scholarly journals Inhibition of tRNA Gene Transcription by the Immunosuppressant Mycophenolic Acid

2019 ◽  
Vol 40 (1) ◽  
Author(s):  
Aneta Jurkiewicz ◽  
Ewa Leśniewska ◽  
Małgorzata Cieśla ◽  
Neuton Gorjão ◽  
Theodoros Kantidakis ◽  
...  
Keyword(s):  
Mycophenolic Acid ◽  
Mammalian Cells ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Sharp Decrease ◽  
Trna Genes ◽  
Anticancer Chemotherapy ◽  
Transcriptional Inhibition ◽  
Cycle Arrest ◽  
Pol Iii

ABSTRACT 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.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

scholarly journals Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

1992 ◽  
Vol 12 (10) ◽  
pp. 4271-4278 ◽  
Author(s):  
D E Syroid ◽  
R I Tapping ◽  
J P Capone
Keyword(s):  
Mammalian Cells ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Lac Repressor ◽  
Trna Genes ◽  
Coding Region ◽  
Lac Operator ◽  
Cell Extracts

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

scholarly journals RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.

1995 ◽  
Vol 15 (9) ◽  
pp. 4873-4883 ◽  
Author(s):  
K S Ullman ◽  
D J Forbes
Keyword(s):  
Rna Polymerase ◽  
Nuclear Structure ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Transcriptional Analysis ◽  
Dna Encoding ◽  
Nuclear Assembly ◽  
Polymerase Iii ◽  
Pol Iii

Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.

scholarly journals Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome

2006 ◽  
Vol 396 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Akhila Parthasarthy ◽  
Karumathil P. Gopinathan
Keyword(s):  
Transcriptional Activation ◽  
Multigene Family ◽  
Rna Polymerase Iii ◽  
Upstream Region ◽  
Trna Genes ◽  
Naked Dna ◽  
Gene Copies ◽  
Direct Binding ◽  
Pol Iii ◽  
Mulberry Silkworm

All of the members of a tRNA1Gly multigene family from the mulberry silkworm, Bombyx mori, have identical coding regions and consequently identical internal promoter elements, but are transcribed at different levels. A moderately expressed copy, tRNA1Gly-4 from within this multigene family, which was transcribed to 30–50% of the highly transcribed gene copies harboured two typical TATAA box sequences in the 5′ upstream region at positions −27 nt and −154 nt with respect to the +1 nt of mature tRNA. Deletion of the distal TATAA sequence at −154 nt brought down the transcription more than 70%, whereas mutation of the proximal element did not affect transcription. tRNA1Gly-4 could be readily assembled into chromatin, with a positioned nucleosome in the upstream region, and the assembled nucleosome formed stable complexes with the transcription factors TFIIIC and TFIIIB. Organization of the gene into nucleosomes also enhanced transcription significantly above that of the naked DNA, reaching transcription levels comparable with those of the highly transcribed copies. This nucleosome-mediated enhancement in transcription was absent when the distal TATAA sequences were deleted, whereas mutation of the proximal TATAA element showed no effect. In the absence of the distal TATAA sequences, assembly into the nucleosome inhibited transcription of tRNA1Gly-4. TFIIIB bound directly through the distal TATAA sequence at −154 nt and the positioned nucleosome facilitated its interaction with TFIIIC. The direct binding of TFIIIB to the DNA provided anchoring of the factor to the template DNA which conferred a higher stability on the TFIIIB–TFIIIC–DNA complex. We have proposed a novel mechanism for the nucleosome-mediated stimulation of pol III (RNA polymerase III) transcription of tRNA genes, a model not presented previously.

scholarly journals Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes.

1994 ◽  
Vol 14 (7) ◽  
pp. 4704-4711 ◽  
Author(s):  
V J Wolf ◽  
T Dang ◽  
P Hartl ◽  
J M Gottesfeld
Keyword(s):  
Rna Polymerase Iii ◽  
Cyclin B ◽  
Trna Genes ◽  
5S Rna ◽  
Transcription System ◽  
Transcription Complexes ◽  
Pol Iii ◽  
Differential Transcription ◽  
5S Rna Genes ◽  
Rna Genes

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.

scholarly journals Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

2015 ◽  
Vol 35 (7) ◽  
pp. 1169-1181 ◽  
Author(s):  
Małgorzata Cieśla ◽  
Ewa Makała ◽  
Marta Płonka ◽  
Rafał Bazan ◽  
Kamil Gewartowski ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Growth Defect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Content Type ◽  
Polymerase Iii ◽  
Cold Sensitive ◽  
Pol Iii ◽  
Synthetic Growth

Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation,rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased inrpc128-1007cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of therpc128-1007mutation and selected the Rbs1 gene, whose overexpression enhancedde novotRNA transcription inrpc128-1007cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. Therpc128-1007 rbs1Δ double mutant shows a synthetic growth defect, indicating thatrpc128-1007andrbs1Δ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.

