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Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays.

Listeria monocytogenes is a pathogenic intracellular microorganism whose infection induces pleiotropic biological changes associated with host cell gene expression regulation. Here we define the gene expression profiles of the human promyelocytic THP1 cell line before and after L. monocytogenes infection. Gene expression was measured on a large scale via oligonucleotide microarrays with probe sets corresponding to 6,800 human genes. We assessed and discussed the reproducibility of the hybridization signatures. In addition to oligonucleotide arrays, we also performed the large scale gene expression measurement with two high-density membranes, assaying for 588 and 18,376 human genes, respectively. This work allowed the reproducible identification of 74 up-regulated RNAs and 23 down-regulated RNAs as a consequence of L. monocytogenes infection of THP1. The reliability of these data was reinforced by performing independent infections. Some of these detected RNAs were consistent with previous results, while some newly identified RNAs encode gene products that may play key roles in L. monocytogenes infection. These findings will undoubtedly enhance the understanding of L. monocytogenes molecular physiology and may help identify new therapeutic targets.

Blotting, Northern↗

Xenopus tropicalis U6 snRNA genes transcribed by Pol III contain the upstream promoter elements used by Pol II dependent U snRNA genes.

We have cloned and sequenced a 977bp DNA fragment, pXTU6-2, that represents the transcription unit for a Xenopus tropicalis U6 RNA gene. This basic repeating unit is reiterated ca.500-fold per haploid genome. Oocyte injections of pXTU6-2 led to the transcription of a mature-sized U6 RNA that, however, lacked internal 2'-O-methylations. These posttranscriptional modifications of U6 RNA might be cytoplasmic and could require its association with U4 RNA to be accomplished. The low alpha- amanitin sensitivity of U6 RNA synthesis in oocytes suggested that U6 RNA is transcribed by RNA polymerase III, consistent with features of the U6 RNA molecule which also contains a Box A- like intragenic control region. Inspection of X. tropicalis, mouse and human U6 DNA upstream sequences revealed the presence of a TATA box as well as of the proximal and enhancer (octamer motif) elements contained in snRNA genes transcribed by RNA polymerase II. We propose that U6 RNAs are synthesized by a specialized transcription complex consisting of RNA polymerase III and transcription factors, some of which are very likely shared with RNA polymerase II promoters.

Animals↗

In vitro polyadenylation is stimulated by the presence of an upstream intron.

The majority of vertebrate pre-mRNAs are both spliced and polyadenylated. To investigate the mechanism whereby processing factors recognize last exons containing both splicing and polyadenylation consensus elements, chimeric precursor RNAs containing a single intron and a poly(A) site were constructed and assayed for in vitro splicing and polyadenylation. Chimeric RNAs underwent splicing and polyadenylation. Both reactions occurred in a single RNA. The presence of an intron enhanced the rate of polyadenylation at a downstream poly(A) site. The extent of stimulation varied from two- to fivefold, depending on the magnesium concentration. Maximal stimulation of polyadenylation by an upstream intron required a 3' splice site but not a 5' splice site, suggesting that the structure of the terminal exon was more important than the presence of a complete upstream intron. We suggest that splicing and polyadenylation factors interact to recognize terminal, poly(A) site-containing exons. Such interaction may explain why all known intron-containing eukaryotic pre-mRNAs generate their 3' ends by polyadenylation.

Base Sequence↗

Expression of the two nested overlapping reading frames of turnip yellow mosaic virus RNA is enhanced by a 5' cap and by 5' and 3' viral sequences.

The translation efficiency of an mRNA molecule is typically determined by its 5'- and/or 3'-untranslated regions (UTRs). Previously, we have found that the 3'-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5' cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5' 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5'-UTR. Maximum expression was observed from RNAs with a cap and both 5' and 3' TYMV sequences. In paired reporter constructs, the 5' 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG69 and AUG206) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5' cap, with a threefold-higher expression occurring from AUG69 than from AUG206 in the presence of the genomic 3'-UTR. Changes that interrupted the cap/3'-UTR synergy (i.e., removal of the cap or TYMV 3'-UTR) resulted in a higher proportion of initiation from AUG206. Mutation of the 3'-UTR to prevent aminoacylation, as well as deletion of 75% of the 5'-UTR, likewise resulted in a lower ratio of expression from AUG69 relative to AUG206. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG69 and AUG206. However, our observations do not support a recent proposal based on in vitro studies in which the 3'-UTR is proposed to direct cap-independent initiation specifically at AUG206 and not at AUG69 (S. Barends et al., Cell 112:123-129, 2003).

