Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Reverse Genetics of Hepatitis C Virus Using an RNA Polymerase I-Mediated Transcription.

The reverse genetics system commonly used for the production of hepatitis C virus (HCV), which is a major causative agent of liver diseases, involves introduction of the viral genomic RNA synthesized in vitro into human hepatoma cells by electroporation. As an alternative methodology, we describe a cell culture system based on transfection with an expression plasmid containing a full-length HCV cDNA clone flanked by RNA polymerase I promoter and terminator sequences to generate infectious virus particles from transfected cells.

Features of yeast RNA polymerase I with special consideration of the lobe binding subunits.

Ribosomal RNAs (rRNAs) are structural components of ribosomes and represent the most abundant cellular RNA fraction. In the yeast Saccharomyces cerevisiae, they account for more than 60 % of the RNA content in a growing cell. The major amount of rRNA is synthesized by RNA polymerase I (Pol I). This enzyme transcribes exclusively the rRNA gene which is tandemly repeated in about 150 copies on chromosome XII. The high number of transcribed rRNA genes, the efficient recruitment of the transcription machinery and the dense packaging of elongating Pol I molecules on the gene ensure that enough rRNA is generated. Specific features of Pol I and of associated factors confer promoter selectivity and both elongation and termination competence. Many excellent reviews exist about the state of research about function and regulation of Pol I and how Pol I initiation complexes are assembled. In this report we focus on the Pol I specific lobe binding subunits which support efficient, error-free, and correctly terminated rRNA synthesis.

Pan-cancer integrated bioinformatic analysis of RNA polymerase subunits reveal RNA Pol I member CD3EAP regulates cell growth by modulating autophagy.

Transcription is a crucial stage in gene expression. An integrated study of 34 RNA polymerase subunits (RNAPS) in the six most frequent cancer types identified several genetic and epigenetic modification. We discovered nine mutant RNAPS with a mutation frequency of more than 1% in at least one tumor type. POLR2K and POLR2H were found to be amplified and overexpressed, whereas POLR3D was deleted and downregulated. Multiple RNAPS were also observed to be regulated by variations in promoter methylation. 5-Aza-2-deoxycytidine mediated re-expression in cell lines verified methylation-driven inhibition of POLR2F and POLR2L expression in BRCA and NSCLC, respectively. Next, we showed that CD3EAP, a Pol I subunit, was overexpressed in all cancer types and was associated with worst survival in breast, liver, lung, and prostate cancers. The knockdown studies showed that CD3EAP is required for cell proliferation and induces autophagy but not apoptosis. Furthermore, autophagy inhibition rescued the cell proliferation in CD3EAP knockdown cells. CD3EAP expression correlated with S and G2 phase cell cycle regulators, and CD3EAP knockdown inhibited the expression of S and G2 CDK/cyclins. We also identified POLR2D, an RNA pol II subunit, as a commonly overexpressed and prognostic gene in multiple cancers. POLR2D knockdown also decreased cell proliferation. POLR2D is related to the transcription of just a subset of RNA POL II transcribe genes, indicating a distinct role. Taken together, we have shown the genetic and epigenetic regulation of RNAPS genes in most common tumors. We have also demonstrated the cancer-specific function of CD3EAP and POLR2D genes.

rRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

Repression of rRNA gene transcription by endothelial SPEN deficiency normalizes tumor vasculature via nucleolar stress.

Human cancers induce a chaotic, dysfunctional vasculature that promotes tumor growth and blunts most current therapies; however, the mechanisms underlying the induction of a dysfunctional vasculature have been unclear. Here, we show that split end (SPEN), a transcription repressor, coordinates rRNA synthesis in endothelial cells (ECs) and is required for physiological and tumor angiogenesis. SPEN deficiency attenuated EC proliferation and blunted retinal angiogenesis, which was attributed to p53 activation. Furthermore, SPEN knockdown activated p53 by upregulating noncoding promoter RNA (pRNA), which represses rRNA transcription and triggers p53-mediated nucleolar stress. In human cancer biopsies, a low endothelial SPEN level correlated with extended overall survival. In mice, endothelial SPEN deficiency compromised rRNA expression and repressed tumor growth and metastasis by normalizing tumor vessels, and this was abrogated by p53 haploinsufficiency. rRNA gene transcription is driven by RNA polymerase I (RNPI). We found that CX-5461, an RNPI inhibitor, recapitulated the effect of Spen ablation on tumor vessel normalization and combining CX-5461 with cisplatin substantially improved the efficacy of treating tumors in mice. Together, these results demonstrate that SPEN is required for angiogenesis by repressing pRNA to enable rRNA gene transcription and ribosomal biogenesis and that RNPI represents a target for tumor vessel normalization therapy of cancer.

