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The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs. [ABSTRACT FROM AUTHOR]

Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids.

The main components of the essential cellular process of eukaryotic ribosome biogenesis are highly conserved from yeast to humans. Among these, the U3 Associated Proteins (UTPs) are a small subunit processome subcomplex that coordinate the first two steps of ribosome biogenesis in transcription and pre-18S processing. While we have identified the human counterparts of most of the yeast Utps, the homologs of yeast Utp9 and Bud21 (Utp16) have remained elusive. In this study, we find that NOL7 is the likely ortholog of Bud21. Previously described as a tumour suppressor through regulation of antiangiogenic transcripts, we now show that NOL7 is required for early pre-rRNA accumulation and pre-18S rRNA processing in human cells. These roles lead to decreased protein synthesis and induction of the nucleolar stress response upon NOL7 depletion. Beyond Bud21’s nonessential role in yeast, we establish human NOL7 as an essential UTP that is necessary to maintain both early pre-rRNA levels and processing.

Abstract The nucleolus is a large nuclear body that serves as the primary site for ribosome biogenesis. Recent studies have suggested that it also plays an important role in organizing chromatin architecture. However, to establish a causal relationship between nucleolar ribosome assembly and chromatin architecture, genetic tools are required to disrupt nucleolar ribosome biogenesis. In this study, we used ATAC-seq to investigate changes in chromatin accessibility upon specific depletion of two ribosome biogenesis components, RPOA-2 and GRWD-1, in the model organism Caenorhabditis elegans. To facilitate the analysis of ATAC-seq data, we introduced two tools: SRAlign, an extensible NGS data processing workflow, and SRAtac, a customizable end-to-end ATAC-seq analysis pipeline. Our results revealed highly comparable changes in chromatin accessibility following both RPOA-2 and GRWD-1 perturbations. However, we observed a weak correlation between changes in chromatin accessibility and gene expression. While our findings corroborate the idea of a feedback mechanism between ribosomal RNA synthesis, nucleolar ribosome large subunit biogenesis, and chromatin structure during the L1 stage of C. elegans development, they also prompt questions regarding the functional impact of these alterations on gene expression.

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs.

Hyperactivation of ribosome biosynthesis (RiBi) is a hallmark of cancer, and targeting ribosome biogenesis has emerged as a potential therapeutic strategy. The depletion of TAF1B, a major component of selectivity factor 1 (SL1), disrupts the pre-initiation complex, preventing RNA polymerase I from binding ribosomal DNA and inhibiting the hyperactivation of RiBi. Here, we investigate the role of TAF1B, in regulating RiBi and proliferation in stomach adenocarcinoma (STAD). We disclosed that the overexpression of TAF1B correlates with poor prognosis in STAD, and found that knocking down TAF1B effectively inhibits STAD cell proliferation and survival in vitro and in vivo. TAF1B knockdown may also induce nucleolar stress, and promote c-MYC degradation in STAD cells. Furthermore, we demonstrate that TAF1B depletion impairs rRNA gene transcription and processing, leading to reduced ribosome biogenesis. Collectively, our findings suggest that TAF1B may serve as a potential therapeutic target for STAD and highlight the importance of RiBi in cancer progression.

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. [ABSTRACT FROM AUTHOR]

The nucleolus is a large nuclear body that serves as the primary site for ribosome biogenesis. Recent studies have suggested that it also plays an important role in organizing chromatin architecture. However, to establish a causal relationship between nucleolar ribosome assembly and chromatin architecture, genetic tools are required to disrupt nucleolar ribosome biogenesis. In this study, we used ATAC-seq to investigate changes in chromatin accessibility upon specific depletion of two ribosome biogenesis components, RPOA-2 and GRWD-1, in the model organism Caenorhabditis elegans. To facilitate the analysis of ATAC-seq data, we introduced two tools: SRAlign, an extensible NGS data processing workflow, and SRAtac, a customizable end-to-end ATAC-seq analysis pipeline. Our results revealed highly comparable changes in chromatin accessibility following both RPOA-2 and GRWD-1 perturbations. However, we observed a weak correlation between changes in chromatin accessibility and gene expression. While our findings corroborate the idea of a feedback mechanism between ribosomal RNA synthesis, nucleolar ribosome large subunit biogenesis, and chromatin structure during the L1 stage of C. elegans development, they also prompt questions regarding the functional impact of these alterations on gene expression. [ABSTRACT FROM AUTHOR]

