Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion.

The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-min protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable "open source" design strategy would enable virtually any laboratory to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment.

Copper-CX-5461: A novel liposomal formulation for a small molecule rRNA synthesis inhibitor.

CX-5461 is currently in Phase I/II clinical trials for advanced hematologic malignancies and triple negative or BRCA-deficient breast cancer. The compound is currently administered to patients intravenously (i.v.) at low pH (3.5) due to solubility challenges. Reliance of low pH to enhance solubility of CX-5461 can adversely impact pharmacokinetics, biodistribution and therapeutic potential. We have addressed this solubility issue through a formulation method that relies on the interactions between CX-5461 and copper. Copper binds CX-5461 through the nitrogens of the pyrazine ring. Here, we describe synthesizing this copper-complexed CX-5461 (Cu(CX-5461)) within liposomes. CX-5461 was added to copper-containing liposomes and incubated at 60 °C for 30 min. The pharmacokinetics of CX-5461 was assessed in mice following a single i.v. injection at 30 mg/kg. Efficacy studies were completed in multiple subcutaneous mouse xenografts as well as in a bone marrow engraftment model of acute myeloid leukemia (AML). The novel Cu(CX-5461) formulation was stable at pH 7.4 and exhibited increased plasma circulation longevity, increasing the total exposure to CX5461 by an order of magnitude. Cu(CX-5461) was more active than CX-5461 in AML models in vivo. In HCT116-B46 and Capan-1 solid tumour models that are BRCA-deficient, the Cu(CX-5461) formulation engendered activity that was comparable to that of the low pH CX-5461 formulation. We have generated the first Cu(CX-5461) formulation suitable for i.v. administration that is more efficacious than the existing low-pH formulation in pre-clinical models of AML. The Cu(CX-5461) formulation may serve as an alternative formulation for CX-5461 in BRCA-deficient cancers.

Nucleolar sub-compartments in motion during rRNA synthesis inhibition: Contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant.

The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules. Here, we used the reproducible reorganization of nucleolar components induced by the inhibition of rRNA synthesis by Actinomycin D (AMD) to address the steps of the spatiotemporal reorganization of FCs and nucleolar condensed chromatin. To reach that goal, we used two complementary approaches: i) time-lapse confocal imaging of cells expressing one or several GFP-tagged proteins (fibrillarin, UBF, histone H2B) and ii) ultrastructural identification of nucleolar components involved in the reorganization. Data obtained by time lapse confocal microscopy were analyzed through detailed 3D imaging. This allowed us to demonstrate that AMD treatment induces no fusion and no change in the relative position of the different nucleoli contained in one nucleus. In contrast, for each nucleolus, we observed step by step gathering and fusion of both FCs and nucleolar condensed chromatin. To analyze the reorganization of FCs and condensed chromatin at a higher resolution, we performed correlative light and electron microscopy electron microscopy (CLEM) imaging of the same cells. We demonstrated that threads of intranucleolar condensed chromatin are localized in a complex 3D network of vacuoles. Upon AMD treatment, these structures coalesce before migrating toward the perinucleolar condensed chromatin, to which they finally fuse. During their migration, FCs, which are all linked to ICC, are pulled by the latter to gather as caps disposed at the periphery of nucleoli.

First-In-Class Inhibitor of Ribosomal RNA Synthesis with Antimicrobial Activity against Staphylococcus aureus.

We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.

Intracellular delivery of oligonucleotides in Helicobacter pylori by fusogenic liposomes in the presence of gastric mucus.

