Human RNA Polymerase Research Articles

Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters

The transcriptional interplay of human RNA polymerase I (RNA Pol I), RNA Pol II, and RNA Pol III remains largely uncharacterized due to limited integrative genomic analyses for all three enzymes. To address this gap, we applied a uniform framework to quantify global RNA Pol I, RNA Pol II, and RNA Pol III occupancies and identify both canonical and noncanonical patterns of gene localization. Most notably, our survey captures unexpected RNA Pol III recruitment at promoters of specific protein-coding genes. We show that such RNA Pol III-occupied promoters are enriched for small nascent RNAs terminating in a run of 4 Ts-a hallmark of RNA Pol III termination indicative of constrained RNA Pol III transcription. Taken further, RNA Pol III disruption generally reduces the expression of RNA Pol III-occupied protein-coding genes, suggesting RNA Pol III recruitment and transcription enhance RNA Pol II activity. These findings resemble analogous patterns of RNA Pol II activity at RNA Pol III-transcribed genes, altogether uncovering a reciprocal form of crosstalk between RNA Pol II and RNA Pol III.

Structural basis for substrate binding and selection by human mitochondrial RNA polymerase

The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.

Identification of 6-Fluorine-Substituted Coumarin Analogues as POLRMT Inhibitors with High Potency and Safety for Treatment of Pancreatic Cancer.

Increasing evidence has demonstrated that oxidative phosphorylation (OXPHOS) is closely associated with the progression of pancreatic cancer (PC). Given its central role in mitochondrial transcription, the human mitochondrial RNA polymerase (POLRMT) is a promising target for developing PC treatments. Herein, structure-activity relationship exploration led to the identification of compound S7, which was the first reported POLRMT inhibitor possessing single-digit nanomolar potency of inhibiting PC cells proliferation. Mechanistic studies showed that compound S7 exerted antiproliferative effects without affecting the cell cycle, apoptosis, mitochondrial membrane potential (MMP), or intracellular reactive oxygen species (ROS) levels specifically in MIA PaCa-2 cells. Notably, compound S7 inhibited tumor growth in MIA PaCa-2 xenograft tumor model with a tumor growth inhibition (TGI) rate of 64.52% demonstrating significant improvement compared to the positive control (44.80%). In conclusion, this work enriched SARs of POLRMT inhibitors, and compound S7 deserved further investigations of drug-likeness as a candidate for PC treatment.

Human RNA Polymerase II Segregates from Genes and Nascent RNA and Transcribes in the Presence of DNA-Bound dCas9.

RNA polymerase II (Pol II) dysfunction is frequently implied in human disease. Understanding its functional mechanism is essential for designing innovative therapeutic strategies. To visualize its supra-molecular interactions with genes and nascent RNA, we generated a human cell line carrying ~335 consecutive copies of a recombinant β-globin gene. Confocal microscopy showed that Pol II was not homogeneously concentrated around these identical gene copies. Moreover, Pol II signals partially overlapped with the genes and their nascent RNA, revealing extensive compartmentalization. Using a cell line carrying a single copy of the β-globin gene, we also tested if the binding of catalytically dead CRISPR-associated system 9 (dCas9) to different gene regions affected Pol II transcriptional activity. We assessed Pol II localization and nascent RNA levels using chromatin immunoprecipitation and droplet digital reverse transcription PCR, respectively. Some enrichment of transcriptionally paused Pol II accumulated in the promoter region was detected in a strand-specific way of gRNA binding, and there was no decrease in nascent RNA levels. Pol II preserved its transcriptional activity in the presence of DNA-bound dCas9. Our findings contribute further insight into the complex mechanism of mRNA transcription in human cells.

Mechanism and spectrum of inhibition of a 4′-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5′-triphosphate (TP) metabolite of a 4ʹ-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4ʹ-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1′-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1ʹ-cyano and 4ʹ-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters.

RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occupancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings reshape our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.

The ancient Z-DNA and Z-RNA specific Zα fold has evolved modern roles in immunity and transcription through the natural selection of flipons.

