Ccr4-not regulates RNA polymerase I transcription and couples nutrient signaling to the control of ribosomal RNA biogenesis.

Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

Is ribosome synthesis controlled by pol I transcription?

Regulation of growth ultimately depends on the control of synthesis of new ribosomes. Ribosome biogenesis is thus a key element of cell biology, which is tightly regulated in response to environmental conditions. In eukaryotic cells, the supply of ribosomal components involves the activities of the three forms of nuclear RNA polymerase (Pol I, Pol II and Pol III). Recently, we demonstrated that upon rapamycin treatment, a partial derepression of Pol I transcription led to a concomitant derepression of Pol II transcription restricted to a small subset of class II genes encompassing the genes encoding all ribosomal proteins, and 19 additional genes. The products of 14 of these 19 genes are principally involved in rDNA structure, ribosome biogenesis or translation, whereas the five remaining genes code for hypothetical proteins. We demonstrate that the proteins encoded by these five genes are required for optimal pre-rRNA processing. In addition, we show that cells in which regulation of Pol I transcription was specifically impaired are either resistant or hypersensitive to different stresses compared to wild-type cells. These results highlight the critical role of the regulation of Pol I activity for the physiology of the cells.

The chromatin remodelling complex WSTF-SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription.

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

Growth factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling.

Synthesis of the 45S rRNA by RNA polymerase I limits cell growth. Knowledge of the mechanism of its regulation is therefore key to understanding growth control. rRNA transcription is believed to be regulated solely at initiation/promoter release. However, we found that stimulation of endogenous 45S rRNA synthesis by epidermal growth factor (EGF) and serum failed to induce an increase in RNA polymerase I engagement on the rRNA genes, despite robust enhancement of 45S rRNA synthesis. Further, endogenous transcription elongation rates were measured and found to be directly proportional to 45S rRNA synthesis. Thus, elongation is a rate-limiting step for rRNA synthesis in vivo. ERK phosphorylation of the HMG boxes of UBF, an RNA polymerase I factor essential for transcription enhancement, was shown to directly regulate elongation by inducing the remodeling of ribosomal gene chromatin. The data suggest a mechanism for coordinating the cotranscriptional assembly of preribosomal particles.

Nuclear actin and myosin I are required for RNA polymerase I transcription.

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

Heterologous overexpression and purification of four common subunits of nuclear RNA polymerases I, II and III of Schizosaccharomyces pombe.

Four subunits of Schizosaccharomyces pombe RNA polymerases I-III shared by all three enzymes (Rpb5, Rpb8, Rpb10 and Rpc10 [Rpb12]) have been overexpressed in Escherichia coli expression vectors pQE or pET as hexahistidine fusions. The recombinant proteins have been purified to near homogeneity using metal-chelate affinity chromatography and gel filtration. Homogeneity and identity of the purified protein preparations was demonstrated by denaturing polyacrylamide gel electrophoresis and TOF-MALDI mass spectrometry. The proteins were obtained in large amounts, and their preparations are currently in use for monoclonal antibody production and physico-chemical studies of these individual components of eukaryotic transcription enzymes.

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.

Differential core histone binding behavior: RNA polymerase I promoter region vs 5S rDNA positioning DNA sequences.

In order to further characterize the previously observed disruptive effect of the RNA polymerase I promoter sequence (Pol I) from Acanthamoeba castellanii on tandemly repeated 5S rDNA positioning sequences from sea urchin (Lytechinus variegatus), we compared the histone-binding ability of the isolated 199-bp Pol I promoter region to that of the 208-bp 5S rDNA and that of nucleosome core particle sequences isolated from chicken erythocytes. We found the 5S rDNA positioning sequence to be more efficient at forming nucleosomes than the RNA polymerase I promoter sequence. Nevertheless, examination of the free-DNA half-depletion points during the titrations suggested that twice as much histone had bound to the RNA polymerase I promoter sequence as to the 5S nucleosome-positioning or core particle sequences. DNA bending analysis suggested two potential DNA bending loci in the RNA polymerase I promoter, whereas only one such locus was predicted for the 5S positioning sequence. Such mixed bending signals on the RNA polymerase I promoter could favor non-nucleosomal deposition of histones on these sequences.

