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PubMed2024

Multiple Forms and Functions of Premature Termination by RNA Polymerase II.

Eukaryotic genomes are widely transcribed by RNA polymerase II (pol II) both within genes and in intergenic regions. POL II elongation complexes comprising the polymerase, the DNA template and nascent RNA transcript must be extremely processive in order to transcribe the longest genes which are over 1 megabase long and take many hours to traverse. Dedicated termination mechanisms are required to disrupt these highly stable complexes. Transcription termination occurs not only at the 3' ends of genes once a full length transcript has been made, but also within genes and in promiscuously transcribed intergenic regions. Termination at these latter positions is termed "premature" because it is not triggered in response to a specific signal that marks the 3' end of a gene, like a polyA site. One purpose of premature termination is to remove polymerases from intergenic regions where they are "not wanted" because they may interfere with transcription of overlapping genes or the progress of replication forks. Premature termination has recently been appreciated to occur at surprisingly high rates within genes where it is speculated to serve regulatory or quality control functions. In this review I summarize current understanding of the different mechanisms of premature termination and its potential functions.

RNA Polymerase II↗
PubMed2026

Transcription elongation by RNA polymerase II: from regulatory complexity to disease.

Transcription elongation by RNA polymerase II (Pol II) was originally considered as the monotonic addition of ribonucleoside triphosphates to the growing nascent RNA chain. However, multiple lines of evidence now indicate that transcription elongation is a regulatory hub in gene expression that requires an increasing number of elongation factors (EFs), dysregulation of which leads to pathologies. In this review, we provide a current view of the elongation phase of Pol II, focusing on mammalian cells. We describe the increasing complexity of the mechanisms that control transcription elongation. We examine the growing set of EFs, their functional roles, and their systemic implications in human disease. Finally, we discuss the emergence of EFs as promising therapeutic targets.

RNA polymerase II↗
PubMed2024

SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II.

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II.

RNA Polymerase II↗
PubMed2024

Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation.

DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.

Humans↗
PubMed2026

Structural Characterization of Native RNA Polymerase II Transcription Complexes and Nucleosomes in Drosophila melanogaster.

Structural studies of eukaryotic RNA polymerase II (Pol II) transcription often rely on in vitro assembly, which may not fully represent native conditions. To investigate Pol II transcription in metazoan cells, we developed a method to isolate native transcription complexes from Drosophila melanogaster embryos using FLAG-tag affinity purification and Micrococcal Nuclease treatment. Cryo-EM and proteomics studies revealed diverse transcription complexes and nucleosomes, including a metazoan Rpb4/Rpb7 stalk-less Pol II elongation complex and a hexameric nucleosome lacking an H2A/H2B dimer. Notably, nucleosome is found only downstream of the nucleosome elongation complex, underscoring it as a major energy barrier and a time-consuming step during Pol II progression through chromatin. Proteomics identified co-purified factors involved in transcription initiation, elongation, and RNA modification. This study provides a framework for investigations of transcription in cells, paving the way for future studies of transient and minor complexes.

Animals↗
PubMed2026

Structural Features of DNA in TATA-Containing and TATA-Less Core Promoters of RNA Polymerase II Differ.

Nucleotide motifs in the core promoters of eukaryotic protein-coding genes transcribed by RNA polymerase II (Pol II) play an important role in the transcription process. We analyzed the role of an octanucleotide located in the TATA box position. Depending on whether this octanucleotide can form a complex with the TATA-binding protein (TBP), the promoter is classified as either TATA-containing or TATA-less. We analyzed the differences in the primary and spatial structures, as well as their dynamics, in TATA-containing and TATA-less promoters of mammals and plants. We divided the complete promoter sets of six organisms (H. sapiens, M. musculus, C. familiaris, A. thaliana, Z. mays, and H. vulgare) from the EPDnew database into TATA-containing and TATA-less fractions. The sizes of the TATA-containing promoter fractions are significantly smaller than those of the TATA-less fractions in all studied organisms, except in A. thaliana, where the sizes of both fractions are approximately equal. We characterized promoter architecture using variation profiles of various base-pair step parameters, minor-groove width, and the conformational dynamics of native DNA. The architectures of TATA-containing and TATA-less promoters differ significantly. The possible mechanistic influence of DNA structural features on the formation of the pre-initiation complex (PIC) in both types of promoters is discussed.