The rising antimicrobial resistance contributes to 25000 annual deaths in Europe. This threat to the public health can only be tackled if novel antimicrobials are developed, combined with a more precise use of the currently available antibiotics through the implementation of fast, specific, diagnostic methods. Nucleic acid mimics (NAMs) that are able to hybridize intracellular bacterial RNA have the potential to become such a new class of antimicrobials and additionally could serve as specific detection probes. However, an essential requirement is that these NAMs should be delivered into the bacterial cytoplasm, which is a particular challenge given the fact that they are charged macromolecules. We consider these delivery challenges in relation to the gastric pathogen Helicobacter pylori, the most frequent chronic infection worldwide. In particular, we evaluate if cationic fusogenic liposomes are suitable carriers to deliver NAMs across the gastric mucus barrier and the bacterial envelope. Our study shows that DOTAP-DOPE liposomes post-PEGylated with DSPE-PEG (DSPE Lpx) can indeed successfully deliver NAMs into Helicobacter pylori, while offering protection to the NAMs from binding and inactivation in gastric mucus isolated from pigs. DSPE Lpx thus offer exciting new possibilities for in vivo diagnosis and treatment of Helicobacter pylori infections.

The Potential of Targeting Ribosome Biogenesis in High-Grade Serous Ovarian Cancer.

Overall survival for patients with ovarian cancer (OC) has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC). HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies. However, deregulation of pathways controlling homologous recombination (HR) and ribosome biogenesis has been observed in a high proportion of HGSOC, raising the possibility that targeting these basic cellular processes may provide improved patient outcomes. The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib has been approved to treat women with defects in HR due to germline BRCA mutations. Recent evidence demonstrated the efficacy of targeting ribosome biogenesis with the specific inhibitor of ribosomal RNA synthesis, CX-5461 in v-myc avian myelocytomatosis viral oncogene homolog (MYC)-driven haematological and prostate cancers. CX-5461 has now progressed to a phase I clinical trial in patients with haematological malignancies and phase I/II trial in breast cancer. Here we review the currently available targeted therapies for HGSOC and discuss the potential of targeting ribosome biogenesis as a novel therapeutic approach against HGSOC.

Antimicrobial Activity, AME Resistance, and A-Site Binding Studies of Anthraquinone-Neomycin Conjugates.

The antibacterial effects of aminoglycosides are based on their association with the A-site of bacterial rRNA and interference with the translational process in the bacterial cell, causing cell death. The clinical use of aminoglycosides is complicated by resistance and side effects, some of which arise from their interactions with the human mitochondrial 12S rRNA and its deafness-associated mutations, C1494U and A1555G. We report a rapid assay that allows screening of aminoglycoside compounds to these classes of rRNAs. These screening tools are important to find antibiotics that selectively bind to the bacterial A-site rather than human, mitochondrial A-sites and its mutant homologues. Herein, we report our preliminary work on the optimization of this screen using 12 anthraquinone-neomycin (AMA-NEO) conjugates against molecular constructs representing five A-site homologues, Escherichia coli, human cytosolic, mitochondrial, C1494U, and A1555G, using a fluorescent displacement screening assay. These conjugates were also tested for inhibition of protein synthesis, antibacterial activity against 14 clinically relevant bacterial strains, and the effect on enzymes that inactivate aminoglycosides. The AMA-NEO conjugates demonstrated significantly improved resistance against aminoglycoside-modifying enzymes (AMEs), as compared with NEO. Several compounds exhibited significantly greater inhibition of prokaryotic protein synthesis as compared to NEO and were extremely poor inhibitors of eukaryotic translation. There was significant variation in antibacterial activity and MIC of selected compounds between bacterial strains, with Escherichia coli, Enteroccocus faecalis, Citrobacter freundii, Shigella flexneri, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Staphylococcus epidermidis, and Listeria monocytogenes exhibiting moderate to high sensitivity (50-100% growth inhibition) whereas Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellla pneumoniae, and MRSA strains expressed low sensitivity, as compared to the parent aminoglycoside NEO.

Analysis of ribosomal inter-subunit sites as targets for complementary oligonucleotides.

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a ß-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC50 values below 1 µM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC50 in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.

Targeted cancer therapy with ribosome biogenesis inhibitors: a real possibility?