Background: TAF1B (TATA Box Binding Protein (TBP)-Associated Factor) is an RNA polymerase regulating rDNA activity, stress response, and cell cycle. However, the function of TAF1B in the progression of hepatocellular carcinoma (HCC) is unknown. Objective: In this study, we intended to characterize the crucial role and molecular mechanisms of TAF1B in modulating nucleolar stress in HCC. Methods: We analyzed the differential expression and prognostic value of TAF1B in hepatocellular carcinoma based on The Cancer Genome Atlas (TCGA) database, tumor and paraneoplastic tissue samples from clinical hepatocellular carcinoma patients, and typical hepatocellular carcinoma. We detected cell proliferation and apoptosis by lentiviral knockdown of TAF1B expression levels in HepG2 and SMMC-7721 cells using clone formation, apoptosis, and Western blotting (WB) detection of apoptosis marker proteins. Simultaneously, we investigated the influence of TAF1B knockdown on the function of the preinitiation complex (PIC) by WB, and co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays verified the interaction between the complexes and the effect on rDNA activity. Immunofluorescence assays measured the expression of marker proteins of nucleolus stress, fluorescence in situ hybridization (FISH) assays checked the rDNA activity, and qRT-PCR assays tested the pre-rRNA levels. Regarding molecular mechanisms, we investigated the role of p53 and miR-101 in modulating nucleolar stress and apoptosis. Finally, the impact of TAF1B knockdown on tumor growth, apoptosis, and p53 expression was observed in xenograft tumors. Result: We identified that TAF1B was highly expressed in hepatocellular carcinoma and associated with poor prognosis in HCC patients. TAF1B depletion modulated nucleolar stress and apoptosis in hepatocellular carcinoma cells through positive and negative feedback from p53-miR-101. RNA polymerase I transcription repression triggered post-transcriptional activation of miR-101 in a p53-dependent manner. In turn, miR-101 negatively feeds back through direct inhibition of the p53-mediated PARP pathway. Conclusion: These findings broaden our comprehension of the function of TAF1B-mediated nucleolar stress in hepatocellular carcinoma and may offer new biomarkers for exploring prospective therapeutic targets in HCC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A -related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies. Smallwood et al. describe a series of individuals with heterozygous variants in POLR1A in association with variable craniofacial, heart, and brain anomalies. Adverse effects of POLR1A perturbation are demonstrated on mouse development in vivo , as well as on rRNA transcription in vitro. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The main components of the essential cellular process of eukaryotic ribosome biogenesis are highly conserved from yeast to humans. Among these, the U3 Associated Proteins (UTPs) are a small subunit processome subcomplex that coordinate the first two steps of ribosome biogenesis in transcription and pre-18S processing. While we have identified the human counterparts of most of the yeast Utps, the homologs of yeast Utp9 and Bud21 (Utp16) have remained elusive. In this study, we find that NOL7 is the likely ortholog of Bud21. Previously described as a tumour suppressor through regulation of antiangiogenic transcripts, we now show that NOL7 is required for early pre-rRNA accumulation and pre-18S rRNA processing in human cells. These roles lead to decreased protein synthesis and induction of the nucleolar stress response upon NOL7 depletion. Beyond Bud21's nonessential role in yeast, we establish human NOL7 as an essential UTP that is necessary to maintain both early pre-rRNA levels and processing. [ABSTRACT FROM AUTHOR]

Simple Summary: Building ribosomes for cellular protein translation is a massive, energy-consuming undertaking. The process is executed by all cells to replenish the relevant pools of proteins required for cellular functions. Cancer cells are strikingly dependent on this activity, as they need continuous protein synthesis for sustained proliferation and growth. Ribosome biogenesis requires the activities of three RNA polymerases, which transcribe essential RNAs that make up the backbone of the ribosome, and hundreds of proteins, which provide structural and functional support. Of the three RNA polymerases, RNA polymerase I executes a critical and rate-limiting step by transcribing three key ribosomal RNAs. Pol I transcription is pervasively deregulated in cancers, enabling unlimited protein synthesis. Here, we review this enzyme and provide examples of current efforts to target Pol I transcription therapeutically. RNA polymerase I is a highly processive enzyme with fast initiation and elongation rates. The structure of Pol I, with its in-built RNA cleavage ability and incorporation of subunits homologous to transcription factors, enables it to quickly and efficiently synthesize the enormous amount of rRNA required for ribosome biogenesis. Each step of Pol I transcription is carefully controlled. However, cancers have highjacked these control points to switch the enzyme, and its transcription, on permanently. While this provides an exceptional benefit to cancer cells, it also creates a potential cancer therapeutic vulnerability. We review the current research on the regulation of Pol I transcription, and we discuss chemical biology efforts to develop new targeted agents against this process. Lastly, we highlight challenges that have arisen from the introduction of agents with promiscuous mechanisms of action and provide examples of agents with specificity and selectivity against Pol I. [ABSTRACT FROM AUTHOR]