3' Untranslated Regions↗

Antisense treatments for biothreat agents.

Antisense oligomers (ASOs) represent a promising technology to treat viral and bacterial infections, and have already been shown to be successful against a variety of pathogens in cell culture studies and nonhuman primate models of infection. For these reasons, antisense technologies are being pursued as treatments against biothreat agents such as Ebola virus, dengue virus and Bacillus anthracis. Several generations of modified oligonucleotides have been developed to maximize nuclease resistance, target affinity, potency, cell entry, and other pharmacokinetic properties. First-generation ASOs contain phosphorothioate modifications to increase stability through nuclease resistance. Further chemical modifications in second-generation ASOs include 2'-O-methyl and 2'-O-methoxy-ethyl oligos, which increase nuclease resistance and oligo:RNA binding affinities. Third-generation ASOs contain a variety of chemical modifications that enhance stability, affinity and bioavailability. A fourth class of oligonucleotide-based compounds consists of small interfering RNAs, which have recently become widely used for gene knockdown in vitro and in vivo. This review focuses on the third-generation phosphorodiamidate morpholino oligomers, which are nonionic and contain a morpholine ring instead of a ribose, as well as phosphorodiamidate linkages in place of phosphorothioates. Multiple antisense oligomer-based therapeutics are being developed for use against biothreat agents, and antisense drugs will likely become a critical member of our arsenal in the defense against highly pathogenic, emerging or genetically engineered pathogens.

Animals↗

[Subviral molecules of RNA associated with plant ss(+)RNA viruses].

Plant ss(+)RNA viruses besides their genome RNAs often are associated with additional subviral RNA molecules which occur naturally or are generated de novo during infection. There are such molecules like: satellite, defective, defective interfering and chimeric RNAs. Subviral RNAs can not replicate and encapsidate by oneself. Helper viruses supply the protein complexes that are necessary to these processes. The subviral molecules are characterized by small size. Recombination, deletion and accumulation of mutation are the main ways of arising subviral elements, although the origin of satRNAs is unknown. The unique feature of subviral RNAs is their ability to modify of infection progress caused by helper virus. They can attenuate or enhance the intensity of disease symptoms. The overall influence on disease development depends on three-component complex consisting of: plant host-virus' strain--subviral RNA. This article is a synthetic review of information concerning subviral RNA molecules of plant viruses, their structure, functions and origin.

Defective Viruses↗

Expression of vimentin and nuclear lamins during the in vitro differentiation of human promyelocytic leukemia cells HL-60.

We have reported previously that the human promyelocytic leukemia cell line HL-60, in its undifferentiated state, is devoid of cytoplasmic intermediate filament proteins and nuclear lamins A and C, but does express lamin B. Using immunofluorescence and immunoblotting techniques, we have further investigated the expression of vimentin and lamins A and C during differentiation of these tumor cells along the macrophage or granulocytic pathway in response to the inducing effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide. Our results show that, while the expression of lamin B remains largely unchanged, the synthesis of vimentin and lamins A and C is dramatically enhanced during the maturation of HL-60 cells along both hemopoietic pathways. Northern blot analysis of cellular RNAs isolated from untreated and TPA-treated HL-60 cell populations as well as from control HeLa cells was performed using two oligonucleotides, one complementary to the 5' region common to human lamin A/C mRNAs and the other to the 5' region of hamster vimentin mRNA. Very low but still detectable amounts of vimentin and lamin A/C mRNAs were found in untreated HL-60 cell population, in accordance with the detection of small quantities of vimentin and lamins A and C in these populations. This is probably due to the presence of a small number of spontaneously differentiating cells. On the other hand, strong signals comparable to those obtained with RNA from control HeLa cells were detected for the three mRNA species from TPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗

Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278-303) and new roles for stem-loops H2 (310-352) and H3 (355-374).