The Zα fold specifically binds to both Z-DNA and Z-RNA, left-handed nucleic acid structures that form under physiological conditions and are encoded by flipons. I trace the Zα fold back to unicellular organisms representing all three domains of life and to the realm of giant nucleocytoplasmic DNA viruses (NCVs). The canonical Zα fold is present in the earliest known holozoan unicellular symbiont Capsaspora owczarzaki and persists in vertebrates and some invertebrates, but not in plants or fungi. In metazoans, starting with porifera, Zα is incorporated into the double-stranded RNA editing enzyme ADAR and reflects an early symbiont relationship with NCV. In vertebrates, Zα is also present in ZBP1 and PKZ proteins that recognize host-derived Z-RNAs to defend against modern-day viruses. A related Zα fold, also likely to bind Z-DNA, is present in proteins thought to modulate gene expression, including a subset of prokaryote arsR proteins and the p15 (PC4) family present in algae. Other Zα variants that probably play a more general role in the reinitiation of transcription include the archaeal and human transcription factor E and the human RNA polymerase 3 subunit C proteins. The roles in immunity and transcription underlie the natural selection of flipons.

Persistence of backtracking by human RNA polymerase II

RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome-wide distribution of more persistent backtracking are unknown. Consequently, we developed a method to directly sequence the extruded, "backtracked" 3' RNA. Our data show that RNA Pol II slides backward more than 20 nt in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where RNA Pol II pauses near promoters and intron-exon junctions and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking can potentially affect diverse gene expression programs.

HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.

Correction Notice: Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M

Correction Notice: Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1–3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.

Exploration of natural compounds against the human mpox virus DNA-dependent RNA polymerase in silico

BackgroundLast year, the human monkeypox virus (hMPXV) emerged as an alarming threat to the community, with a detectable outbreak outside the African continent for the first time. According to The American Centers for Disease Control and Prevention (CDC), the virus is reported globally, with 86,746 confirmed cases (until April 08, 2023). DNA-dependent RNA polymerase (DdRp) is an essential protein for viral replication; hence it is a promising drug target for developing antiviral drugs against DNA viruses. Therefore, this study was conducted to search for natural compounds that could provide scaffolds for RNA polymerase inhibitors. MethodsIn this study, the DdRp structure of hMPXV was modeled and used to screen the natural compounds database (COCONUT). The virtual screening revealed 15 compounds able to tightly bind to the active site of the DdRp (binding energies less than −7.0 kcal/mol) compared to the physiological nucleotide, guanosine triphosphate (GTP). Molecular dynamics simulation was then performed on the top four hits and compared to GTP ResultsThe results revealed the potential of four compounds (comp289, comp295, comp441, and comp449) in binding the hMPXV DdRp active site with a comparable binding affinity (−17.06 ± 2.96, −11.6 ± 5.34, −14.85 ± 2.66, and −10.79 ± 4.49 kcal/mol) with GTP (−21.03 ± 7.55 kcal/mol) ConclusionThese findings may also pave the way for developing new hMPXV inhibitors based on natural product scaffolds. These results need further experimental validation but promising as it was validated by unbiased all-atom MD simulations and binding free energy calculations.

Discovery of a Novel, Potent, Orally Active, and Safe Inhibitor Targeting Human Mitochondrial RNA Polymerase.

High oxidative phosphorylation (OXPHOS) happens in some tumors, which depends on OXPHOS for energy supply, particularly in slow-cycling tumor cells. Therefore, targeting human mitochondrial RNA polymerase (POLRMT) to inhibit mitochondrial gene expression emerges as a potential therapeutic strategy to eradicate tumor cells. In this work, exploration and optimization of the first-in-class POLRMT inhibitor IMT1B and its SAR led to the identification of a novel compound D26, which exerted a strong antiproliferative effect on several cancer cells and decreased mitochondrial-related genes expression. In addition, mechanism studies demonstrated that D26 arrested cell cycle at the G1 phase and had no effect on apoptosis, depolarized mitochondria, or reactive oxidative stress generation in A2780 cells. Importantly, D26 exhibited more potent anticancer activity than the lead IMT1B in A2780 xenograft nude mice and had no observable toxic effect. All results suggest that D26 deserves to be further investigated as a potent and safe antitumor candidate.

Modeling and simulating human mitochondrial RNA polymerase nucleotide addition cycle using computational microscope.