Mechanism of inhibition of RNA polymerase I transcription by DNA-dependent protein kinase.

DNA-dependent protein kinase represses RNA polymerase I (Pol I) transcription in vitro. To investigate the mechanism underlying transcriptional repression, we compared Pol I transcription in extracts from cells that either contain or lack the catalytic subunit of DNA-PK (DNA-PKcs). ATP-dependent repression of Pol I transcription was observed in extracts from DNA-PKcs-containing but not -deficient cells, required templates with free DNA ends, and was overcome by exogenous SL1, the factor that nucleates initiation complex formation. Order-of-addition experiments demonstrate that DNA-PKcs does not inactivate component(s) of the Poll transcription machinery. Instead, phosphorylated Ku protein competes with SL1 for binding to the rDNA promoter and, as a consequence, prevents initiation complex formation. The results reveal a novel mechanism of transcriptional regulation by DNA-PK. Once targeted to DNA, autophosphorylated Ku may displace positive- or negative-acting factors from their target sites, thereby repressing or activating transcription in a gene-specific manner.

Differential roles of phosphorylation in the formation of transcriptional active RNA polymerase I.

Regulation of rDNA transcription depends on the formation and dissociation of a functional complex between RNA polymerase I (pol I) and transcription initiation factor Rrn3p. We analyzed whether phosphorylation is involved in this molecular switch. Rrn3p is a phosphoprotein that is predominantly phosphorylated in vivo when it is not bound to pol I. In vitro, Rrn3p is able both to associate with pol I and to enter the transcription cycle in its nonphosphorylated form. By contrast, phosphorylation of pol I is required to form a stable pol I-Rrn3p complex for efficient transcription initiation. Furthermore, association of pol I with Rrn3p correlates with a change in the phosphorylation state of pol I in vivo. We suggest that phosphorylation at specific sites of pol I is a prerequisite for proper transcription initiation and that phosphorylation/dephosphorylation of pol I is one possibility to modulate cellular rDNA transcription activity.

Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

The transcript release factor PTRF augments ribosomal gene transcription by facilitating reinitiation of RNA polymerase I.

Termination of murine rDNA transcription by RNA polymerase I (Pol I) requires pausing of Pol I by terminator-bound TTF-I (transcription termination factor for Pol I), followed by dissociation of the ternary complex by PTRF (Pol I and transcript release factor). To examine the functional correlation between transcription termination and initiation, we have compared transcription on terminator-containing and terminator-less rDNA templates. We demonstrate that terminated RNA molecules are more efficiently synthesized than run-off transcripts, indicating that termination facilitates reinitiation. Transcriptional enhancement is observed in multiple- but not single-round transcription assays measuring either promoter-dependent or promoter-independent Pol I transcription. Increased synthesis of terminated transcripts is observed in crude extracts but not in a PTRF-free reconstituted transcription system, indicating that PTRF-mediated release of pre-rRNA is responsible for transcriptional enhancement. Consistent with PTRF serving an important role in modulating the efficiency of rRNA synthesis, PTRF exhibits pronounced charge heterogeneity, is phosphorylated at multiple sites and fractionates into transcriptionally active and inactive forms. The results suggest that regulation of PTRF activity may be an as yet unrecognized means to control the efficiency of ribosomal RNA synthesis.

Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1.

We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBF-mediated transcription. In the present study, we have examined the mechanism by which Rb represses UBF-dependent rDNA transcription and determined if other Rb-like proteins have similar effects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb.

Expression and purification of recombinant hRPABC25, hRPABC17, and hRPABC14.4, three essential subunits of human RNA polymerases I, II, and III.