The effects of many chemotherapeutic drugs on ribosome biogenesis have been underestimated for a long time. Indeed, many drugs currently used for cancer treatment--and which are known to either damage DNA or hinder DNA synthesis--have been shown to exert their toxic action mainly by inhibiting rRNA synthesis or maturation. Moreover, there are new drugs that have been proposed recently for cancer chemotherapy, which only hinder ribosome biogenesis without any genotoxic activity. Even though ribosome biogenesis occurs in both normal and cancer cells, whether resting or proliferating, there is evidence that the selective inhibition of ribosome biogenesis may, in some instances, result in a selective damage to neoplastic cells. The higher sensitivity of cancer cells to inhibitors of rRNA synthesis appears to be the consequence of either the loss of the mechanisms controlling the cell cycle progression or the acquisition of activating oncogene and inactivating tumor suppressor gene mutations that up-regulate the ribosome biogenesis rate. This article reviews those cancer cell characteristics on which the selective cancer cell cytotoxicity induced by the inhibitors of ribosome biogenesis is based.

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells.

The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated.

Protein synthesis inhibition activity by strawberry tissue protein extracts during plant life cycle and under biotic and abiotic stresses.

Ribosome-inactivating proteins (RIPs), enzymes that are widely distributed in the plant kingdom, inhibit protein synthesis by depurinating rRNA and many other polynucleotidic substrates. Although RIPs show antiviral, antifungal, and insecticidal activities, their biological and physiological roles are not completely understood. Additionally, it has been described that RIP expression is augmented under stressful conditions. In this study, we evaluated protein synthesis inhibition activity in partially purified basic proteins (hereafter referred to as RIP activity) from tissue extracts of Fragaria × ananassa (strawberry) cultivars with low (Dora) and high (Record) tolerance to root pathogens and fructification stress. Association between the presence of RIP activity and the crop management (organic or integrated soil), growth stage (quiescence, flowering, and fructification), and exogenous stress (drought) were investigated. RIP activity was found in every tissue tested (roots, rhizomes, leaves, buds, flowers, and fruits) and under each tested condition. However, significant differences in RIP distribution were observed depending on the soil and growth stage, and an increase in RIP activity was found in the leaves of drought-stressed plants. These results suggest that RIP expression and activity could represent a response mechanism against biotic and abiotic stresses and could be a useful tool in selecting stress-resistant strawberry genotypes.

Using sequence-specific oligonucleotides to inhibit bacterial rRNA.

The majority of antibiotics used in the clinic target bacterial protein synthesis. However, the widespread emergence of bacterial resistance to existing drugs creates a need to discover or develop new therapeutic agents. Ribosomal RNA (rRNA) has been a target for numerous antibiotics that bind to functional rRNA regions such as the peptidyl transferase center, polypeptide exit tunnel, and tRNA binding sites. Even though the atomic resolution structures of many ribosome-antibiotic complexes have been solved, improving the ribosome-acting drugs is difficult because the large rRNA has a complicated 3D architecture and is surrounded by numerous proteins. Computational approaches, such as structure-based design, often fail when applied to rRNA binders because electrostatics dominate the interactions and the effect of ions and bridging waters is difficult to account for in the scoring functions. Improving the classical anti-ribosomal agents has not proven particularly successful and has not kept pace with acquired resistance. So one needs to look for other ways to combat the ribosomes, finding either new rRNA targets or totally different compounds. There have been some efforts to design translation inhibitors that act on the basis of the sequence-specific hybridization properties of nucleic acid bases. Indeed oligonucleotides hybridizing with functional regions of rRNA have been shown to inhibit translation. Also, some peptides have been shown to be reasonable inhibitors. In this review we describe these nonconventional approaches to screening for ribosome inhibition and function of particular rRNA regions. We discuss inhibitors against rRNA that may be designed according to nucleotide sequence and higher order structure.

Crystal structures of a bioactive 6'-hydroxy variant of sisomicin bound to the bacterial and protozoal ribosomal decoding sites.

