scholarly journals Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription

2020 ◽  
Vol 48 (11) ◽  
pp. 5891-5906 ◽  
Author(s):  
Cristina Sorino ◽  
Valeria Catena ◽  
Tiziana Bruno ◽  
Francesca De Nicola ◽  
Stefano Scalera ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Cellular Proliferation ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Response To Stress ◽  
Polymerase I

Abstract Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.

scholarly journals Structure and function of ribosomal RNA gene chromatin

2008 ◽  
Vol 36 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Joanna L. Birch ◽  
Joost C.B.M. Zomerdijk
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pol I ◽  
And Function ◽  
Polymerase I ◽  
Cell Growth And Proliferation ◽  
Cellular Control

Transcription of the major ribosomal RNAs by Pol I (RNA polymerase I) is a key determinant of ribosome biogenesis, driving cell growth and proliferation in eukaryotes. Hundreds of copies of rRNA genes are present in each cell, and there is evidence that the cellular control of Pol I transcription involves adjustments to the number of rRNA genes actively engaged in transcription, as well as to the rate of transcription from each active gene. Chromatin structure is inextricably linked to rRNA gene activity, and the present review highlights recent advances in this area.

scholarly journals The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

NAR Cancer ◽  
2020 ◽  
Vol 2 (4) ◽  
Author(s):  
Jean-Clément Mars ◽  
Michel G Tremblay ◽  
Mélissa Valere ◽  
Dany S Sibai ◽  
Marianne Sabourin-Felix ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Transcription Initiation ◽  
Ribosome Biogenesis ◽  
Chemotherapeutic Agent ◽  
Rna Polymerase I ◽  
Initiation Complex ◽  
Promoter Recruitment ◽  
Polymerase I

Abstract In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

scholarly journals Ribosomal RNA Transcription Regulation in Breast Cancer

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 502
Author(s):  
Cecelia M. Harold ◽  
Amber F. Buhagiar ◽  
Yan Cheng ◽  
Susan J. Baserga
Keyword(s):  
Breast Cancer ◽  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Rna Polymerase I ◽  
Cancer Therapies ◽  
Rdna Transcription ◽  
Global Protein Synthesis ◽  
Novel Drugs ◽  
Polymerase I

Ribosome biogenesis is a complex process that is responsible for the formation of ribosomes and ultimately global protein synthesis. The first step in this process is the synthesis of the ribosomal RNA in the nucleolus, transcribed by RNA Polymerase I. Historically, abnormal nucleolar structure is indicative of poor cancer prognoses. In recent years, it has been shown that ribosome biogenesis, and rDNA transcription in particular, is dysregulated in cancer cells. Coupled with advancements in screening technology that allowed for the discovery of novel drugs targeting RNA Polymerase I, this transcriptional machinery is an increasingly viable target for cancer therapies. In this review, we discuss ribosome biogenesis in breast cancer and the different cellular pathways involved. Moreover, we discuss current therapeutics that have been found to affect rDNA transcription and more novel drugs that target rDNA transcription machinery as a promising avenue for breast cancer treatment.

scholarly journals Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

2018 ◽  
Author(s):  
Tommy Darrière ◽  
Michael Pilsl ◽  
Marie-Kerguelen Sarthou ◽  
Adrien Chauvier ◽  
Titouan Genty ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Growth Defect ◽  
Pol I ◽  
Polymerase I ◽  
Extragenic Suppressors

AbstractMost transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

scholarly journals Increased numbers of nucleoli in a genome-wide RNAi screen reveal proteins that link the cell cycle to RNA polymerase I transcription

2021 ◽  
Vol 32 (9) ◽  
pp. 956-973 ◽  
Author(s):  
Lisa M. Ogawa ◽  
Amber F. Buhagiar ◽  
Laura Abriola ◽  
Bryan A. Leland ◽  
Yulia V. Surovtseva ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Ribosome Biogenesis ◽  
Rna Polymerase I ◽  
Wide Field ◽  
Genome Wide ◽  
A Genome ◽  
Nucleolar Number ◽  
Polymerase I ◽  
Cycle Regulation

