LINC01116-dependent upregulation of RNA polymerase I transcription drives oncogenic phenotypes in lung adenocarcinoma.

BACKGROUND: Hyperactive RNA Polymerase I (Pol I) transcription is canonical in cancer, associated with malignant proliferation, poor prognosis, epithelial-mesenchymal transition, and chemotherapy resistance. Despite its significance, the molecular mechanisms underlying Pol I hyperactivity remain unclear. This study aims to elucidate the role of long noncoding RNAs (lncRNAs) in regulating Pol I transcription in lung adenocarcinoma (LUAD). METHODS: Bioinformatics analyses were applied to identify lncRNAs interacting with Pol I transcriptional machinery. Fluorescence in situ hybridization was employed to examine the nucleolar localization of candidate lncRNA in LUAD cells. RNA immunoprecipitation assay validated the interaction between candidate lncRNA and Pol I components. Chromatin isolation by RNA purification and Chromatin Immunoprecipitation (ChIP) were utilized to confirm the interactions of candidate lncRNA with Pol I transcriptional machinery and the rDNA core promoter. Functional analyses, including lncRNA knock-in and knockdown, inhibition of Pol I transcription, quantitative PCR, cell proliferation, clonogenicity, apoptosis, cell cycle, wound-healing, and invasion assays, were performed to determine the effect of candidate lncRNA on Pol I transcription and associated malignant phenotypes in LUAD cells. ChIP assays and luminometry were used to investigate the transcriptional regulation of the candidate lncRNA. RESULTS: We demonstrate that oncogenic LINC01116 scaffolds essential Pol I transcription factors TAF1A and TAF1D, to the ribosomal DNA promoter, and upregulate Pol I transcription. Crucially, LINC01116-driven Pol I transcription activation is essential for its oncogenic activities. Inhibition of Pol I transcription abrogated LINC01116-induced oncogenic phenotypes, including increased proliferation, cell cycle progression, clonogenicity, reduced apoptosis, increased migration and invasion, and drug sensitivity. Conversely, LINC01116 knockdown reversed these effects. Additionally, we show that LINC01116 upregulation in LUAD is driven by the oncogene c-Myc, a known Pol I transcription activator, indicating a functional regulatory feedback loop within the c-Myc-LINC01116-Pol I transcription axis. CONCLUSION: Collectively, our findings reveal, for the first time, that LINC01116 enhances Pol I transcription by scaffolding essential transcription factors to the ribosomal DNA promoter, thereby driving oncogenic activities in LUAD. We propose the c-Myc-LINC01116-Pol I axis as a critical oncogenic pathway and a potential therapeutic target for modulating Pol I transcription in LUAD.

Topological stress triggers persistent DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment.

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.

POLR1D silencing suppresses lung cancer cells proliferation and migration via inhibition of PI3K-Akt pathway.

AIM: The most common type of cancer that leads to death worldwide is lung cancer. Despite significant surgery and chemotherapy improvements, lung cancer patient's survival rate is still poor. The RNA polymerase I subunit D (POLR1D) gene can induce various cancers. A current study reported that POLR1D plays a vital role in cancer prognosis. However, its biological function in the development of lung cancer remains unclear. METHODS: Reverse transcription PCR (RT-PCR) measured the relative POLR1D protein expression level in lung cancer cell lines. Lung cancer cell proliferation, migration, and invasion were analyzed by performing cell counting kit-8 (CCK-8), and transwell. The phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/AKT) signaling pathway-related protein expressions were examined by Western blotting assay. RESULTS: POLR1D protein expression was elevated in lung cancer. Lung cancer cell loss-of-function tests showed that POLR1D silencing could attenuate cell viability both in SK-MES-1 and in H2170 cells. Furthermore, silencing POLR1D inhibited SK-MES-1 and H2170 cells proliferation, migration, and invasion. Moreover, SK-MES-1 and H2170 cells' migration and invasion capacity were potentially suppressed by the knockdown of POLR1D. The progression of multiple cancers has been implicated in the PI3K/AKT pathway. Here, we observed that POLR1D silencing suppressed lung cancer progression by inhibition of the PI3K-Akt pathway. CONCLUSIONS: The study speculated that POLR1D might provide a new potential therapeutic possibility for treating lung cancer patients via targeting PI3K/AKT.

Targeting EMT using low-dose Teniposide by downregulating ZEB2-driven activation of RNA polymerase I in breast cancer.