Genomic RNAs from retroviruses are packaged as dimers of two identical RNA molecules. In Moloney murine leukemia virus, a stem-loop structure (H1) located in the encapsidation domain Psi (nucleotides 215-564) was postulated to trigger RNA dimerization through base pairing between auto complementary sequences. The Psi domain also contains two other stem-loop structures (H2 and H3) that are essential for RNA packaging. Since it was suspected than H1 is not the only element involved in RNA dimerization, we systematically investigated the dimerization capacity of several subdomains of the first 725 nucleotides of genomic RNA. The efficiency of dimerization of the various RNAs was estimated by measuring their apparent dissociation constants, and the specificity was tested by competition experiments. Our results indicate that the specificity of dimerization of RNA nucleotides 1-725 is driven by motifs H1-H3 in domain Psi. To define the relative contributions of these elements, RNA deletion mutants containing different combinations of H1-H3 were constructed and further analyzed in competition and kinetic experiments. Our results confirm the importance of H1 in triggering dimerization and shed new light on the mechanism of dimerization. H1 is required to provide a stable dimer, probably through the formation of extended intermolecular interactions. However, H1-mediated association is a slow process that is kinetically enhanced by H3, and to a lesser extent by H2. We suggest that they facilitate the recognition between the two RNAs, most likely through their conserved GACG loops. Our results reinforce the idea that dimerization and packaging are two closely related processes.

Animals↗

The effect of base sequence on the stability of RNA and DNA single base bulges.

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.

Base Sequence↗

Transcriptional coactivators CBP and p300 cooperatively enhance HNF-1alpha-mediated expression of the albumin gene in hepatocytes.

Transcriptional coactivators, CREB-binding protein (CBP) and p300, exhibit high homology in structure and similar functions. In the present study, we analyzed the function of CBP and p300 proteins as transcriptional coactivators in the expression of albumin in hepatocytes. The expression levels of CBP and p300 were high in fetal hepatocytes, but low in adult ones. Immunoprecipitation assays showed that both CBP and p300 interacted with hepatocyte nuclear factor-1alpha (HNF-1alpha) in primary hepatocytes. Furthermore, CBP and p300 were co-precipitated without HNF-1alpha. Chromatin immunoprecipitation (ChIP) assays revealed that both CBP and p300 are located in the albumin promoter region in hepatocytes. These results suggested that HNF-1alpha, CBP and p300 were incorporated into a preinitiation complex of RNA polymerase II at the albumin promoter. Luciferase reporter assays showed that CBP and p300 cooperatively triggered HNF-1alpha-mediated transcription of the albumin promoter. In addition, inhibition of CBP or p300 using small interfering RNAs (siRNAs) resulted in a reduction in albumin expression. These results suggest that both CBP and p300 are required for enhanced expression of albumin.

Albumins↗

A comparative study of the regulation of cytochrome P-450 and glutathione transferase gene expression in rat liver.