Purpose: Ribosome biogenesis is a key process in all living organisms, energetically expensive and tightly regulated. Currently, little is known about the components of the ribosomal RNA (rRNA) transcription machinery that are present in intestinal parasites, such as Giardia duodenalis, Cryptosporidium parvum, and Entamoeba histolytica. Thus, in the present work, an analysis was carried out looking for the components of the rRNA transcription machinery that are conserved in intestinal parasites and if these could be used to design new treatment strategies. Methods: The different components of the rRNA transcription machinery were searched in the studied parasites with the NCBI BLAST tool in the EuPathDB Bioinformatics Resource Center database. The sequences of the RRN3 and POLR1F orthologs were aligned and important regions identified. Subsequently, three-dimensional models were built with different bioinformatic tools and a structural analysis was performed. Results: Among the protozoa examined, C. parvum is the parasite with the fewest identifiable components of the rRNA transcription machinery. TBP, RRN3, POLR1A, POLR1B, POLR1C, POLR1D, POLR1F, POLR1H, POLR2E, POLR2F and POLR2H subunits were identified in all species studied. Furthermore, the interaction regions between RRN3 and POLR1F were found to be conserved and could be used to design drugs that inhibit rRNA transcription in the parasites studied. Conclusion: The inhibition of the rRNA transcription machinery in parasites might be a new therapeutic strategy against these microorganisms. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer cells upregulate RNA polymerase I (Pol I) activity to increase ribosome abundance in support of rapid cell growth and proliferation. During the last decade, Pol I has emerged as a promising anti-cancer target. Eukaryotes express three closely related RNA polymerases (Pols I, II, and III), responsible for synthesis of all the genome-encoded RNA required by the cell. Effective therapeutic development requires that the treatment be selective for Pol I, without inhibition of Pols II or III. This study evaluates the specificity of the compound BMH-21 on transcription by Pols I, II, and III using purified in vitro transcription assays. These results reveal that Pol I is uniquely sensitive to inhibition by BMH-21, compared to Pols II and III. These findings support ongoing preclinical development of BMH-21 and its derivatives for potential therapeutic applications. Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols' elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I. [ABSTRACT FROM AUTHOR]

Background: POLR1D is a subunit of RNA Polymerases I and III, which synthesize ribosomal RNAs. Dysregulation of these polymerases cause several types of diseases, including ribosomopathies. The craniofacial disorder Treacher Collins Syndrome (TCS) is a ribosomopathy caused by mutations in several subunits of RNA Polymerase I, including POLR1D. Here, we characterized the effect of a missense mutation in POLR1D and RNAi knockdown of POLR1D on Drosophila development. Results: We found that a missense mutation in Drosophila POLR1D (G30R) reduced larval rRNA levels, slowed larval growth, and arrested larval development. Remarkably, the G30R substitution is at an orthologous glycine in POLR1D that is mutated in a TCS patient (G52E). We showed that the G52E mutation in human POLR1D, and the comparable substitution (G30E) in Drosophila POLR1D, reduced their ability to heterodimerize with POLR1C in vitro. We also found that POLR1D is required early in the development of Drosophila neural cells. Furthermore, an RNAi screen revealed that POLR1D is also required for development of non‐neural Drosophila cells, suggesting the possibility of defects in other cell types. Conclusions: These results establish a role for POLR1D in Drosophila development, and present Drosophila as an attractive model to evaluate the molecular defects of TCS mutations in POLR1D. Key Findings: POLR1D is required for Drosophila larval growth and developmental progression.A conserved glycine in POLR1D is required for Drosophila and human development.Loss of POLR1D function in Drosophila neural cells causes developmental defects.An RNAi screen reveals a requirement for POLR1D function in the proper development of a variety of cell typesDrosophila is an attractive model to evaluate the genetic and molecular defects of TCS mutations in POLR1D. [ABSTRACT FROM AUTHOR]