Parasitic infections recognized as neglected tropical diseases are a source of concern for several regions of the world. Aminoglycosides are potent antimicrobial agents that have been extensively studied by biochemical and structural studies in prokaryotes. However, the molecular mechanism of their potential antiprotozoal activity is less well understood. In the present study, we have examined the in vitro inhibitory activities of some aminoglycosides with a 6'-hydroxy group on ring I and highlight that one of them, 6'-hydroxysisomicin, exhibits promising activity against a broad range of protozoan parasites. Furthermore, we have conducted X-ray analyses of 6'-hydroxysisomicin bound to the target ribosomal RNA A-sites in order to understand the mechanisms of both its antibacterial and antiprotozoal activities at the molecular level. The unsaturated ring I of 6'-hydroxysisomicin can directly stack on G1491, which is highly conserved in bacterial and protozoal species, through π-π interaction and fits closer to the guanidine base than the typically saturated and hydroxylated ring I of other structurally related aminoglycosides. Consequently, the compound adopts a lower energy conformation within the bacterial and protozoal A-sites and makes pseudo pairs to either A or G at position 1408. The A-site-selective binding mode strongly suggests that 6'-hydroxysisomicin is a potential lead for the design of next-generation aminoglycosides targeting a wide variety of infectious diseases.

Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation.

The tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

Nucleolar follistatin promotes cancer cell survival under glucose-deprived conditions through inhibiting cellular rRNA synthesis.

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

Nuclear-localized calcineurin homologous protein CHP1 interacts with upstream binding factor and inhibits ribosomal RNA synthesis.

Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca(2+)-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibroblasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca(2+). Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.

Molecular determinants of microbial resistance to thiopeptide antibiotics.

Ribosomally produced thiopeptide antibiotics are highly promising lead compounds targeting the GTPase-associated region (GAR) of the bacterial ribosome. A representative panel of GAR mutants suspected to confer resistance against thiopeptide antibiotics was reconstituted in vitro and quantitatively studied with fluorescent probes. It was found that single-site mutations of the ribosomal 23S rRNA binding site region directly affect thiopeptide affinity. Quantitative equilibrium binding data clearly identified A1067 as the base contributing most strongly to the binding environment. The P25 residue on the ribosomal protein L11 was essential for binding of the monocyclic thiopeptides micrococcin and promothiocin B, confirming that the mutation of this residue in the producer organism confers self-resistance. For the bicyclic thiopeptides thiostrepton and nosiheptide, all studied single-site resistance mutations on the L11 protein were still fully capable of ligand binding in the upper pM range, both in the RNA-protein complex and in isolated 70S ribosomes. These single-site mutants were then specifically reconstituted in Bacillus subtilis, confirming their efficacy as resistance-conferring. It is thus reasoned that, in contrast to modifications of the 23S rRNA in the GAR, mutations of the L11 protein do not counteract binding of bicyclic thiopeptides, but allow the ribosome to bypass the protein biosynthesis blockade enforced by these antibiotics in the wild type.

Maintaining ATP levels via the suppression of PERK-mediated rRNA synthesis at ER stress.

Currently, [(3)H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [alpha-(33)P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1alpha or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2alpha. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2alpha knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.

Variability in inhibition of host RNA synthesis by entero- and cardioviruses.

Both entero- and cardioviruses have been shown to suppress host mRNA synthesis. Enteroviruses are also known to inhibit the activity of rRNA genes, whereas this ability of cardioviruses is under debate. This study reported that mengovirus (a cardiovirus) suppressed rRNA synthesis but less efficiently than poliovirus (an enterovirus). In contrast to poliovirus infection, the incorporation of BrUTP, fluorouridine and [14C]uridine in rRNA precursors was observed even during the late stages of mengovirus infection, although at a significantly reduced level. The cleavage of TATA-binding protein, considered to be one of the central events in poliovirus-induced transcription shutoff, was not detected in mengovirus-infected cells, indicating a difference in the mechanisms of host RNA synthesis inhibition caused by these viruses. The results also showed that functional leader protein is redundant for the suppression of host RNA synthesis by cardiovirus.