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

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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The RNA polymerase I transcription machinery

Biochemical Society Symposium ◽

10.1042/bss0730203 ◽

2006 ◽

Vol 73 ◽

pp. 203-216 ◽

Cited By ~ 82

Author(s):

Jackie Russell ◽

Joost C.B.M. Zomerdijk

Keyword(s):

Rna Polymerase ◽

Mammalian Cells ◽

Growth Conditions ◽

Rna Polymerase I ◽

Cellular Growth ◽

Rrna Synthesis ◽

Transcription Machinery ◽

Pol I ◽

A Cell ◽

Polymerase I

The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

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Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription

Journal of General Virology ◽

10.1099/vir.0.80817-0 ◽

2005 ◽

Vol 86 (8) ◽

pp. 2315-2322 ◽

Cited By ~ 28

Author(s):

Rajeev Banerjee ◽

Mary K. Weidman ◽

Sonia Navarro ◽

Lucio Comai ◽

Asim Dasgupta

Keyword(s):

Rna Polymerase ◽

Rna Polymerase I ◽

Rrna Synthesis ◽

Selectivity Factor ◽

Pol I ◽

Binding Factor ◽

Infected Cells ◽

Upstream Binding Factor ◽

Polymerase I

Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3Cpro appeared to cleave the TATA-binding protein-associated factor 110 (TAF110), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF110 and purified 3Cpro indicated that the Q265G266 and Q805G806 sites were cleaved by 3Cpro. Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells.

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A34.5, a nonessential component of yeast RNA polymerase I, cooperates with subunit A14 and DNA topoisomerase I to produce a functional rRNA synthesis machine.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.1787 ◽

1997 ◽

Vol 17 (4) ◽

pp. 1787-1795 ◽

Cited By ~ 44

Author(s):

O Gadal ◽

S Mariotte-Labarre ◽

S Chedin ◽

E Quemeneur ◽

C Carles ◽

...

Keyword(s):

Rna Polymerase ◽

Topoisomerase I ◽

Synthetic Lethality ◽

Rna Polymerase I ◽

Rrna Synthesis ◽

Dna Topoisomerase I ◽

Dna Topoisomerase ◽

Pol Ii ◽

Pol I ◽

Polymerase I

A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes. Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification. Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C. Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal. Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis. A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality. A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa344 ◽

2020 ◽

Vol 48 (11) ◽

pp. 5891-5906 ◽

Cited By ~ 1

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.

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The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

eLife ◽

10.7554/elife.20832 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 32

Author(s):

Eva Torreira ◽

Jaime Alegrio Louro ◽

Irene Pazos ◽

Noelia González-Polo ◽

David Gil-Carton ◽

...

Keyword(s):

Cell Growth ◽

Rna Polymerase ◽

Ribosome Biogenesis ◽

Mutational Analysis ◽

Rna Polymerase I ◽

Initiation Factor ◽

Rdna Transcription ◽

Pol I ◽

Polymerase I

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

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

10.1101/307199 ◽

2018 ◽

Cited By ~ 2

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.

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Coilin participates in the suppression of RNA polymerase I in response to cisplatin-induced DNA damage

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0731 ◽

2011 ◽

Vol 22 (7) ◽

pp. 1070-1079 ◽

Cited By ~ 33

Author(s):

Andrew S. Gilder ◽

Phi M. Do ◽

Zunamys I Carrero ◽

Angela M. Cosman ◽

Hanna J. Broome ◽

...

Keyword(s):

Dna Damage ◽

Rna Polymerase ◽

Rna Polymerase I ◽

Rrna Synthesis ◽

Γ Irradiation ◽

Small Nuclear Ribonucleoprotein ◽

Pol I ◽

Upstream Binding Factor ◽

Polymerase I ◽

Nuclear Ribonucleoprotein

Coilin is a nuclear phosphoprotein that concentrates within Cajal bodies (CBs) and impacts small nuclear ribonucleoprotein (snRNP) biogenesis. Cisplatin and γ-irradiation, which cause distinct types of DNA damage, both trigger the nucleolar accumulation of coilin, and this temporally coincides with the repression of RNA polymerase I (Pol I) activity. Knockdown of endogenous coilin partially overrides the Pol I transcriptional arrest caused by cisplatin, while both ectopically expressed and exogenous coilin accumulate in the nucleolus and suppress rRNA synthesis. In support of this mechanism, we demonstrate that both cisplatin and γ-irradiation induce the colocalization of coilin with RPA-194 (the largest subunit of Pol I), and we further show that coilin can specifically interact with RPA-194 and the key regulator of Pol I activity, upstream binding factor (UBF). Using chromatin immunoprecipitation analysis, we provide evidence that coilin modulates the association of Pol I with ribosomal DNA. Collectively, our data suggest that coilin acts to repress Pol I activity in response to cisplatin-induced DNA damage. Our findings identify a novel and unexpected function for coilin, independent of its role in snRNP biogenesis, establishing a new link between the DNA damage response and the inhibition of rRNA synthesis.