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3′ end was fastest when the 3′ terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step. [Display omitted] • Pol I employs one universal kinetic mechanism independent of base identity. • UMP is incorporated substantially faster than GMP, CMP, and AMP. • Fast incorporation of UMP may be important for termination. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation. [Display omitted] • The ribosomal protein L22 binds to the MDM4 pre-mRNA and promotes exon 6 skipping • Consensus sequences in intron 6 are required for L22 binding to the MDM4 pre-mRNA • MDM4 splicing regulation by L22 causes p53 activation upon nucleolar stress • L22 also regulates the splicing of L22L1 and UBAP2L Jansen et al. show that the ribosomal protein L22 binds to the MDM4 pre-mRNA at intron 6 to promote exon 6 skipping. This leads to reduced levels of functional MDM4 when L22 relocalizes to the nucleoplasm upon nucleolar stress, thus representing a mechanism of p53 activation in this context. [ABSTRACT FROM AUTHOR]

Transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I) is the rate-limiting step in ribosome biogenesis and a major determinant of cellular growth rates. Unlike virtually every other eukaryote, which express identical rRNA from large tandem arrays of dozens to hundreds of identical rRNA genes in every cell, the genome of the human malaria parasite Plasmodium falciparum contains only a handful single-copy 47S rRNA loci that differ substantially from one another in length, sequence and expression in different cell-types. We found that growth of malaria parasite was acutely sensitive to the Pol I inhibitors 9-hydroxyellipticine and BMH-21 and demonstrate that they greatly reduce the transcription of 47S rRNAs as well as transcription of other non-coding RNA genes. Surprisingly, we found that the various types of Pol I-transcribed genes differed by more than two orders of magnitude in their susceptibility to these inhibitors and explore the implications of these findings for regulation of rRNA in P. falciparum ., Competing Interests: Conflicts of Interest: The authors declare no conflicts of interest.

Simple Summary: Cells need to produce ribosomes to sustain continuous proliferation and expand in numbers, a feature that is even more prominent in uncontrollably proliferating cancer cells. Certain cancer cell types are expected to depend more on ribosome biogenesis based on their genetic background, and this potential vulnerability can be exploited in designing effective, targeted cancer therapies. This review provides information on anti-cancer molecules that target the ribosome biogenesis machinery and indicates avenues for future research. Rapid growth and unrestrained proliferation is a hallmark of many cancers. To accomplish this, cancer cells re-wire and increase their biosynthetic and metabolic activities, including ribosome biogenesis (RiBi), a complex, highly energy-consuming process. Several chemotherapeutic agents used in the clinic impair this process by interfering with the transcription of ribosomal RNA (rRNA) in the nucleolus through the blockade of RNA polymerase I or by limiting the nucleotide building blocks of RNA, thereby ultimately preventing the synthesis of new ribosomes. Perturbations in RiBi activate nucleolar stress response pathways, including those controlled by p53. While compounds such as actinomycin D and oxaliplatin effectively disrupt RiBi, there is an ongoing effort to improve the specificity further and find new potent RiBi-targeting compounds with improved pharmacological characteristics. A few recently identified inhibitors have also become popular as research tools, facilitating our advances in understanding RiBi. Here we provide a comprehensive overview of the various compounds targeting RiBi, their mechanism of action, and potential use in cancer therapy. We discuss screening strategies, drug repurposing, and common problems with compound specificity and mechanisms of action. Finally, emerging paths to discovery and avenues for the development of potential biomarkers predictive of therapeutic outcomes across cancer subtypes are also presented. [ABSTRACT FROM AUTHOR]