Promoter Regions, Genetic↗
PubMed2026

Targeting super elongation complex-driven RNA polymerase II elongation reduces plaque vulnerability.

Atherosclerotic plaque rupture is a major cause of myocardial infarction and stroke, yet the mechanisms governing plaque stability remain incompletely understood. Endothelial activation can trigger endothelial-to-mesenchymal transition, a program linked to endothelial dysfunction and lesion vulnerability. Here we investigated whether transcriptional pause release and RNA polymerase II elongation constitute an early regulatory layer that promotes endothelial-to-mesenchymal transition and atherosclerosis. Analysis of human plaque single-cell transcriptomics indicated increased expression of super elongation complex components in endothelial cells with a transition signature. In primary human endothelial cell models, pharmacological inhibition of the super elongation complex attenuated the induction of mesenchymal markers. AFF4, pCDK9, and pSMAD2/3 showed physical interaction during endothelial transition. Genome-wide profiling of RNA polymerase II occupancy revealed reduced promoter-proximal pausing during early transition, accompanied by a rapid increase in nascent transcriptional elongation rates. Super elongation complex inhibition restored pausing and suppressed fast-responding transition-associated target genes. In a human cardiac organoid model, inhibition of the super elongation complex prevented EndMT-induced fibrillar collagen deposition and prevented the loss of beating rate. In a hyperlipidemic Pcsk9 gain-of-function mouse model, super elongation complex inhibition administered both prophylactically and therapeutically after established atherosclerosis reduced plaque burden and reduced features of plaque vulnerability. Finally, analysis of 1048 human plaque segments from the Athero-Express biobank showed significant associations between the elongation axis and multiple vulnerability-related plaque traits. Together, these findings identify rapid transcriptional elongation as a mechanistic driver of endothelial plasticity and features of plaque vulnerability and support targeting the elongation machinery as a potential strategy to reduce features of plaque vulnerability in atherosclerotic disease.

Humans↗
PubMed2026

hnRNPK condensates facilitate enhancer-promoter looping and RNA polymerase II recruitment.

Enhancer RNAs interact with promoter-derived RNAs to dictate enhancer-promoter looping, but the RNA-binding protein that mediates this process has remained unidentified. Here we identify hnRNPK as a general structural regulator that preferentially binds to nascent RNAs transcribed from enhancer and promoter regions, promoting enhancer-promoter looping and transcriptional activation. We further show that hnRNPK forms phase-separated, cavity-containing condensates that encapsulate RNA polymerase II (Pol II) via its RPB3 subunit, facilitating chromatin looping and potentially enabling recruitment of Pol II from enhancers to promoters through protein dimerization. Notably, a mutation associated with Au-Kline syndrome in hnRNPK (c.953+1dupG) alters its condensates from a liquid-like to a gel-like state, leading to developmental defects in knock-in mice. Fibroblasts derived from these mutants display reduced enhancer-promoter looping and decreased Pol II recruitment at promoters of key developmental genes. These findings suggest that hnRNPK is a structural regulator of enhancer-promoter communication and highlight the importance of RNA-RNA interactions mediated by RNA-binding proteins in transcriptional regulation.

RNA Polymerase II↗
PubMed2025

A single cluster of RNA Polymerase II molecules is stably associated with active genes.

In eukaryotic nuclei, transcription is associated with the clustering of RNA Polymerase II (RNAPII) molecules. The mechanisms underlying cluster formation, their interactions with genes, and their impact on transcriptional activity remain heavily debated. Here we take advantage of the naturally occurring increase in transcriptional activity during Zygotic Genome Activation (ZGA) in Drosophila melanogaster embryos to characterize the functional roles of RNAPII clusters in a developmental context. Using single-molecule tracking and lattice light-sheet microscopy, we find that RNAPII cluster formation depends on transcription initiation, and that cluster lifetimes depend on transcriptional activity when not constrained by interphase duration. We show that single clusters are stably associated with active gene loci during transcription and that cluster intensities are strongly correlated with transcriptional output. Collectively our data and simulations on cluster formation kinetics show that RNAPII clusters reflect local accumulations of transcriptionally engaged polymerases and do not form through higher-order mechanisms such as phase separation.