Synthesis of the ribosomal RNA precursor in human cells: mechanisms, factors and regulation.

The ribosomal RNA precursor (pre-rRNA) comprises three of the four ribosomal RNAs and is synthesized by RNA polymerase (Pol) I. Here, we describe the mechanisms of Pol I transcription in human cells with a focus on recent insights gained from structure-function analyses. The comparison of Pol I-specific structural and functional features with those of other Pols and with the excessively studied yeast system distinguishes organism-specific from general traits. We explain the organization of the genomic rDNA loci in human cells, describe the Pol I transcription cycle regarding structural changes in the enzyme and the roles of human Pol I subunits, and depict human rDNA transcription factors and their function on a mechanistic level. We disentangle information gained by direct investigation from what had apparently been deduced from studies of the yeast enzymes. Finally, we provide information about how Pol I mutations may contribute to developmental diseases, and why Pol I is a target for new cancer treatment strategies, since increased rRNA synthesis was correlated with rapidly expanding cell populations.

Distinctive Participation of Transcription-Coupled and Global Genome Nucleotide Excision Repair of Pyrimidine Dimers in the Transcribed Strand of Yeast rRNA Genes.

UV light causes the formation of pyrimidine dimers (PDs). Transcription-coupled (TC) nucleotide excision repair (NER) and global genome (GG) NER remove PDs from the transcribed strand (TS) of active genes and the inactive genome, respectively. TC-NER is triggered by elongating RNA polymerases that are blocked at PDs. The yeast rRNA genes are densely loaded with RNA polymerase-I. After UV irradiation, their density increases at the 5'-end of the gene, which results from continuous transcription initiation, followed by elongation and pausing/release at the first encountered PD, from the transcription start site. RNA polymerase-I posed at downstream PDs are released from the TS and are replaced by nucleosomes. Consequently, discrete chromatin structures are formed in the damaged transcribed rRNA genes. Singular assignation of the two NER sub-pathways could therefore be required to eliminate PDs from the TS. To advance our understanding of NER in the dynamic structure of transcribed chromatin, we investigated the repair of PDs at nucleotide resolution in separate rRNA gene coding regions. In the TS, the TC-NER efficiency reflected the density of RNA polymerase-I, and PDs were removed faster in the 5'-end than in the 3'-end of the gene. GG-NER removed PDs from the TS where RNA polymerase-I was transiently replaced by a nucleosome. The two NER sub-pathways inversely participated to remove PDs from the TS. In the non-TS of both nucleosome and non-nucleosome rRNA gene coding regions, GG-NER was solely responsible to remove UV-induced DNA lesions.

RNA polymerase I subunit RPA43 activates rRNA expression and cell proliferation but inhibits cell migration.

The products synthesized by RNA polymerase I (Pol I) play fundamental roles in several cellular processes, including ribosomal biogenesis, protein synthesis, cell metabolism, and growth. Deregulation of Pol I products can cause various diseases such as ribosomopathies, leukaemia, and solid tumours. However, the detailed mechanism of Pol I-directed transcription remains elusive, and the roles of Pol I subunits in rRNA synthesis and cellular activities still need clarification. In this study, we found that RPA43 expression levels positively correlate with Pol I product accumulation and cell proliferation, indicating that RPA43 activates these processes. Unexpectedly, RPA43 depletion promoted HeLa cell migration, suggesting that RPA43 functions as a negative regulator in cell migration. Mechanistically, RPA43 positively modulates the recruitment of Pol I transcription machinery factors to the rDNA promoter by activating the transcription of the genes encoding Pol I transcription machinery factors. RPA43 inhibits cell migration by dampening the expression of c-JUN and Integrin. Collectively, we found that RPA43 plays opposite roles in cell proliferation and migration except for driving Pol I-dependent transcription. These findings provide novel insights into the regulatory mechanism of Pol I-mediated transcription and cell proliferation and a potential pathway to developing anti-cancer drugs using RPA43 as a target.