Microalgae are used in food and feed, and they are considered a potential feedstock for sustainably produced chemicals and biofuel. However, production of microalgal-derived chemicals is not yet economically feasible. Genetic engineering could bridge the gap to industrial application and facilitate the production of novel products from microalgae. Here, we report the discovery of a novel gene expression system in the oleaginous microalga Nannochloropsis that exploits the highly efficient transcriptional activity of RNA polymerase I and an internal ribosome entry site for translation. We identified the nucleolus as a genomic safe harbor for Pol I transcription and used it to construct transformant strains with consistently strong transgene expression. The new expression system provides an outstanding tool for genetic and metabolic engineering of microalgae and thus will probably make substantial contributions to microalgal research. This work describes the development of a novel system for genetic engineering of a eukaryotic microalga. It was shown that eukaryotic RNA polymerase I can be exploited for expression of protein-coding genes by employing an internal ribosome entry site to allow translation. Importantly, this study identified the nucleolus as a genomic safe harbor for consistent and strong gene expression. [ABSTRACT FROM AUTHOR]

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest., Competing Interests: Declaration of interests A.C. is founder, equity holder, and consultant, and M.J.A. is Chief Scientific Officer at DarwinHealth Inc., a company that has licensed some of the algorithms used in this manuscript from Columbia University. Columbia University is also an equity holder in DarwinHealth Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle, but the molecular underpinnings of their involvement, particularly in mammalian cells, remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control.

Evolutionarily conserved kinases and phosphatases regulate RNA polymerase II (RNAPII) transcript synthesis by modifying the phosphorylation status of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII. Proper levels of Rpb1-CTD phosphorylation are required for RNA co-transcriptional processing and to coordinate transcription with other nuclear processes, such as chromatin remodeling and histone modification. Whether other RNAPII subunits are phosphorylated and influences their role in gene expression is still an unanswered question. Much less is known about RNAPI and RNAPIII phosphorylation, whose subunits do not contain functional CTDs. However, diverse studies have reported that several RNAPI and RNAPIII subunits are susceptible to phosphorylation. Some of these phosphorylation sites are distributed within subunits common to all three RNAPs whereas others are only shared between RNAPI and RNAPIII. This suggests that the activities of all RNAPs might be finely modulated by phosphorylation events and raises the idea of a tight coordination between the three RNAPs. Supporting this view, the transcription by all RNAPs is regulated by signaling pathways that sense different environmental cues to adapt a global RNA transcriptional response. This review focuses on how the phosphorylation of RNAPs might regulate their function and we comment on the regulation by phosphorylation of some key transcription factors in the case of RNAPI and RNAPIII. Finally, we discuss the existence of possible common mechanisms that could coordinate their activities.

RNA polymerase I (RNAPI) and RNAPIII are multi-heterogenic protein complexes that specialize in the transcription of highly abundant non-coding RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA). In terms of subunit number and structure, RNAPI and RNAPIII are more complex than RNAPII that synthesizes thousands of different mRNAs. Specific subunits of the yeast RNAPI and RNAPIII form associated subcomplexes that are related to parts of the RNAPII initiation factors. Prior to their delivery to the nucleus where they function, RNAP complexes are assembled at least partially in the cytoplasm. Yeast RNAPI and RNAPIII share heterodimer Rpc40-Rpc19, a functional equivalent to the αα homodimer which initiates assembly of prokaryotic RNAP. In the process of yeast RNAPI and RNAPIII biogenesis, Rpc40 and Rpc19 form the assembly platform together with two small, bona fide eukaryotic subunits, Rpb10 and Rpb12. We propose that this assembly platform is co-translationally seeded while the Rpb10 subunit is synthesized by cytoplasmic ribosome machinery. The translation of Rpb10 is stimulated by Rbs1 protein, which binds to the 3′-untranslated region of RPB10 mRNA and hypothetically brings together Rpc19 and Rpc40 subunits to form the αα-like heterodimer. We suggest that such a co-translational mechanism is involved in the assembly of RNAPI and RNAPIII complexes.

RNA polymerase I (Pol I) transcription takes place at the border of the fibrillar center (FC) and the dense fibrillar component (DFC) in the nucleolus. Here, we report that individual spherical FC/DFC units are coated by the DEAD-box RNA helicase DDX21 in human cells. The long noncoding RNA (lncRNA) SLERT binds to DDX21 RecA domains to promote DDX21 to adopt a closed conformation at a substoichiometric ratio through a molecular chaperoneÐlike mechanism resulting in the formation of hypomultimerized and loose DDX21 clusters that coat DFCs, which is required for proper FC/DFC liquidity and Pol I processivity. Our results suggest that SLERT is an RNA regulator that controls the biophysical properties of FC/DFCs and thus ribosomal RNA production. [ABSTRACT FROM AUTHOR]