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Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011354 ◽

2019 ◽

Vol 295 (5) ◽

pp. 1288-1299 ◽

Cited By ~ 1

Author(s):

Catherine E. Scull ◽

Andrew M. Clarke ◽

Aaron L. Lucius ◽

David Alan Schneider

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Dna Sequence ◽

Transcription Elongation ◽

Gc Content ◽

Rna Polymerase I ◽

Pol I ◽

Polymerase I

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo. Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.

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Targeting the RNA Polymerase I Transcription for Cancer Therapy Comes of Age

Cells ◽

10.3390/cells9020266 ◽

2020 ◽

Vol 9 (2) ◽

pp. 266 ◽

Cited By ~ 10

Author(s):

Rita Ferreira ◽

John S. Schneekloth ◽

Konstantin I. Panov ◽

Katherine M. Hannan ◽

Ross D. Hannan

Keyword(s):

Rna Polymerase ◽

Cancer Progression ◽

Ribosome Biogenesis ◽

Therapeutic Potential ◽

Cancer Therapeutics ◽

Active Role ◽

Rna Polymerase I ◽

Cancer Etiology ◽

Pol I ◽

Polymerase I

Transcription of the ribosomal RNA genes (rDNA) that encode the three largest ribosomal RNAs (rRNA), is mediated by RNA Polymerase I (Pol I) and is a key regulatory step for ribosomal biogenesis. Although it has been reported over a century ago that the number and size of nucleoli, the site of ribosome biogenesis, are increased in cancer cells, the significance of this observation for cancer etiology was not understood. The realization that the increase in rRNA expression has an active role in cancer progression, not only through increased protein synthesis and thus proliferative capacity but also through control of cellular check points and chromatin structure, has opened up new therapeutic avenues for the treatment of cancer through direct targeting of Pol I transcription. In this review, we discuss the rational of targeting Pol I transcription for the treatment of cancer; review the current cancer therapeutics that target Pol I transcription and discuss the development of novel Pol I-specific inhibitors, their therapeutic potential, challenges and future prospects.

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Multiple Protein-Protein Interactions by RNA Polymerase I-Associated Factor PAF49 and Role of PAF49 in rRNA Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6338-6349.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6338-6349 ◽

Cited By ~ 30

Author(s):

Kazuo Yamamoto ◽

Mika Yamamoto ◽

Ken-ichi Hanada ◽

Yasuhisa Nogi ◽

Toshifumi Matsuyama ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase I ◽

Rrna Synthesis ◽

Associated Factor ◽

Isolation And Characterization ◽

Cell Extracts ◽

Rrna Transcription ◽

Pol I ◽

Polymerase I

ABSTRACT We previously demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. Here, we report the isolation and characterization of another Pol I-associated factor, PAF49. Mouse PAF49 shows striking homology to the human nucleolar protein ASE-1, so that they are considered orthologues. PAF49 and PAF53 were copurified with a subpopulation of Pol I during purification from cell extracts. Physical association of PAF49 with Pol I was confirmed by a coimmunoprecipitation assay. PAF49 was shown to interact with PAF53 through its N-terminal segment. This region of PAF49 also served as the target for TAFI48, the 48-kDa subunit of selectivity factor SL1. Concomitant with this interaction, the other components of SL1 also coimmunoprecipitated with PAF49. Specific transcription from the mouse rRNA promoter in vitro was severely impaired by anti-PAF49 antibody, which was overcome by addition of recombinant PAF49 protein. Moreover, overexpression of a deletion mutant of PAF49 significantly reduced pre-rRNA synthesis in vivo. Immunolocalization analysis revealed that PAF49 accumulated in the nucleolus of growing cells but dispersed to nucleoplasm in growth-arrested cells. These results strongly suggest that PAF49/ASE-1 plays an important role in rRNA transcription.

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