Human mitochondrial RNA polymerase (POLRMT) is essential to the transcription of mitochondrial DNA and cell metabolism. This work contributes to an all-atom structural dynamics model for individual steps of the nucleotide addition cycle of POLRMT. We aim to establish a computational model using all-atom molecular dynamics to reveal Watson-Crick base pair regulation of natural nucleotide and nucleotide analog binding and insertion in POLRMT, and a model for the post-catalytic mechanochemical coupling between product release and POLRMT translocation. The models are currently constructed combining the available high-resolution structures of POLRMT in the open and closed conformations with the bound NTP and Mg2+ ions detected in structurally similar T7 bacteriophage RNA polymerase, and by additionally using curve interpolation to place a missing non-template DNA segment. The models have been evaluated for protein subdomain stability and the presence of the appropriate protein conformation, open or closed, via subdomain motion regulation on the active site. Ongoing work will establish a model for the post-translocation mechanochemical regulations that reset for a new nucleotide addition cycle. Since POLRMT is structurally similar to viral RNA polymerases, the studies are particularly important for antiviral drug design, where nucleotide analog drug candidates are expected to be well screened for POLRMT toxicity.

Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M.

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

The POLR3G Subunit of Human RNA Polymerase III Regulates Tumorigenesis and Metastasis in Triple-Negative Breast Cancer

RNA polymerase (Pol) III transcribes short untranslated RNAs that contribute to the regulation of gene expression. Two isoforms of human Pol III have been described that differ by the presence of the POLR3G/RPC32α or POLR3GL/RPC32β subunits. POLR3G was found to be expressed in embryonic stem cells and at least a subset of transformed cells, whereas POLR3GL shows a ubiquitous expression pattern. Here, we demonstrate that POLR3G is specifically overexpressed in clinical samples of triple-negative breast cancer (TNBC) but not in other molecular subtypes of breast cancer. POLR3G KO in the MDA-MB231 TNBC cell line dramatically reduces anchorage-independent growth and invasive capabilities in vitro. In addition, the POLR3G KO impairs tumor growth and metastasis formation of orthotopic xenografts in mice. Moreover, KO of POLR3G induces expression of the pioneer transcription factor FOXA1 and androgen receptor. In contrast, the POLR3G KO neither alters proliferation nor the expression of epithelial-mesenchymal transition marker genes. These data demonstrate that POLR3G expression is required for TNBC tumor growth, invasiveness and dissemination and that its deletion affects triple-negative breast cancer-specific gene expression.

DNA Damage-Induced RNAPII Degradation and Its Consequences in Gene Expression.

RPB1, the major and catalytic subunit of human RNA Polymerase II (RNAPII), is specifically degraded by the ubiquitin-proteasome system upon induction of DNA damage by different agents, such as ultraviolet (UV) light. The "last resort" model of RNAPII degradation states that a persistently stalled RNAPII is degraded at the site of the DNA lesion in order to facilitate access to Nucleotide Excision Repair (NER) factors, thereby promoting repair in template strands of active genes. Recent identification and mutation of the lysine residue involved in RPB1 ubiquitylation and degradation unveiled the relevance of RNAPII levels in the control of gene expression. Inhibition of RNAPII degradation after UV light exposure enhanced RNAPII loading onto chromatin, demonstrating that the mere concentration of RNAPII shapes the gene expression response. In this review, we discuss the role of RNAPII ubiquitylation in NER-dependent repair, recent advances in RPB1 degradation mechanisms and its consequences in gene expression under stress, both in normal and repair deficient cells.

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Differential dependencies of human RNA polymerase II promoters on TBP, TAF1, TFIIBand XPB.

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

2,6-Diaminopurine (Z) is a naturally occurring adenine (A) analog that bacteriophages employ in place of A in their genetic alphabet. Recent discoveries of biogenesis pathways of Z in bacteriophages have stimulated substantial research interest in this DNA modification. Here, we systematically examined the effects of Z on the efficiency and fidelity of DNA transcription. Our results showed that Z exhibited no mutagenic yet substantial inhibitory effects on transcription mediated by purified T7 RNA polymerase and by human RNA polymerase II in HeLa nuclear extracts and in human cells. A structurally related adenine analog, 2-aminopurine (2AP), strongly blocked T7 RNA polymerase but did not impede human RNA polymerase II in vitro or in human cells, where no mutant transcript could be detected. The lack of mutagenic consequence and the presence of a strong blockage effect of Z on transcription suggest a role of Z in transcriptional regulation. Z is also subjected to removal by transcription-coupled nucleotide-excision repair (TC-NER), but not global-genome NER in human cells. Our findings provide new insight into the effects of Z on transcription and its potential biological functions.