The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.

Expression of RNA polymerase I catalytic core is influenced by RPA12.

RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135. Furthermore, we find that despite these alterations not only does the Pol I core complex between RPA194 and RPA135 remain intact upon RPA12 knockdown, but the transcription of Pol I and its engagement with chromatin remain unaffected. The BMH-21-mediated degradation of RPA194 was independent of RPA12 suggesting that RPA12 affects the basal expression, but not the drug-inducible turnover of RPA194. These studies add to knowledge defining regulatory factors for the expression of this Pol I catalytic subunit.

Distinct Interaction Modes for the Eukaryotic RNA Polymerase Alpha-like Subunits.

Eukaryotic DNA-dependent RNA polymerases (Pols I-III) encode two distinct alpha-like heterodimers where one is shared between Pols I and III, and the other is unique to Pol II. Human alpha-like subunit mutations are associated with several diseases including Treacher Collins Syndrome (TCS), 4H leukodystrophy, and primary ovarian sufficiency. Yeast is commonly used to model human disease mutations, yet it remains unclear whether the alpha-like subunit interactions are functionally similar between yeast and human homologs. To examine this, we mutated several regions of the yeast and human small alpha-like subunits and used biochemical and genetic assays to establish the regions and residues required for heterodimerization with their corresponding large alpha-like subunits. Here we show that different regions of the small alpha-like subunits serve differential roles in heterodimerization, in a polymerase- and species-specific manner. We found that the small human alpha-like subunits are more sensitive to mutations, including a "humanized" yeast that we used to characterize the molecular consequence of the TCS-causingPOLR1D G52E mutation. These findings help explain why some alpha subunit associated disease mutations have little to no effect when made in their yeast orthologs and offer a better yeast model to assess the molecular basis of POLR1D associated disease mutations.

The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression.

Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.

The RNA export factor TbMex67 connects transcription and RNA export in Trypanosoma brucei and sets boundaries for RNA polymerase I.

TbMex67 is the major mRNA export factor known to date in trypanosomes, forming part of the docking platform within the nuclear pore. To explore its role in co-transcriptional mRNA export, recently reported in Trypanosoma brucei, pulse labelling of nascent RNAs with 5-ethynyl uridine (5-EU) was performed with cells depleted of TbMex67 and complemented with a dominant-negative mutant (TbMex67-DN). RNA polymerase (Pol) II transcription was unaffected, but the procyclin loci, which encode mRNAs transcribed by Pol I from internal sites on chromosomes 6 and 10, showed increased levels of 5-EU incorporation. This was due to Pol I readthrough transcription, which proceeded beyond the procyclin and procyclin-associated genes up to the Pol II transcription start site on the opposite strand. Complementation by TbMex67-DN also increased Pol I-dependent formation of R-loops and γ-histone 2A foci. The DN mutant exhibited reduced nuclear localisation and binding to chromatin compared to wild-type TbMex67. Together with its interaction with chromatin remodelling factor TbRRM1 and Pol II, and transcription-dependent association of Pol II with nucleoporins, our findings support a role for TbMex67 in connecting transcription and export in T. brucei. In addition, TbMex67 stalls readthrough by Pol I in specific contexts, thereby limiting R-loop formation and replication stress.

A homozygous POLR1A variant causes leukodystrophy and affects protein homeostasis.