Transcription of eukaryotic genes is performed by RNA polymerases I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. Eukaryotic RNA polymerases are large macromolecular complexes composed of multiple subunits. Among these subunits, five are shared by all RNA polymerases and are essential for cell growth and viability. Remarkably, the human common subunits are structurally conserved and functionally interchangeable with their yeast homologues and are believed to play an important role in the assembly of the three transcription complexes. To understand the structure and function of human RNA polymerases, we overexpressed the common subunits hRPABC25, hRPABC17, and hRPABC14.4 as hexahistidine fusions in Escherichia coli. The recombinant proteins were purified using metal-chelate affinity chromatography on Ni-NTA resin and gel filtration. Depending on the subunit, the yield was 5-17 mg of purified recombinant protein per liter of culture medium. The purified proteins were of high quality and sufficient quantity for structural studies, as demonstrated by the successful crystallization of hRPABC17 and hRPABC14.4. The expression and purification of the common subunits hRPABC25, hRPABC17, and hRPABC14. 4 will make possible their structural analysis with X-ray crystallography and nuclear magnetic resonance, providing important insights into the structure and function of the three human RNA polymerases.

Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth.

The retinoblastoma family of proteins, also known as pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130. Pocket proteins control growth processes in many cell types, and this has been linked to the ability of pocket proteins to interact with a multitude of cellular proteins that regulate gene expression at various levels. By regulating gene expression, pocket proteins control cell cycle progression, cell cycle entry and exit, cell differentiation and apoptosis. This review will focus on the mechanisms of regulation of pocket proteins and how modulation of pocket protein levels and phosphorylation status regulate association with their cellular targets. The coordinated regulation of pocket proteins provides the cells with a competence mechanism for passage through certain cell growth and differentiation transitions.

Rpo26p, a subunit common to yeast RNA polymerases, is essential for the assembly of RNA polymerases I and II and for the stability of the largest subunits of these enzymes.

Eukaryotic nuclear RNA polymerases (RNAPs) are composed of two large subunits and a number of small polypeptides, some of which are common among these enzymes. To understand the function of Rpo26p, one of the five subunits common to yeast RNAPs, 34 different mutations have been isolated in RP026 that cause cell death in a strain carrying a temperature-sensitive (ts) mutation in the gene (RP021) encoding the largest subunit of RNAPII. These mutant alleles were grouped into three phenotypic classes (null, ts, and neutral) on the basis of the phenotype they imposed in combination with wild-type RP021. The function of Rpo26p was addressed by biochemical analysis of the ts rpo26-31 allele. The steady-state level of rpo26-31p was reduced at high temperature; this was accompanied by a decrease in the level of at least two other subunits, the largest subunits of RNAPI (A190p) and RNAPII (Rpo21p). Pulse-chase metabolic labeling and immunoprecipitation of RNAPII showed that at high temperature, rpo26-31 did not lead to dissociation of Rpo26p from the polymerase but prevented the assembly of RNAPII. Overexpression of rpo26-31 partially suppressed the ts phenotype and led to accumulation of the mutant subunit. However, overexpression only marginally suppressed the assembly defect of RNAPII. Furthermore, A190p and Rpo21p continued to accumulate at low levels under these conditions. We suggest that Rpo26p is essential for the assembly of RNAPI and RNAPII and for the stability of the largest subunits of these enzymes.

DNA length is a critical parameter for eukaryotic transcription in vivo.

The organization of eukaryotic chromosomes into topological domains has led to the assumption that DNA topology and perhaps supercoiling are involved in eukaryotic nuclear processes. Xenopus oocytes provide a model system for studying the role of DNA topology in transcription. Linear plasmid templates for RNA polymerases (Pols) I and II are not transcribed in Xenopus oocytes, while circular templates are transcriptionally active. Here we show that circularity is not required for transcription of Pol I or Pol II promoters if the linear template is sufficiently long (> 17 to 19 kb). The Xenopus rRNA (Pol I) promoter is active in central positions on a long linear template but is not transcribed when located near an end. Because supercoils generated by transcription could be retained by viscous drag against the long template, these results are consistent with a supercoiling requirement for this promoter. Surprisingly, the herpes simplex virus thymidine kinase (Pol II) promoter is active even 100 bp from the end of the long template, indicating that template length fulfills a critical parameter for transcription that is not consistent with a supercoiling requirement. These results show that DNA length has unrecognized importance for transcription in vivo.

