Regulation of RNA Polymerase I Transcription in Development, Disease, and Aging.

Ribosome biogenesis is a complex and highly energy-demanding process that requires the concerted action of all three nuclear RNA polymerases (Pol I-III) in eukaryotes. The three largest ribosomal RNAs (rRNAs) originate from a precursor transcript (pre-rRNA) that is encoded by multicopy genes located in the nucleolus. Transcription of these rRNA genes (rDNA) by Pol I is the key regulation step in ribosome production and is tightly controlled by an intricate network of signaling pathways and epigenetic mechanisms. In this article, we give an overview of the composition of the basal Pol I machinery and rDNA chromatin. We discuss rRNA gene regulation in response to environmental signals and developmental cues and focus on perturbations occurring in diseases linked to either excessive or limited rRNA levels. Finally, we discuss the emerging view that rDNA integrity and activity may be involved in the aging process.

Structural Basis of RNA Polymerase I Transcription Initiation.

Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

SLERT Regulates DDX21 Rings Associated with Pol I Transcription.

Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.

Control of ribosomal RNA synthesis by hematopoietic transcription factors.

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.

Beyond rRNA: nucleolar transcription generates a complex network of RNAs with multiple roles in maintaining cellular homeostasis.

Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes ∼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.

Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates.

Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.

A high-resolution protein architecture of the budding yeast genome.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.

RNA Polymerase I Activators Count and Adjust Ribosomal RNA Gene Copy Number.

Ribosome is the most abundant RNA-protein complex in a cell, and many copies of the ribosomal RNA gene (rDNA) have to be maintained. However, arrays of tandemly repeated rDNA genes can lose the copies by intra-repeat recombination. Loss of the rDNA copies of Saccharomyces cerevisiae is counteracted by gene amplification whereby the number of rDNA repeats stabilizes around 150 copies, suggesting the presence of a monitoring mechanism that counts and adjusts the number. Here, we report that, in response to rDNA copy loss, the upstream activating factor (UAF) for RNA polymerase I that transcribes the rDNA is released and directly binds to a RNA polymerase II-transcribed gene, SIR2, whose gene products silence rDNA recombination, to repress. We show that the amount of UAF determines the rDNA copy number that is stably maintained. UAF ensures rDNA production not only by rDNA transcription activation but also by its copy-number maintenance.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Establishment and maintenance of alternative chromatin states at a multicopy gene locus.

In eukaryotes, each of the more than 100 copies of ribosomal RNA (rRNA) genes exists in either an RNA polymerase I transcribed open chromatin state or a nucleosomal, closed chromatin state. Open rRNA genes guarantee the cell's supply with structural components of the ribosome, whereas closed rRNA genes ensure genomic integrity. We report that the observed balance between open and closed rRNA gene chromatin states in proliferating yeast cells is due to a dynamic equilibrium of transcription-dependent removal and replication-dependent assembly of nucleosomes. Pol I transcription is required for the association of the HMG box protein Hmo1 with open rRNA genes, counteracting replication-independent nucleosome deposition and maintaining the open rRNA gene chromatin state outside of S phase. The findings indicate that the opposing effects of replication and transcription lead to a de novo establishment of chromatin states for rRNA genes during each cell cycle.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

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.

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.

A nucleolar long "non-coding" RNA encodes a novel protein that functions in response to stress.

Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.

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.

Transcription elongation mechanisms of RNA polymerases I, II, and III and their therapeutic implications.

Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.

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.

Mendelian randomization analyses implicate biogenesis of translation machinery in human aging.

Reduced provision of protein translation machinery promotes healthy aging in a number of animal models. In humans, however, inborn impairments in translation machinery are a known cause of several developmental disorders, collectively termed ribosomopathies. Here, we use casual inference approaches in genetic epidemiology to investigate whether adult, tissue-specific biogenesis of translation machinery drives human aging. We assess naturally occurring variation in the expression of genes encoding subunits specific to the two RNA polymerases (Pols) that transcribe ribosomal and transfer RNAs, namely Pol I and III, and the variation in expression of ribosomal protein (RP) genes, using Mendelian randomization. We find each causally associated with human longevity (ß = -0.15 ± 0.047, P = 9.6 × 10-4, q = 0.015; ß = -0.13 ± 0.040, P = 1.4 × 10-3, q = 0.023; ß = -0.048 ± 0.016, P = 3.5 × 10-3, q = 0.056, respectively), and this does not appear to be mediated by altered susceptibility to a single disease. We find that reduced expression of Pol III, RPs, or Pol I promotes longevity from different organs, namely visceral adipose, liver, and skeletal muscle, echoing the tissue specificity of ribosomopathies. Our study shows the utility of leveraging genetic variation in expression to elucidate how essential cellular processes impact human aging. The findings extend the evolutionary conservation of protein synthesis as a critical process that drives animal aging to include humans.

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.

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.