The nucleolus is a dynamic nuclear condensate and site of ribosome biogenesis. Using wide-field fluorescence microscopy, we screened for proteins that when depleted cause an increase in nucleolar number. Our results uncovered an unexpected subset of proteins that link the nucleolus, cell cycle regulation, and RNA polymerase I transcription.

scholarly journals Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Benjamin Albert ◽  
Jorge Perez-Fernandez ◽  
Isabelle Léger-Silvestre ◽  
Olivier Gadal
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Chromatin Structure ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Elongation Factors ◽  
Pol I ◽  
Polymerase I

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

scholarly journals Human Nopp140, Which Interacts with RNA Polymerase I: Implications for rRNA Gene Transcription and Nucleolar Structural Organization

1999 ◽  
Vol 19 (12) ◽  
pp. 8536-8546 ◽  
Author(s):  
Hung-Kai Chen ◽  
Chi-Yun Pai ◽  
Jing-Yi Huang ◽  
Ning-Hsing Yeh
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Gene Transcription ◽  
Ectopic Expression ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Amino Terminal ◽  
Polymerase I

ABSTRACT Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

scholarly journals Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein.

1997 ◽  
Vol 17 (8) ◽  
pp. 4230-4237 ◽  
Author(s):  
R Voit ◽  
K Schäfer ◽  
I Grummt
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Susceptibility Gene ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rdna Transcription ◽  
Polymerase I

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.

Cytoplasmic to nuclear translocation of RNA polymerase I is required for lipopolysaccharide-induced nucleolar RNA synthesis and subsequent DNA synthesis in murine B lymphocytes

1989 ◽  
Vol 92 (1) ◽  
pp. 101-109
Author(s):  
M.S. Halleck ◽  
R.A. Schlegel ◽  
K.M. Rose
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
B Lymphocytes ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Nuclear Translocation ◽  
Rna Polymerase I ◽  
Nuclear Accumulation ◽  
Rrna Synthesis ◽  
Polymerase I

The synthesis of ribosomal RNA (rRNA) in murine B lymphocytes is markedly elevated in response to mitogens such as lipopolysaccharide (LPS). First, to investigate the mechanism involved, antibodies directed against RNA polymerase I, the enzyme responsible for transcription of ribosomal genes, were introduced into the cytoplasm of lymphocytes via red cell-mediated microinjection and the ability of cells to synthesize RNA was examined. Simultaneous immunofluorescence/autoradiography revealed that 7% or less of the cells injected with specific antibodies prior to stimulation were actively synthesizing rRNA 15 or 40 h following LPS addition. In contrast 19% and 27% of cells injected with control IgG were active at these times. Non-ribosomal RNA synthesis was unaffected by the presence of anti-RNA polymerase I antibodies. Since antibodies injected into the cytoplasm were limited to that compartment, these data suggest that rRNA synthesis induced by LPS requires translocation of cytoplasmic RNA polymerase I into the nucleus. Second, to test whether synthesis of rRNA is required for entry into S phase, the effect of anti-RNA polymerase I antibodies on DNA synthesis in response to LPS was evaluated. Only 7% of cells containing anti-RNA polymerase I antibodies had initiated DNA synthesis 40 h after LPS addition whereas 25% of cells containing control IgG were actively synthesizing DNA at that time. These results suggest that nuclear accumulation of RNA polymerase I and increased rRNA synthesis are required for LPS-induced DNA synthesis in B lymphocytes.

scholarly journals Defining the Influence of the A12.2 Subunit on Transcription Elongation and Termination by RNA Polymerase I In Vivo

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1939
Author(s):  
Andrew M. Clarke ◽  
Abigail K. Huffines ◽  
Yvonne J. K. Edwards ◽  
Chad M. Petit ◽  
David A. Schneider
Keyword(s):  
Rna Polymerase ◽  
Ribosome Biogenesis ◽  
Transcription Elongation ◽  
5S Rdna ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rdna Gene ◽  
Pol I ◽  
Nucleotide Addition ◽  
Polymerase I

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination.

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