RNA polymerase I (Pol I) is responsible for the synthesis of the majority of ribosomal RNA molecules in eukaryotes. Pol I subunit 12 (RPA12) is involved in the transcriptional termination and lipid metabolism in yeast. However, its role in human cells hasn't been investigated so far. Here, we show that RPA12 is present in the nucleolus and nucleoplasm of HeLa cells. RPA12 can act as a positive factor to regulate Pol I-mediated transcription and the proliferation of 293T and HeLa cells. Unexpectedly, RPA12 can repress HeLa cell migration, indicating that RPA12 plays opposite roles in cell proliferation and migration. This study provides a novel insight into the role of RPA12 in human cells. • RPA12 is localized to both nucleolus and nucleoplasm. • RPA12 acts as a positive factor to regulate Pol I transcription. • RPA12 promotes cell proliferation. • RPA12 represses cell migration. [ABSTRACT FROM AUTHOR]

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence. [Display omitted] • Pol I demonstrates intrinsic sensitivity to the sequence of the DNA template. • Rate constants governing nucleotide addition by Pol I are heterogeneous in vitro compared to Pols II and III. • Altering the length of the RNA primer does not alter the heterogeneity of nucleotide addition by Pol I. [ABSTRACT FROM AUTHOR]

Most RNA polymerases contain zinc, yet the precise function of zinc and its influence of polymerases stability are unknown. A recent study provides evidence that zinc levels control the stability of RNA polymerase I in vivo and that the enzyme might serve as a zinc reservoir for other proteins.

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., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PIs) has revolutionized the treatment of MM. However, resistance to PIs is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461, has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here, we show that CX-5461 has potent anti-myeloma activity in PI-resistant MM preclinical models in vitro and in vivo . In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell-cycle arrest and apoptotic cell death. Combining CX-5461 with PI does not further enhance the anti-myeloma activity of CX-5461 in vivo . In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk∗MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM., Competing Interests: R.D.H. is a Chief Scientific Advisor to Pimera, Inc., (© 2024 The Author(s).)

Key signaling pathways (such as phosphoinositide 3-kinase, Myc, and RAS) act as sensors of energy, stress, and nutrient availability and integrate these inputs to directly control ribosome production and gene expression at the translational level. This activity is normally directly coupled to cell growth, division, and survival. However, it remains poorly understood the extent to which changes in ribosome number and nucleolar integrity downstream of these key signaling pathways contribute to their oncogenic activity. Emerging studies provide interesting insight into how deregulations in RNA polymerase I activity may lead to tumorigenesis and suggest that new drugs targeting ribosomal DNA transcription may hold great promise for the treatment of cancer.

Hyperactivation of RNA polymerase I (Pol I) transcription of ribosomal RNA (rRNA) genes (rDNA) is a key determinant of growth and proliferation and a consistent feature of cancer cells. We have demonstrated that inhibition of rDNA transcription by the Pol I transcription inhibitor CX-5461 selectively kills tumor cells in vivo. Moreover, the first-in human trial of CX-5461 has demonstrated CX-5461 is well-tolerated in patients and has single-agent anti-tumor activity in hematologic malignancies. However, the mechanisms underlying tumor cell sensitivity to CX-5461 remain unclear. Understanding these mechanisms is crucial for the development of predictive biomarkers of response that can be utilized for stratifying patients who may benefit from CX-5461. The rDNA repeats exist in four different and dynamic chromatin states: inactive rDNA can be either methylated silent or unmethylated pseudo-silent; while active rDNA repeats are described as either transcriptionally competent but non-transcribed or actively transcribed, depending on the level of rDNA promoter methylation, loading of the essential rDNA chromatin remodeler UBF and histone marks status. In addition, the number of rDNA repeats per human cell can reach hundreds of copies. Here, we tested the hypothesis that the number and/or chromatin status of the rDNA repeats, is a critical determinant of tumor cell sensitivity to Pol I therapy. We systematically examined a panel of ovarian cancer (OVCA) cell lines to identify rDNA chromatin associated biomarkers that might predict sensitivity to CX-5461. We demonstrated that an increased proportion of active to inactive rDNA repeats, independent of rDNA copy number, determines OVCA cell line sensitivity to CX-5461. Further, using zinc finger nuclease genome editing we identified that reducing rDNA copy number leads to an increase in the proportion of active rDNA repeats and confers sensitivity to CX-5461 but also induces genome-wide instability and sensitivity to DNA damage. We propose that the proportion of active to inactive rDNA repeats may serve as a biomarker to identify cancer patients who will benefit from CX-5461 therapy in future clinical trials. The data also reinforces the notion that rDNA instability is a threat to genomic integrity and cellular homeostasis.