A cDNA clone for the Ya subunit of glutathione transferase from rat liver was constructed in E. coli. The clone hybridized to Ya and Yc subunit messenger RNAs. On the basis of experiments involving cell-free translation and hybridization to the cloned probe, it was shown that prototype inducers of cytochrome P-450 such as phenobarbitone and 3-methylcholanthrene as well as inhibitors such as CoCl2 and 3-amino-1,2,4-triazole enhanced the glutathione transferase (Ya+Yc) messenger RNA contents in rat liver. A comparative study with the induction of cytochrome P-450 (b+e) by phenobarbitone revealed that the drug manifested a striking increase in the nuclear pre-messenger RNAs for the cytochrome at 12 hr, but did not significantly affect the same in the case of glutathione transferase (Ya+Yc). 3-Amino-1,2,4-triazole and CoCl2 blocked the phenobarbitone mediated increase in cytochrome P-450 (b+e) nuclear pre-messenger RNAs. These compounds did not significantly affect the glutathione transferase (Ya+Yc) nuclear pre-messenger RNA levels. The polysomal, poly (A)- containing messenger RNAs for cytochrome P-450 (b+e) increased by 12-15 fold after phenobarbitone administration, reached a maximum around 16 hr and then decreased sharply. In comparison, the increase in the case a glutathione transferase (Ya+Yc) messenger RNAs was sluggish and steady and a value of 3-4 fold was reached around 24 hr. Run-off transcription rates for cytochrome P-450 (b+e) increased by nearly 15 fold in 4 hr after phenobarbitone administration, whereas the increase for glutathione transferase (Ya+Yc) was only 2.0 fold. At 12 hr after the drug administration, the glutathione transferase (Ya+Yc) transcription rates were near normal. Administration of 3-amino-1,2,4-triazole and CoCl2 blocked the phenobarbitone-mediated increase in the transcription of cytochrome P-450 (b+e) messenger RNAs. These compounds at best had only marginal effects on the transcription of glutathione transferase (Ya+Yc) messenger RNAs. The half-life of cytochrome P-450 (b+e) messenger RNA was estimated to be 3-4 hr, whereas that for glutathione transferase (Ya+Yc) was found to be 8-9 hr. Administration of phenobarbitone enhanced the half-life of glutathione transferase (Ya+Yc) messenger RNA by nearly two fold. It is suggested that while transcription activation may play a primary role in the induction of cytochrome P-450 (b+e), the induction of glutathione transferase (Ya+Yc) may essentially involve stabilization of the messenger RNAs.

Amitrole↗

Rat hepatic stellate cells contribute to the acute-phase response with increased expression of alpha1(I) and alpha1(IV) collagens, tissue inhibitor of metalloproteinase-1, and matrix-metalloproteinase-2 messenger RNAs.

The acute-phase response (APR) represents a systemic reaction of the organism to multiple nonspecific inflammatory stimuli. In general, it is protective for the host, and hepatocytes are the main cells responding with alterations in the expression of a set of liver-specific proteins named the acute-phase proteins. We have previously shown that although a turpentine-induced APR is not fibrogenic per se, it enhances collagen deposition in rats treated with CCl(4) and up-regulates expression of hepatic alpha1(I) collagen and tissue inhibitor of metalloproteinases 1 (TIMP-1) messenger RNAs (mRNAs). In this report we extended our studies and showed that turpentine induced, in a time-dependent manner, expression of alpha1(I) and alpha1(IV) collagens, TIMP-1, and matrix-metalloproteinase 2 (MMP-2) mRNAs. We further showed that expression of these mRNAs occurs in hepatic stellate cells, but not in hepatocytes obtained 6 hours after the induction of an APR episode. These changes were accompanied by increased blood levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) without noticeable immediate changes in the expression of their respective mRNAs in the liver. In contrast to CCl(4)-induced liver damage, turpentine alone, whether administered as a single dose or as a weekly dose for 3 weeks did not up-regulate expression of transforming growth factor beta1 (TGF-beta1) mRNA and did not result in excess collagen deposition. Overall, these findings suggest that collagen deposition in the livers of rats with repeated APR episodes may be enhanced only when given together with a fibrogenic stimulus that activates hepatic stellate cells (HSCs) and/or up-regulates TGF-beta1 mRNA expression.

Acute-Phase Reaction↗

The effect of dibromo-dulcitol and dianhydro-dulcitol (galactitol) on RNA synthesis in ascites tumor cells.

The mechanism of action on RNA synthesis of anticancer dibromo-dulcitol (DBD, NSC-104800) and dianhydro-dulcitol (DAD, or elsewhere dianhydrogalactitol, DAG, NSC-132313) was investigated. Rats, bearing Yoshida or Novikoff hepatoma ascites tumor cells sensitive to these drugs were treated with doses equivalent to half the LD50 value. Nucleolar RNA (noRNA) and nuclear RNA (nRNA) were pulse labelled with 3H-uridine, isolated and fractionated on sucrose density gradient. After 18 h treatment with either drug and after 3 h with DAD noRNA synthesis increased and the rate of ribosomal RNA (rRNA) precursor processing was enhanced. Investigation of low-molecular weight nRNAs (LMW nRNAs) (separated by polyacrylamide gel electrophoresis) showed increased synthesis and/or accumulation of RNA species (5S RNA, uridylic acid rich RNAs) related to rRNA synthesis. The tritium labelled drugs were bound to distinct fractions of nRNA, separated by sucrose density gradient ultracentrifugation, both in vivo and in vitro. This fact may be explained by the formation of intra-, or intermolecular crosslinking of pre-messenger RNA. The enhanced RNA synthesis might be interpreted as an alteration in the functions of nuclear proteins, involved in the regulation of gene transcription and processing of RNA precursors.

