I subunits has provided evidence that only 7–10% of Pol I in the nucleolus is engaged in transcription [18,19]. Each ribosomal gene is transcribed by approx. 100 Pol I molecules at any given time [20–22]. Consequently, far greater than 100 Pol I complexes per active repeat must be present in FCs but not engaged in transcription. This large amount of unengaged Pol I is incompatible with a model in which it is recruited solely through interaction with PICs on promoters. How then is Pol I recruited? An initial clue was provided by ChIP (chromatin immunoprecipitation) experiments, which demonstrated that a significant amount of Pol I is associated with sequences present in the 30 kb intergenic spacer of the human ribosomal gene repeat . Pol I associated with the transcribed region of the repeat is presumably engaged in transcription, whereas that associated with the intergenic spacer is not. This raised the possibility that UBF mediates recruitment of Pol I either directly through contacts with Pol I or indirectly through the action of an unidentified factor. A prediction of this hypothesis is that pseudo-NORs (described above) would sequester Pol I. Immunofluorescence microscopy and ChIP demonstrated that this was indeed the case . Furthermore, Pol I was recruited to pseudo-NORs that are located outside nucleoli (Figure 2). In the live cell imaging experiments described above, Misteli and co-workers made the somewhat controversial claim that subunits of Pol I were recruited as individual polypeptides to the FC [18,19]. More recent work in yeast  and studies of the dynamics of Pol I subunits in human cells  have undermined these claims.
Pseudo-NORs sequester Pol I
As with Pol I, ChIP experiments demonstrated that the majority of SL1 in nucleoli is not engaged in PICs on the promoter but is associated with sequences in the intergenic spacer . Given the specificity of the SL1 interaction with a promoter DNA, it is likely that protein–protein interactions mediate this association. Components of SL1 (TAF110 and TBP) are also sequestered by pseudo-NORs. Finally, we demonstrated that TIF-IA/Rrn3 is also sequestered by pseudo-NORs . Thus it would appear that every component of the Pol I transcription machinery is recruited to pseudo-NORs and by inference to FCs by interaction with UBF-associated chromatin. The most reasonable assumption is that these factors are recruited to sites in the intergenic spacer through protein–protein contacts with UBF. SL1 subunits, TAFI48 and TBP, have been shown individually to interact with UBF [25,26]. The Pol I subunit PAF53 (polymerase-associated factor 53) has been shown to contact UBF directly . The yeast homologue of PAF53, RPA49, interacts with the presumptive yeast homologue of UBF, hmo1p . Direct contacts between TIF-IA/Rrn3 and UBF have not been described. Presumably TIF-IA/Rrn3 is recruited to pseudo-NORs by interactions with Pol I or SL1 [15,29].
As outlined above, studies using cell free systems led to a model in which SL1 and UBF combine to form stable PICs that support multiple rounds of transcription initiation. The intellectual appeal of this model was that if PIC formation is the rate-limiting step in transcription initiation, it could then explain the high rate of transcription initiation on ribosomal genes. If transcription of rDNA in vivo is mediated by stable PICs that support repeated rounds of initiation, why then is there a stockpile of unengaged factors in the FC? This raises the possibility of an alternative model for transcription initiation in vivo, in which PICs are recycled after each round of initiation. Indeed, evidence from yeast supports this view . In this model the high concentration of factors present in the FC, recruited by UBF bound to the intergenic spacer, would greatly enhance the rate of PIC formation on the promoter.
Recruitment of the Pol I transcription machinery and the initiation of ribosomal gene transcription is the initial step in the formation of the nucleolus and ribosome biogenesis. Subsequent steps include processing of pre-RNA and both pseudouridylation and methylation of rRNA. Functional links between these processes and ribosomal gene transcription are beginning to emerge . Presumably these links facilitate the co-ordinate regulation of all the steps required for the generation of ribosomes, one of the major metabolic activities of all eukaryotic cells.
The Nucleus and Gene Expression: A Focus Topic at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by W. Bickmore (Edinburgh, U.K.), R. Borts (Leicester, U.K.), J. Cáceres (Edinburgh, U.K.), A. Maxwell (John Innes Centre, Norwich, U.K.), S. Newbury (Newcastle upon Tyne, U.K.), D. Wigley (Cancer Research London, U.K.) and A. Willis (Nottingham, U.K.).
nucleolar organizer region
- Pol I
RNA polymerase I
TATA box binding protein
TBP-associated factor I
transcription initiation factor IA
upstream binding factor