Journal Article↗
PubMed2026

The CTNNB1-TRIM28 complex governs hormone-induced RNA polymerase II dynamics in kidney epithelial cells.

Arginine vasopressin maintains water homeostasis by regulating epithelial water permeability through complex transcriptional mechanisms in kidney collecting duct cells. Although CTNNB1 (β-catenin) functions as a transcriptional coregulator in vasopressin-responsive gene transcription, its role remains poorly understood. To identify CTNNB1-dependent components mediating the vasopressin-responsive transcription, we profiled transcriptomic changes following Ctnnb1 knockdown in mouse kidney collecting duct cells using RNA sequencing (RNA-Seq). RNA-Seq and promoter enrichment analyses identified bromodomain-containing proteins (TRIM28, TRIM33, BRD4, CREBBP, and EP300) as components of a CTNNB1-dependent complex regulating RNA Polymerase II (Pol II) activity. Biochemical analyses revealed physical interactions between TRIM28, CTNNB1, Pol II, and CDK9. Functionally, Trim28 knockdown blunted vasopressin-induced expression of the Aqp2 gene. Quantitative genomic binding assays demonstrated that TRIM28 is required for robust genomic occupancy and stabilization of Pol II at the transcription start site of Aqp2. Additionally, dynamic formation of phase-separated nuclear TRIM28 condensates in response to vasopressin suggests that TRIM28-associated machinery functions at specialized chromatin hubs. These findings reveal that TRIM28 facilitates Pol II recruitment, pause release, and elongation upon vasopressin stimulation. Our study establishes the CTNNB1-TRIM28 machinery as a critical transcriptional scaffold that controls Pol II dynamics and chromatin structure, thereby driving osmotic water reabsorption and urine concentration.

Animals↗
PubMed2025

Integrator promotes the association of TFIID and RNA polymerase II to maintain pluripotency during development.

The mechanisms by which the expression of pluripotency and Polycomb networks are harmonized to allow the transition from pluripotency to a differentiated state have not been fully elucidated. Integrator complex regulates transcription pause release and RNA processing in metazoans. We show that Integrator is required for stemness and plays a critical role as early as day 2 in embryonic development. While the catalytic endonuclease activity enhances cellular reprogramming, Integrator recruits RNA polymerase II (RNAPII) to promoters and super enhancers of pluripotency and Polycomb genes. Integrator coordinates expression of pluripotency and Polycomb networks by fostering the association of RNAPII and basal transcription factors. We pinpoint a critical role for TATA-binding protein-associated factors (TAFs) in Integrator entry into the preinitiation complex. Taken together, beyond its role in RNAPII pause release, Integrator recruitment of RNAPII ensures an orderly cellular differentiation during development.

RNA Polymerase II↗
PubMed2023

Cell type-specific loops linked to RNA polymerase II elongation in human neural differentiation.

DNA is folded into higher-order structures that shape and are shaped by genome function. The role for long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNAPolII) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitors (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNAPolII initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops, promoter-promoter loops, or unlooped. Genes transitioning from repression to RNAPolII initiation exhibit slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNAPolII-mediated elongation during neural cell fate transitions.

Preprint↗
PubMed2025

BRD2 bridges TFIID and MOF-H4K16ac-containing nucleosomes to promote transcriptional initiation.

Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA polymerase II (RNA Pol II). Small-molecule inhibitors that block interaction between BET bromodomains and acetylated histones have been developed for disease therapeutics. However, the BET protein BRD4 does not require bromodomains to perform its major transcriptional elongation control, and mechanisms by which other BET proteins regulate RNA Pol II remain insufficiently understood. Addressing the disparity between pan-BET degraders and BRD4-specific depletion, we report that the BET protein BRD2 generally functions to promote transcriptional initiation in a bromodomain-dependent manner at both promoters and enhancers in human cell lines. We demonstrate that BRD2 bromodomains preferentially bind to histone H4 harboring MOF-mediated H4K16ac, while the BRD2 C-terminal domain facilitates recruitment of TFIID. Our studies provide mechanistic insight into distinct roles for BRD2 and BRD4 in transcriptional initiation and elongation control for proper regulation of gene expression.