Metastatic dissemination from the primary tumor is a complex process that requires crosstalk between tumor cells and the surrounding milieu and involves the interplay between numerous cellular-signaling programs. Epithelial-mesenchymal transition (EMT) remains at the forefront of orchestrating a shift in numerous cellular programs, such as stemness, drug resistance, and apoptosis that allow for successful metastasis. Till date, there is limited success in therapeutically targeting EMT. Utilizing a high throughput screen of FDA-approved compounds, we uncovered a novel role of the topoisomerase inhibitor, Teniposide, in reversing EMT. Here, we demonstrate Teniposide as a potent modulator of the EMT program, specifically through an IRF7-NMI mediated response. Furthermore, Teniposide significantly reduces the expression of the key EMT transcriptional regulator, Zinc Finger E-Box Binding Homeobox 2 (ZEB2). ZEB2 downregulation by Teniposide inhibited RNA polymerase I (Pol I) activity and rRNA biogenesis. Importantly, Teniposide treatment markedly reduced pulmonary colonization of breast cancer cells. We have uncovered a novel role of Teniposide, which when used at a very low concentration, mitigates mesenchymal-like invasive phenotype. Overall, its ability to target EMT and rRNA biogenesis makes Teniposide a viable candidate to be repurposed as a therapeutic option to restrict breast cancer metastases.

Transcription factor regulation of ribosomal RNA in hematopoiesis.

PURPOSE OF REVIEW: Ribosomal RNAs (rRNAs) are transcribed within nucleoli from rDNA repeats by RNA Polymerase I (Pol I). There is variation in rRNA transcription rates across the hematopoietic tree, and leukemic blast cells have prominent nucleoli, indicating abundant ribosome biogenesis. The mechanisms underlying these variations are poorly understood. The purpose of this review is to summarize findings of rDNA binding and Pol I regulation by hematopoietic transcription factors. RECENT FINDINGS: Our group recently used custom genome assemblies optimized for human and mouse rDNA mapping to map nearly 2200 ChIP-Seq datasets for nearly 250 factors to rDNA, allowing us to identify conserved occupancy patterns for multiple transcription factors. We confirmed known rDNA occupancy of MYC and RUNX factors, and identified new binding sites for CEBP factors, IRF factors, and SPI1 at canonical motif sequences. We also showed that CEBPA degradation rapidly leads to reduced Pol I occupancy and nascent rRNA in mouse myeloid cells. SUMMARY: We propose that a number of hematopoietic transcription factors bind rDNA and potentially regulate rRNA transcription. Our model has implications for normal and malignant hematopoiesis. This review summarizes the literature, and outlines experimental considerations to bear in mind while dissecting transcription factor roles on rDNA.

p53 inhibitor or antioxidants reduce the severity of ethmoid plate deformities in zebrafish Type 3 Treacher Collins syndrome model.

Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.

Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.

VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma.

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV-) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles' heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

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.

Distinct states of nucleolar stress induced by anticancer drugs.

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed 'nucleolar normality score' was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I- recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

Ribosomes are cell structures within a compartment called the nucleolus that are required to make proteins, which are essential for cell function. Due to their uncontrolled growth and division, cancer cells require many proteins and therefore have a particularly high demand for ribosomes. Due to this, some anti-cancer drugs deliberately target the activities of the nucleolus. However, it was not clear if anti-cancer drugs with other targets also disrupt the nucleolus, which may result in side effects. Previously, it had been difficult to study how nucleoli work, partly because in human cells they vary naturally in shape, size, and number. Potapova et al. used fluorescent microscopy to develop a new way of assessing nucleoli based on the location and ratio of certain proteins. These measurements were used to calculate a "nucleolar normality score". Potapova et al. then tested over a thousand anti-cancer drugs in healthy and cancerous human cells. Around 10% of the tested drugs changed the nucleolar normality score when compared to placebo treatment, indicating that they caused nucleolar stress. For most of these drugs, the nucleolus was not the intended target, suggesting that disrupting it was an unintended side effect. Drugs inhibiting proteins called cyclin-dependent kinases caused the most drastic changes in the size and shape of nucleoli, disrupting them completely. These kinases are known to be involved in activating enzymes required for general transcription. Potapova et al. showed that they also are involved in production of ribosomal RNA, revealing an additional role in coordinating ribosome assembly. Taken together, the findings suggest that evaluating the effect of new anti-cancer drugs on the nucleolus could help to develop future treatments with less toxic side effects. The experiments also reveal new avenues for researching how cyclin-dependent kinases control the production of RNA more generally.

Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops.

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS-R-loop-H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis.

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.

PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53.

The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth.

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.

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.

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.

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.

Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.

Identification of an E3 ligase that targets the catalytic subunit of RNA Polymerase I upon transcription stress.

RNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp-Cullin-F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.