RNA polymerase I transcribes ribosomal DNA to produce precursor 47S rRNA. Post-transcriptional processing of this rRNA generates mature 28S, 18S and 5.8S rRNAs, which form the ribosomes, together with 5S rRNA, assembly factors and ribosomal proteins. We previously reported a homozygous variant in the catalytic subunit of RNA polymerase I, POLR1A, in two brothers with leukodystrophy and progressive course. However, the disease mechanism remained unknown. In this report, we describe another missense variant POLR1A NM_015425.3:c.1925C>A; p.(Thr642Asn) in homozygosity in two unrelated patients. Patient 1 was a 16-year-old male and Patient 2 was a 2-year-old female. Both patients manifested neurological deficits, with brain MRIs showing hypomyelinating leukodystrophy and cerebellar atrophy; and in Patient 1 additionally with hypointensity of globi pallidi and small volume of the basal ganglia. Patient 1 had progressive disease course, leading to death at the age of 16.5 years. Extensive in vitro experiments in fibroblasts from Patient 1 documented that the mutated POLR1A led to aberrant rRNA processing and degradation, and abnormal nucleolar homeostasis. Proteomics data analyses and further in vitro experiments documented abnormal protein homeostasis, and endoplasmic reticulum stress responses. We confirm that POLR1A biallelic variants cause neurodegenerative disease, expand the knowledge of the clinical phenotype of the disorder, and provide evidence for possible pathological mechanisms leading to POLR1A-related leukodystrophy.

Targeting mutant dicer tumorigenesis in pleuropulmonary blastoma via inhibition of RNA polymerase I.

DICER1 mutations predispose to increased risk for various cancers, particularly pleuropulmonary blastoma (PPB), the commonest lung malignancy of childhood. There is a paucity of directly actionable molecular targets as these tumors are driven by loss-of-function mutations of DICER1. Therapeutic development for PPB is further limited by a lack of biologically and physiologically-representative disease models. Given recent evidence of Dicer's role as a haploinsufficient tumor suppressor regulating RNA polymerase I (Pol I), Pol I inhibition could abrogate mutant Dicer-mediated accumulation of stalled polymerases to trigger apoptosis. Hence, we developed a novel subpleural orthotopic PPB patient-derived xenograft (PDX) model that retained both RNase IIIa and IIIb hotspot mutations and recapitulated the cardiorespiratory physiology of intra-thoracic disease, and with it evaluated the tolerability and efficacy of first-in-class Pol I inhibitor CX-5461. In PDX tumors, CX-5461 significantly reduced H3K9 di-methylation and increased nuclear p53 expression, within 24 hours' exposure. Following treatment at the maximum tolerated dosing regimen (12 doses, 30 mg/kg), tumors were smaller and less hemorrhagic than controls, with significantly decreased cellular proliferation, and increased apoptosis. As demonstrated in a novel intrathoracic tumor model of PPB, Pol I inhibition with CX-5461 could be a tolerable and clinically-feasible therapeutic strategy for mutant Dicer tumors, inducing antitumor effects by decreasing H3K9 methylation and enhancing p53-mediated apoptosis.

RNA Polymerases I and III in development and disease.

Ribosomes are macromolecular machines that are globally required for the translation of all proteins in all cells. Ribosome biogenesis, which is essential for cell growth, proliferation and survival, commences with transcription of a variety of RNAs by RNA Polymerases I and III. RNA Polymerase I (Pol I) transcribes ribosomal RNA (rRNA), while RNA Polymerase III (Pol III) transcribes 5S ribosomal RNA and transfer RNAs (tRNA) in addition to a wide variety of small non-coding RNAs. Interestingly, despite their global importance, disruptions in Pol I and Pol III function result in tissue-specific developmental disorders, with craniofacial anomalies and leukodystrophy/neurodegenerative disease being among the most prevalent. Furthermore, pathogenic variants in genes encoding subunits shared between Pol I and Pol III give rise to distinct syndromes depending on whether Pol I or Pol III function is disrupted. In this review, we discuss the global roles of Pol I and III transcription, the consequences of disruptions in Pol I and III transcription, disorders arising from pathogenic variants in Pol I and Pol III subunits, and mechanisms underpinning their tissue-specific phenotypes.