Comparative transcriptional analyses require appropriate and precise normalization. Here we describe a modified transcription run-on (TRO) method, named quantitative TRO (qTRO), that allows quantification of nascent transcription activity. The most critical improvement it introduces is a new standardization method for RNA isolation and hybridization steps, enabling transcription activity quantification and comparative biological analysis. We used this technique with chromatin immunoprecipitation to investigate RNA polymerase I (RNAPI) transcription activity and its rDNA gene profiles in Saccharomyces cerevisiae. We designed a set of new oligonucleotide probes complementary to nascent ribosomal RNA (rRNA) transcripts and standardized their hybridization strength. The qTRO method could be successfully implemented to study RNAPI transcription in response to oxidative stress and in two mutant strains with impaired rRNA synthesis.

Ribosomes are universally important in biology and their production is dysregulated by developmental disorders, cancer, and virus infection. Although presumed required for protein synthesis, how ribosome biogenesis impacts virus reproduction and cell-intrinsic immune responses remains untested. Surprisingly, we find that restricting ribosome biogenesis stimulated human cytomegalovirus (HCMV) replication without suppressing translation. Interfering with ribosomal RNA (rRNA) accumulation triggered nucleolar stress and repressed expression of 1392 genes, including High Mobility Group Box 2 (HMGB2), a chromatin-associated protein that facilitates cytoplasmic double-stranded (ds) DNA-sensing by cGAS. Furthermore, it reduced cytoplasmic HMGB2 abundance and impaired induction of interferon beta (IFNB1) mRNA, which encodes a critical anti-proliferative, proinflammatory cytokine, in response to HCMV or dsDNA in uninfected cells. This establishes that rRNA accumulation regulates innate immune responses to dsDNA by controlling HMGB2 abundance. Moreover, it reveals that rRNA accumulation and/or nucleolar activity unexpectedly regulate dsDNA-sensing to restrict virus reproduction and regulate inflammation. (145 words)

Upstream Activation Factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA Polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the rDNA promoter and interacts with two other Pol I initiation factors, the TATA-Binding Protein (TBP) and Core Factor (CF). We used an integrated combination of chemical crosslinking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provides roles for previously identified UAF domains that include the Rrn5 SANT and Histone Fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal Winged Helix domain and a disordered Tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual functioning UAF complex. [ABSTRACT FROM AUTHOR]

RNA polymerase transcribes certain genomic loci with higher errors rates. These transcription error-enriched genomic loci (TEELs) have implications in disease. Current deep-sequencing methods cannot distinguish TEELs from post-transcriptional modifications, stochastic transcription errors, and technical noise, impeding efforts to elucidate the mechanisms linking TEELs to disease. Here, we describe background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) to discern genomic regions enriched for transcription misincorporations. EmPC-seq innovatively combines a nuclear run-on assay for capturing nascent RNA before post-transcriptional modifications, a circular-sequencing step that sequences the same nascent RNA molecules multiple times to improve accuracy, and a statistical model for distinguishing error-enriched regions among stochastic polymerase errors. Applying EmPC-seq to the ribosomal RNA transcriptome, we show that TEELs of RNA polymerase I are not randomly distributed but clustered together, with higher error frequencies at nascent transcript 3′ ends. Our study establishes a reliable method of identifying TEELs with nucleotide precision, which can help elucidate their molecular origins. Unlabelled Image • EmPC-seq combines experimental, bioinformatics, and statistical methods to identify a high confidence set of transcriptional mutation-prone positions within genes, eliminating typically confounding factors arising from coverage biases, sequencing noise, alignment artifacts, and background mutations, • EmPC-seq affirmatively shows that transcriptional mutation-prone regions cluster together. • EmPC-seq allows single transcript analyses to investigate of the molecular origin of transcriptional errors, and we show that transcriptional mutations are enriched at the active site. • EmPC-seq can be widely applicable for the study of transcription mutations in various model systems including virus, bacteria, and mammalian cells. [ABSTRACT FROM AUTHOR]