Maf1 ameliorates cardiac hypertrophy by inhibiting RNA polymerase III through ERK1/2.

Rationale: An imbalance between protein synthesis and degradation is one of the mechanisms of cardiac hypertrophy. Increased transcription in cardiomyocytes can lead to excessive protein synthesis and cardiac hypertrophy. Maf1 is an RNA polymerase III (RNA pol III) inhibitor that plays a pivotal role in regulating transcription. However, whether Maf1 regulates of cardiac hypertrophy remains unclear. Methods: Cardiac hypertrophy was induced in vivo by thoracic aortic banding (AB) surgery. Both the in vivo and in vitro gain- and loss-of-function experiments by Maf1 knockout (KO) mice and adenoviral transfection were used to verify the role of Maf1 in cardiac hypertrophy. RNA pol III and ERK1/2 inhibitor were utilized to identify the effects of RNA pol III and ERK1/2. The possible interaction between Maf1 and ERK1/2 was clarified by immunoprecipitation (IP) analysis. Results: Four weeks after surgery, Maf1 KO mice exhibited significantly exacerbated AB-induced cardiac hypertrophy characterized by increased heart size, cardiomyocyte surface area, and atrial natriuretic peptide (ANP) expression and by exacerbated pulmonary edema. Also, the deficiency of Maf1 causes more severe cardiac dilation and dysfunction than wild type (WT) mice after pressure overload. In contrast, compared with adenoviral-GFP injected mice, mice injected with adenoviral-Maf1 showed significantly ameliorated AB-induced cardiac hypertrophy. In vitro study has demonstrated that Maf1 could significantly block phenylephrine (PE)-induced cardiomyocyte hypertrophy by inhibiting RNA pol III transcription. However, application of an RNA pol III inhibitor markedly improved Maf1 knockdown-promoted cardiac hypertrophy. Moreover, ERK1/2 was identified as a regulator of RNA pol III, and ERK1/2 inhibition by U0126 significantly repressed Maf1 knockdown-promoted cardiac hypertrophy accompanied by suppressed RNA pol III transcription. Additionally, IP analysis demonstrated that Maf1 could directly bind ERK1/2, suggesting Maf1 could interact with ERK1/2 and then inhibit RNA pol III transcription so as to attenuate the development of cardiac hypertrophy. Conclusions: Maf1 ameliorates PE- and AB-induced cardiac hypertrophy by inhibiting RNA pol III transcription via ERK1/2 signaling suppression.

Inhibition of tRNA Gene Transcription by the Immunosuppressant Mycophenolic Acid.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling.

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila.

The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that Ras signalling promotes Pol III function and tRNA synthesis by phosphorylating, and inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.

Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles.

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

RNA polymerase III limits longevity downstream of TORC1.

Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity. This raises the possibility that Pol III is involved in ageing. Here we show that Pol III limits lifespan downstream of TORC1. We find that a reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target.

High-resolution RNA allelotyping along the inactive X chromosome: evidence of RNA polymerase III in regulating chromatin configuration.

We carried out padlock capture, a high-resolution RNA allelotyping method, to study X chromosome inactivation (XCI). We examined the gene reactivation pattern along the inactive X (Xi), after Xist (X-inactive specific transcript), a prototype long non-coding RNA essential for establishing X chromosome inactivation (XCI) in early embryos, is conditionally deleted from Xi in somatic cells (Xi∆Xist). We also monitored the behaviors of X-linked non-coding transcripts before and after XCI. In each mutant cell line, gene reactivation occurs to ~6% genes along Xi∆Xist in a recognizable pattern. Genes with upstream regions enriched for SINEs are prone to be reactivated. SINE is a class of retrotransposon transcribed by RNA polymerase III (Pol III). Intriguingly, a significant fraction of Pol III transcription from non-coding regions is not subjected to Xist-mediated transcriptional silencing. Pol III inhibition affects gene reactivation status along Xi∆Xist, alters chromatin configuration and interferes with the establishment XCI during in vitro differentiation of ES cells. These results suggest that Pol III transcription is involved in chromatin structure re-organization during the onset of XCI and functions as a general mechanism regulating chromatin configuration in mammalian cells.

Transcribing RNA polymerase III observed by electron cryomicroscopy.

Electron cryomicroscopy reconstructions of elongating RNA polymerase (Pol) III at 3.9 Å resolution and of unbound Pol III (apo Pol III) in two distinct conformations at 4.6 Å and 4.7 Å resolution allow the construction of complete atomic models of Pol III and provide new functional insights into the adaption of Pol III to fulfill its specific transcription tasks.

Telomeric transcriptome from Chironomus riparius (Diptera), a species with noncanonical telomeres.

Although there are alternative telomere structures, most telomeres contain DNA arrays of short repeats (6-26 bp) maintained by telomerase. Like other diptera, Chironomus riparius has noncanonical telomeres and three subfamilies, TsA, TsB and TsC, of longer sequences (176 bp) are found at their chromosomal ends. Reverse transcription PCR was used to show that different RNAs are transcribed from these sequences. Only one strand from TsA sequences seems to render a noncoding RNA (named CriTER-A); transcripts from both TsB strands were found (CriTER-B and αCriTER-B) but no TsC transcripts were detected. Interestingly, these sequences showed a differential transcriptional response upon heat shock, and they were also differentially affected by inhibitors of RNA polymerase II and RNA polymerase III. A computer search for transcription factor binding sites revealed putative regulatory cis-elements within the transcribed sequence, reinforcing the experimental evidence which suggests that the telomeric repeat might function as a promoter. This work describes the telomeric transcriptome of an insect with non-telomerase telomeres, confirming the evolutionary conservation of telomere transcription. Our data reveal differences in the regulation of telomeric transcripts between control and stressful environmental conditions, supporting the idea that telomeric RNAs could have a relevant role in cellular metabolism in insect cells.

Citrus MAF1, a repressor of RNA polymerase III, binds the Xanthomonas citri canker elicitor PthA4 and suppresses citrus canker development.

Transcription activator-like (TAL) effectors from Xanthomonas species pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the carboxyl-terminal domain of the sweet orange (Citrus sinensis) RNA polymerase II (Pol II) and inhibit the activity of CsCYP, a cyclophilin associated with the carboxyl-terminal domain of the citrus RNA Pol II that functions as a negative regulator of cell growth. Here, we show that PthA4 specifically interacted with the sweet orange MAF1 (CsMAF1) protein, an RNA polymerase III (Pol III) repressor that controls ribosome biogenesis and cell growth in yeast (Saccharomyces cerevisiae) and human. CsMAF1 bound the human RNA Pol III and rescued the yeast maf1 mutant by repressing tRNA(His) transcription. The expression of PthA4 in the maf1 mutant slightly restored tRNA(His) synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNA interference plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis, and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and the presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and Pol III to coordinately increase the transcription of host genes involved in ribosome biogenesis and cell proliferation.