scholarly journals Epigenetic silencing of clustered tRNA genes in Arabidopsis

2020 ◽  
Vol 48 (18) ◽  
pp. 10297-10312
Author(s):  
Guillaume Hummel ◽  
Alexandre Berr ◽  
Stéfanie Graindorge ◽  
Valérie Cognat ◽  
Elodie Ubrig ◽  
...  
Keyword(s):  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Biological Processes ◽  
Predominant Role ◽  
Genomic Features ◽  
Transfer Rnas ◽  
Regulatory Processes ◽  
Repressive Chromatin ◽  
Wide Range ◽  
Pol Iii

Abstract Beyond their key role in translation, cytosolic transfer RNAs (tRNAs) are involved in a wide range of other biological processes. Nuclear tRNA genes (tDNAs) are transcribed by the RNA polymerase III (RNAP III) and cis-elements, trans-factors as well as genomic features are known to influence their expression. In Arabidopsis, besides a predominant population of dispersed tDNAs spread along the 5 chromosomes, some clustered tDNAs have been identified. Here, we demonstrate that these tDNA clusters are transcriptionally silent and that pathways involved in the maintenance of DNA methylation play a predominant role in their repression. Moreover, we show that clustered tDNAs exhibit repressive chromatin features whilst their dispersed counterparts contain permissive euchromatic marks. This work demonstrates that both genomic and epigenomic contexts are key players in the regulation of tDNAs transcription. The conservation of most of these regulatory processes suggests that this pioneering work in Arabidopsis can provide new insights into the regulation of RNA Pol III transcription in other organisms, including vertebrates.

scholarly journals Role of maturation-promoting factor (p34cdc2-cyclin B) in differential expression of the Xenopus oocyte and somatic-type 5S RNA genes

1994 ◽  
Vol 14 (7) ◽  
pp. 4704-4711
Author(s):  
V J Wolf ◽  
T Dang ◽  
P Hartl ◽  
J M Gottesfeld
Keyword(s):  
Rna Polymerase Iii ◽  
Cyclin B ◽  
Trna Genes ◽  
5S Rna ◽  
Transcription System ◽  
Transcription Complexes ◽  
Pol Iii ◽  
Differential Transcription ◽  
5S Rna Genes ◽  
Rna Genes

Transcription of 5S rRNA and tRNA genes by RNA polymerase III (pol III) in cytosolic extracts of unfertilized Xenopus eggs and in a reconstituted system derived from Xenopus oocytes is repressed by the action of one or more mitotic protein kinases. Repression is due to the phosphorylation of a component of the pol III transcription apparatus. We find that the maturation/mitosis-promoting factor kinase (MPF, p34cdc2-cyclin B) can directly mediate this repression in vitro. Affinity-purified MPF and immune complexes formed with antibodies to the protein subunits of MPF (p34cdc2 and cyclin B) retain both histone H1 kinase activity and the capacity to repress transcription in the reconstituted transcription system. Transcription complexes of oocyte-type 5S RNA genes and tRNA genes are quantitatively more sensitive to MPF repression than the corresponding transcription complexes of the somatic-type 5S RNA gene. The differential transcription of oocyte- and somatic-type genes observed during early Xenopus embryogenesis has been reproduced with the reconstituted transcription system and affinity-purified MPF. This differential transcription may be due to the instability of transcription complexes on the oocyte-type genes and the heightened sensitivity of soluble transcription factors to inactivation by mitotic phosphorylation. Our results suggest that MPF may play a role in vivo in the establishment of the embryonic pattern of pol III gene expression.

scholarly journals The Ty1 integrase nuclear localization signal is necessary and sufficient for retrotransposon targeting to tRNA genes

2019 ◽  
Author(s):  
Amna Asif-Laidin ◽  
Christine Conesa ◽  
Amandine Bonnet ◽  
Camille Grison ◽  
Indranil Adhya ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Integration Site ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Site Specificity ◽  
Localization Signal ◽  
Pol Iii ◽  
Necessary And Sufficient ◽  
Subsequent Integration

SUMMARYIntegration of transposable elements into the genome is mutagenic. Mechanisms that target integration into relatively safe locations and minimize deleterious consequences for cell fitness have emerged during evolution. In budding yeast, the integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)-transcribed genes requires the interaction between the AC40 subunit of Pol III and Ty1 integrase (IN1). Here we show that the IN1-AC40 interaction involves a short linker sequence in the bipartite nuclear localization signal (bNLS) of IN1. Mutations in this sequence do not impact the frequency of Ty1 retromobility, instead they decrease the recruitment of IN1 to Pol III-transcribed genes and the subsequent integration of Ty1 at these loci. The replacement of Ty5 retrotransposon targeting sequence by the IN1 bNLS induces Ty5 integration into Pol III-transcribed genes. Therefore, the IN1 bNLS is both necessary and sufficient to confer integration site specificity on Ty1 and Ty5 retrotransposons.

scholarly journals Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Mingen Lin ◽  
Chenghao Huang ◽  
Wenfeng Ren ◽  
Jun Chen ◽  
Ningshao Xia ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colony Formation ◽  
Rna Polymerase Iii ◽  
Tumor Development ◽  
5S Rrna ◽  
Trna Genes ◽  
Related Factor ◽  
Mechanism Analysis ◽  
Primary Mouse ◽  
Pol Iii

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

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