Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation.

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

Basic mechanisms in RNA polymerase I transcription of the ribosomal RNA genes.

RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription factors guide Pol I to the rDNA promoter and contribute to multiple rounds of transcription initiation, promoter escape, elongation and termination. In addition, many accessory factors are now known to assist at each stage of this transcription cycle, some of which allow the integration of transcriptional activity with metabolic demands. The organisation and accessibility of rDNA chromatin also impinge upon Pol I output, and complex mechanisms ensure the appropriate maintenance of the epigenetic state of the nucleolar genome and its effective transcription by Pol I. The following review presents our current understanding of the components of the Pol I transcription machinery, their functions and regulation by associated factors, and the mechanisms operating to ensure the proper transcription of rDNA chromatin. The importance of such stringent control is demonstrated by the fact that deregulated Pol I transcription is a feature of cancer and other disorders characterised by abnormal translational capacity.

TAF1B is a TFIIB-like component of the basal transcription machinery for RNA polymerase I.

Transcription by eukaryotic RNA polymerases (Pols) II and III and archaeal Pol requires structurally related general transcription factors TFIIB, Brf1, and TFB, respectively, which are essential for polymerase recruitment and initiation events. A TFIIB-like protein was not evident in the Pol I basal transcription machinery. We report that TAF1B, a subunit of human Pol I basal transcription factor SL1, is structurally related to TFIIB/TFIIB-like proteins, through predicted amino-terminal zinc ribbon and cyclin-like fold domains. SL1, essential for Pol I recruitment to the ribosomal RNA gene promoter, also has an essential postpolymerase recruitment role, operating through TAF1B. Therefore, a TFIIB-related protein is implicated in preinitiation complex assembly and postpolymerase recruitment events in Pol I transcription, underscoring the parallels between eukaryotic Pol I, II, and III and archaeal transcription machineries.

RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle.

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I-specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

FACT facilitates chromatin transcription by RNA polymerases I and III.

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Structure and function of ribosomal RNA gene chromatin.

Transcription of the major ribosomal RNAs by Pol I (RNA polymerase I) is a key determinant of ribosome biogenesis, driving cell growth and proliferation in eukaryotes. Hundreds of copies of rRNA genes are present in each cell, and there is evidence that the cellular control of Pol I transcription involves adjustments to the number of rRNA genes actively engaged in transcription, as well as to the rate of transcription from each active gene. Chromatin structure is inextricably linked to rRNA gene activity, and the present review highlights recent advances in this area.

A novel TBP-associated factor of SL1 functions in RNA polymerase I transcription.

In mammalian RNA polymerase I transcription, SL1, an assembly of TBP and associated factors (TAFs), is essential for preinitiation complex formation at ribosomal RNA gene promoters in vitro. We provide evidence for a novel component of SL1, TAF(I)41 (MGC5306), which functions in Pol I transcription. TAF(I)41 resides at the rDNA promoter in the nucleolus and co-purifies and co-immunoprecipitates with SL1. TAF(I)41 immunodepletion from nuclear extracts dramatically reduces Pol I transcription; addition of SL1 restores the ability of these extracts to support Pol I transcription. In cells, siRNA-mediated decreased expression of TAF(I)41 leads to loss of SL1 from the rDNA promoter in vivo, with concomitant loss of Pol I from the rDNA and reduced synthesis of the pre-rRNA. Extracts from these cells support reduced levels of Pol I transcription; addition of SL1 to the extracts raises the level of Pol I transcription. These data suggest that TAF(I)41 is integral to transcriptionally active SL1 and imply a role for SL1, including the TAF(I)41 subunit, in Pol I recruitment and, therefore, preinitiation complex formation in vivo.

Casein kinase 2 associates with initiation-competent RNA polymerase I and has multiple roles in ribosomal DNA transcription.

Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Ibeta isoform and not with Pol Ialpha. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Ibeta-associated CK2 can phosphorylate topoisomerase IIalpha in Pol Ibeta, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.

UBF activates RNA polymerase I transcription by stimulating promoter escape.

Ribosomal RNA gene transcription by RNA polymerase I (Pol I) is the driving force behind ribosome biogenesis, vital to cell growth and proliferation. The key activator of Pol I transcription, UBF, has been proposed to act by facilitating recruitment of Pol I and essential basal factor SL1 to rDNA promoters. However, we found no evidence that UBF could stimulate recruitment or stabilization of the pre-initiation complex (PIC) in reconstituted transcription assays. In this, UBF is fundamentally different from archetypal activators of transcription. Our data imply that UBF exerts its stimulatory effect on RNA synthesis, after PIC formation, promoter opening and first phosphodiester bond formation and before elongation. We provide evidence to suggest that UBF activates transcription in the transition between initiation and elongation, at promoter escape by Pol I. This novel role for UBF in promoter escape would allow control of rRNA synthesis at active rDNA repeats, independent of and complementary to the promoter-specific targeting of SL1 and Pol I during PIC assembly. We posit that stimulation of promoter escape could be a general mechanism of activator function.