Animals↗

Differential subtraction display: a unified approach for isolation of cDNAs from differentially expressed genes.

We have developed a novel efficient approach, termed differential subtraction display, for the identification of differentially expressed genes. Several critical parameters for the reproducibility and enhanced sensitivity of display, as well as steps to reduce the number of false positive cDNA species, have been defined. These include- (a) use of standardized oligo(dT)-primed cDNA pools rather than total RNA as the starting material for differential display, (b) critical role of optimal cDNA input for each distinct class of primers, (c) phenomena of primer dominance and interference, and (d) design of a novel set of enhanced specificity anchor primers. Introduction of an efficient subtractive hybridization step prior to cloning of cDNA species enriches the bona fide cDNA species that are either exclusively present in one sample (+/-) or show altered expression (up-/down-regulation) in RNA samples from two different tissues or cell types. This approach, in comparison to differential display, has several advantages in terms of reproducibility and enhanced sensitivity of display coupled to the cloning of enriched bona fide cDNA species corresponding to differentially expressed RNAs.

Animals↗

Copresence of prostaglandin EP2 and EP3 receptors on gastric enterochromaffin-like cell carcinoid in African rodents.

BACKGROUND & AIMS: Prostaglandins (PGs) have important roles in the regulation of gastric acid secretion. The aim of this study was to examine the possible presence of PG receptors on the gastric enterochromaffin-like (ECL) carcinoid of Mastomys natalensis, which might be a useful model of normal ECL cells. METHODS: A [3H]PGE2 binding experiment was performed by using the ECL tumor membrane, and intracellular signal transduction was studied in the cells. In addition, Northern blot analysis using EP2 and EP3 receptor complementary DNAs was conducted. RESULTS: [3H]PGE2 specifically bound to the tumor cell membrane, and the binding was displaced by various PGs with a potency order of PGE1 = PGE2 > enprostil > PGF2 alpha. Although PGE1 and PGE2 stimulated 5'-cyclic adenosine monophosphate (cAMP) production, neither PGF2 alpha nor enprostil had any effect. On the other hand, all of PGE1, PGE2, PGF2 alpha, and enprostil attenuated the forskolin-induced cAMP production. Moreover, enprostil inhibited histamine release induced by forskolin. However, on pertussis toxin treatment, PGE2 paradoxically enhanced the forskolin-induced increase of cAMP production. Finally, the presence of EP2 and EP3 receptor messenger RNAs was confirmed by RNA blot analysis. CONCLUSIONS: The ECL carcinoid tumor cells of Mastomys seem to possess two subtypes of PGE receptor: EP2 linked to cAMP production and EP3 coupled with inhibitory guanosine 5'-triphosphate-binding proteins mediating the inhibition of cAMP production.

Animals↗

A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells.

RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells.

Animals↗

High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon.

The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.

Animals↗

c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription.

The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis. c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation. Proteasome inhibition leads to c-Myc accumulation within nucleoli, indicating that c-Myc might have a nucleolar function. Here we show that the proteins c-Myc and Max interact in nucleoli and are associated with ribosomal DNA. This association is increased upon activation of quiescent cells and is followed by recruitment of the Myc cofactor TRRAP, enhanced histone acetylation, recruitment of RNA polymerase I (Pol I), and activation of rDNA transcription. Using small interfering RNAs (siRNAs) against c-Myc and an inhibitor of Myc-Max interactions, we demonstrate that c-Myc is required for activating rDNA transcription in response to mitogenic signals. Furthermore, using the ligand-activated MycER (ER, oestrogen receptor) system, we show that c-Myc can activate Pol I transcription in the absence of Pol II transcription. These results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.

Animals↗