Humans↗
PubMed2025

Macromolecular and cytological changes in fission yeast G0 nuclei.

When starved of nitrogen, cells of the fission yeast Schizosaccharomyces pombe enter a quiescent 'G0' state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here, we use confocal microscopy, immunoblots and electron cryotomography to investigate the cytological, biochemical and ultrastructural differences between S. pombe proliferating, G1-arrested and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation during nitrogen starvation results in cells that have larger nuclei and therefore chromatin that is less compact. However, these histone-hyperacetylated cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganization at the macromolecular level.

Schizosaccharomyces↗
PubMed2024

Chromatin Transcription Elongation - A Structural Perspective.

In eukaryotic cells, transcription by RNA polymerase II occurs in the context of chromatin, requiring the transcription machinery to navigate through nucleosomes as it traverses gene bodies. Recent advances in structural biology have provided unprecedented insights into the mechanisms underlying transcription elongation. This review presents a structural perspective on transcription through chromatin, focusing on the latest findings from high-resolution structures of transcribing RNA polymerase II-nucleosome complexes. I discuss how RNA polymerase II, in concert with elongation factors such as SPT4/5, SPT6, ELOF1, and the PAF1 complex, engages with and transcribes through nucleosomes. The review examines the stepwise unwrapping of nucleosomal DNA as polymerase advances, the roles of elongation factors in facilitating this process, and the mechanisms of nucleosome retention and transfer during transcription. This structural perspective provides a foundation for understanding the intricate interplay between the transcription machinery and chromatin, offering insights into how cells balance the need for genetic accessibility with the maintenance of genome stability and epigenetic regulation.

Chromatin↗
PubMed2025

Efficient Expression of Oropouche Virus Nonstructural Proteins NSs and NSm.

Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.

Viral Nonstructural Proteins↗
PubMed2025

CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing.

Pre-mRNA splicing is kinetically coupled to transcription as shown by the widespread effects of transcription speed on alternative splicing (AS) outcomes. The molecular basis for such kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate-dependent alternative folding pathways of the nascent pre-messenger RNA (pre-mRNA). To search for RNA structures in nascent pre-mRNA, we modified Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids (SPLASH) [J. G. Ashley Aw et al., Mol. Cell 62, 603-617 (2016)] for use with Chromatin Associated RNA. We applied this method called Chromatin Associated RNA (CAR)-SPLASH to cells expressing wild-type and slow mutant RNA polymerase II and identified >3,000 intramolecular RNA duplexes of which >400 are proximal to splice sites. Antisense oligonucleotide (ASO) disruption of several such duplexes that sequester splice sites has a major impact on AS outcomes, even though the ASOs do not directly disrupt splice sites. ASO disruption of these regulatory elements that we designate "RNA kinetic switches" modified AS of NISCH Exon 18, GAK Exon 7, and MEGF8 Exon 14 in a way that depends on the rate of transcription elongation. We propose that these switches mediate kinetic coupling via the effects of transcription speed on folding of nascent RNA structures that modulate AS and that many nascent RNA structures can thereby serve as targets for splice-modifying ASOs.

RNA Precursors↗
PubMed2025

DNA topoisomerase II promotes N6-adenosine mRNA methylation.

DNA topoisomerase II (TOP2) is an enzyme that regulates DNA topology, primarily by removing DNA supercoiling. This function is crucial during transcription, as the movement of RNA polymerase II (RNAPII) generates torsional stress. However, the specific role of TOP2 in the regulation of gene expression remains to be fully elucidated, as both TOP2 inhibitors and poisons have been shown to upregulate specific genes. In this study, we show that TOP2 poisoning negatively affects transcription elongation of genes repressed at the level of promoter-proximal pausing. Importantly, this effect is counteracted by defective mRNA N6-adenosine methylation (m6A), which results in altered RNA turnover and pre-mRNA splicing. We propose that TOP2 serves a dual function, supporting the maintenance of basal transcription elongation while simultaneously promoting m6A modification in pre-mRNAs to reduce the overall gene expression output.

RNA Methylation↗