RNA polymerase I-specific subunit CAST/hPAF49 has a role in the activation of transcription by upstream binding factor.

Eukaryotic RNA polymerases are large complexes, 12 subunits of which are structurally or functionally homologous across the three polymerase classes. Each class has a set of specific subunits, likely targets of their cognate transcription factors. We have identified and characterized a human RNA polymerase I (Pol I)-specific subunit, previously identified as ASE-1 (antisense of ERCC1) and as CD3epsilon-associated signal transducer (CAST), and here termed CAST or human Pol I-associated factor of 49 kDa (hPAF49), after mouse orthologue PAF49. We provide evidence for growth-regulated Tyr phosphorylation of CAST/hPAF49, specifically in initiation-competent Pol Ibeta complexes in HeLa cells, at a conserved residue also known to be important for signaling during T-cell activation. CAST/hPAF49 can interact with activator upstream binding factor (UBF) and, weakly, with selectivity factor 1 (SL1) at the rDNA (ribosomal DNA repeat sequence encoding the 18S, 5.8S, and 28S rRNA genes) promoter. CAST/hPAF49-specific antibodies and excess CAST/hPAF49 protein, which have no effect on basal Pol I transcription, inhibit UBF-activated transcription following functional SL1-Pol I-rDNA complex assembly and disrupt the interaction of UBF with CAST/hPAF49, suggesting that interaction of this Pol I-specific subunit with UBF is crucial for activation. Drawing on parallels between mammalian and Saccharomyces cerevisiae Pol I transcription machineries, we advance one model for CAST/hPAF49 function in which the network of interactions of Pol I-specific subunits with UBF facilitates conformational changes of the polymerase, leading to stabilization of the Pol I-template complex and, thereby, activation of transcription.

The RNA polymerase I transcription machinery.

The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

TBP-TAF complex SL1 directs RNA polymerase I pre-initiation complex formation and stabilizes upstream binding factor at the rDNA promoter.

Knowledge of the role of components of the RNA polymerase I transcription machinery is paramount to understanding regulation of rDNA expression. We describe key findings for the roles of essential transcription factor SL1 and activator upstream binding factor (UBF). We demonstrate that human SL1 can direct accurate Pol I transcription in the absence of UBF and can interact with the rDNA promoter independently and stably, consistent with studies of rodent SL1 but contrary to previous reports of human SL1. UBF itself does not bind stably to rDNA but rapidly associates and dissociates. We show that SL1 significantly reduces the rate of dissociation of UBF from the rDNA promoter. Our findings challenge the idea that UBF activates transcription through recruitment of SL1 at the rDNA promoter and suggest that the rate of pre-initiation complex (PIC) formation is primarily determined by the rate of association of SL1, rather than UBF, with the promoter. Therefore, we propose that SL1 directs PIC formation, functioning in core promoter binding, RNA polymerase I recruitment, and UBF stabilization and that SL1-promoter complex formation is a necessary prerequisite to the assembly of functional and stable PICs that include the UBF activator in mammalian cells.

RNA-polymerase-I-directed rDNA transcription, life and works.

In the extensive network of interdependent biochemical processes required for cell growth and division, there is mounting evidence that ribosomal DNA transcription by RNA polymerase I (pol I) not only drives cell growth via its direct role in production of the ribosomal RNA (rRNA) component of the protein-synthesis machinery, but that it is also crucial in determining the fate of the cell. Considerable progress has been made in recent years towards understanding both the function of components of the pol I transcription machinery and how cells accomplish the tight control of pol I transcription, balancing the supply of rRNA with demand under different growth conditions.

Phosphatidylinositol 3-kinase and mTOR signaling pathways regulate RNA polymerase I transcription in response to IGF-1 and nutrients.

Regulation of ribosomal RNA gene transcription by RNA polymerase I (Pol I) is fundamental to ribosome biogenesis and therefore protein translation capacity and cell growth, yet little is known of the key signaling cascades involved. We show here that insulin-like growth factor-1 (IGF-1)-induced Pol I transcription in HEK293 cells is entirely dependent on phosphatidylinositol 3-kinase (PI3K) activity and, additionally, is modulated by the mammalian target of rapamycin (mTOR), which coordinates Pol I transcription with the availability of amino acids. The mitogen-activated protein kinase (MAPK) pathway is weakly stimulated by IGF-1 in these cells and partly contributes to Pol I transcription regulation. Activation of Pol I transcription by IGF-1 results from enhancement of the activity of the Pol I transcription machinery and increased occupancy by SL1 of the endogenous tandemly repeated ribosomal promoters in vivo. The inputs from PI3K, mTOR, and MAPK pathways converge to direct appropriate rRNA gene expression by Pol I in the nucleolus of mammalian cells in response to environmental cues, such as growth factors and nutrients.