RB [corrected] as a modulator of transcription.

pRB interacts with a number of transcription factors and can both directly and indirectly modulate transcriptional activity. Growth suppression by pRB is tightly linked to its ability to form complexes with E2F which are capable of repressing transcription of certain genes required for S phase. The ability of pRB to enhance the activity of several non-E2F transcription factors might suggest a mechanism by which pRB could coordinately regulate sets of genes at or near the restriction point. Specifically, complexes consisting of underphosphorylated pRB and E2F, by virtue of transcriptional repression of promoters containing E2F sites, would act to block entry into S phase. At the same time, distinct complexes of underphosphorylated pRB and transcription factors such as the glucocorticoid receptor, ATF-2, or MyoD, might lead to an increase in the transcription of genes required for differentiation or for additional growth inhibitory functions (e.g. TGF-beta 1). Changes in the activities of various cyclin-dependent kinase complexes would lead to phosphorylation of pRB and thus coordinate a release of S phase genes from repression with a loss of activation of differentiation genes. While this model is speculative, the role of pRB as a transcriptional modulator, as well as its interactions with cell-cycle regulatory kinases, places it in a position to integrate extracellular and intracellular growth signals and to transduce those signals into changes in gene transcription which ultimately influence cell growth and differentiation.

Gene RPA43 in Saccharomyces cerevisiae encodes an essential subunit of RNA polymerase I.

Yeast RNA polymerase I contains 14 distinct polypeptides, including A43, a component of about 43 kDa. The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the peptidic sequence of two tryptic fragments isolated from A43. Gene inactivation leads to a lethal phenotype that is rescued by a plasmid containing the 35S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of 35S rRNA by RNA polymerase II in the presence of galactose. A screening for mutants rescued by the presence of GAL7-35SrDNA identified a nonsense rpa43 allele truncating the protein at amino acid position 217. [3H]Uridine pulse labeling showed that this mutation abolishes 35S rRNA synthesis without significant effects on the synthesis of 5 S RNA and tRNAs. These properties establish that A43 is an essential component of RNA polymerase I. This highly hydrophilic phosphoprotein has a strongly acidic carboxyl-terminal domain, and shows no homology to entries in current sequence data banks, including all the genetically identified components of the other two yeast RNA polymerases. RPA43 mapped next to RPA190, encoding the largest subunit of polymerase I. These genes are divergently transcribed and may thus share upstream regulatory elements ensuring their co-regulation.

Characterization of a protein that interacts with the rat ribosomal gene promoter and modulates RNA polymerase I transcription.

An RNA polymerase I core promoter binding factor (CPBF) was purified to apparent homogeneity from rat adenocarcinoma ascites cells by chromatographic fractionation on a series of columns including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides with molecular masses of 44,000 and 39,000 daltons. The binding of the factor to the promoter was demonstrated by Southwestern blotting, UV cross-linking and electrophoretic mobility shift assay. The specificity of its binding to the core promoter was confirmed by competitive electrophoretic mobility shift assay using several unlabeled oligo probes in the assay. The addition of increasing amounts of purified CPBF to the in vitro transcription reaction that contains a limiting quantity of the factor resulted in dramatic stimulation of RNA polymerase I (pol I) transcription of rat ribosomal RNA gene. The transcription stimulatory activity associated with the purified CPBF fractions co-purified with the core promoter binding activity in an electrophoretic mobility shift assay. Finally, in a reconstitution transcription system which is devoid of the factor and is incapable of ribosomal gene transcription, purified CPBF could trans-activate the pol I promoter. These data indicate that CPBF is a novel pol I promoter binding factor required for ribosomal gene transcription.