Real-time imaging of RNA polymerase I activity in living human cells.

RNA polymerase I (Pol I) synthesizes about 60% of cellular RNA by transcribing multiple copies of the ribosomal RNA gene (rDNA). The transcriptional activity of Pol I controls the level of ribosome biogenesis and cell growth. However, there is currently a lack of methods for monitoring Pol I activity in real time. Here, we develop LiveArt (live imaging-based analysis of rDNA transcription) to visualize and quantify the spatiotemporal dynamics of endogenous ribosomal RNA (rRNA) synthesis. LiveArt reveals mitotic silencing and reactivation of rDNA transcription, as well as the transcriptional kinetics of interphase rDNA. Using LiveArt, we identify SRFBP1 as a potential regulator of rRNA synthesis. We show that rDNA transcription occurs in bursts and can be altered by modulating burst duration and amplitude. Importantly, LiveArt is highly effective in the screening application for anticancer drugs targeting Pol I transcription. These approaches pave the way for a deeper understanding of the mechanisms underlying nucleolar functions.

MiR-330-5p and miR-1270 target essential components of RNA polymerase I transcription and exhibit a novel tumor suppressor role in lung adenocarcinoma.

Upregulation of RNA polymerase I (Pol I) transcription and the overexpression of Pol I transcriptional machinery are crucial molecular alterations favoring malignant transformation. However, the causal molecular mechanism(s) of this aberration remain largely unknown. Here, we found that Pol I transcription and its core machinery are upregulated in lung adenocarcinoma (LUAD). We show that the loss of miRNAs (miR)-330-5p and miR-1270 expression contributes to the upregulation of Pol I transcription in LUAD. Constitutive overexpression of these miRs in LUAD cell lines suppressed the expression of core components of Pol I transcription, and reduced global ribosomal RNA synthesis. Importantly, miR-330-5p/miR-1270-mediated repression of Pol I transcription exerted multiple tumor suppressive functions including reduced proliferation, cell cycle arrest, enhanced apoptosis, reduced migration, increased drug sensitivity, and reduced tumor burden in a mouse xenograft model. Mechanistically, the downregulation of miR-330-5p and miR-1270 is regulated by Pol I subunit-derived circular RNA circ_0055467 and DNA hypermethylation, respectively. This study uncovers a novel miR-330-5p/miR-1270 mediated post-transcriptional regulation of Pol I transcription, and establish tumor suppressor properties of these miRs in LUAD. Ultimately, our findings provide a rationale for the therapeutic targeting of Pol I transcriptional machinery for LUAD.

TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth.

Eukaryotic RNA polymerase I (Pol I) products play fundamental roles in ribosomal assembly, protein synthesis, metabolism and cell growth. Abnormal expression of both Pol I transcription-related factors and Pol I products causes a range of diseases, including ribosomopathies and cancers. However, the factors and mechanisms governing Pol I-dependent transcription remain to be elucidated. Here, we report that transcription factor IIB-related factor 1 (BRF1), a subunit of transcription factor IIIB required for RNA polymerase III (Pol III)-mediated transcription, is a nucleolar protein and modulates Pol I-mediated transcription. We showed that BRF1 can be localized to the nucleolus in several human cell types. BRF1 expression correlates positively with Pol I product levels and tumour cell growth in vitro and in vivo. Pol III transcription inhibition assays confirmed that BRF1 modulates Pol I-directed transcription in an independent manner rather than through a Pol III product-to-45S pre-rRNA feedback mode. Mechanistically, BRF1 binds to the Pol I transcription machinery components and can be recruited to the rDNA promoter along with them. Additionally, alteration of BRF1 expression affects the recruitment of Pol I transcription machinery components to the rDNA promoter and the expression of TBP and TAF1A. These findings indicate that BRF1 modulates Pol I-directed transcription by controlling the expression of selective factor 1 subunits. In summary, we identified a novel role of BRF1 in Pol I-directed transcription, suggesting that BRF1 can independently regulate both Pol I- and Pol III-mediated transcription and act as a key coordinator of Pol I and Pol III.