The Oropouche virus (OROV) is an emerging orthobunyavirus that has caused outbreaks in South and Central America. Its nonstructural protein (NSs) is a key virulence factor that suppresses host antiviral responses, particularly type I interferon (IFN-I) signaling. We examined whether OROV NSs can modulate IFN-I signaling downstream of IFN alpha receptor (IFNAR) activation and assessed its effect on host transcription. NSs function was evaluated by analyzing RNA polymerase II (RNAPII) abundance, phosphorylation, nucleolar localization, effects of proteasome inhibition, and characterization of basic residue motifs resembling nucleolar localization signals. OROV NSs dampens IFN-I signaling downstream of IFNAR activation and suppresses host transcription through nucleolar targeting and RNAPII degradation. NSs expression reduced total and phosphorylated RNAPII levels in human cells, leading to global transcriptional suppression, which was reversed by proteasome inhibition, indicating proteasome-mediated degradation. NSs was partially localized to the nucleolus, where it disrupted the distribution of the nucleolar protein fibrillarin. Mutations in its two basic residue motifs restored fibrillarin localization and alleviated transcriptional repression. OROV NSs suppresses host transcription via nucleolar targeting and RNAPII degradation and partially modulates IFN-I signaling downstream of IFNAR activation, representing an evolutionarily acquired immune evasion mechanism. These findings enhance our understanding of OROV pathogenesis and help to identify NSs as a potential antiviral target.

Structural basis of RNA polymerase II transcription on the histone H3-H4 octasome.

Cyclin-dependent kinase 1 (CDK1) regulates multiple cellular processes that HSV-1 can exploit to promote its own replication, particularly during the early steps of lytic infection. We investigated whether CDK1 inhibition disrupts immediate-early (IE) gene expression and analyzed the host phosphoproteome early in infection to identify putative host factors and mechanisms that facilitate HSV-1 IE gene expression and are controlled by CDK1. Human foreskin fibroblasts (HFFs) were pre-treated with a CDK1 inhibitor and showed a 1000-fold reduction in HSV-1 replication and significant reductions in IE mRNAs and protein levels at 4 hpi. We characterized cells after CDK1 inhibition and HSV-1 infection at 3 hpi by tandem mass spectrometry and identified >5500 phosphopetides (~2600 proteins), analyzing differential phosphorylation and protein-protein interactions. We validated CDK1 inhibition by detecting phosphorylation-specific decreases in known CDK1 substrates, as well as Robust Kinase Activity Inference. Rank- and network-based analyses of our dataset highlighted several candidate proteins, linking their CDK-directed phosphorylation to HSV-1 IE gene expression. Notably, the C-terminal domain of the large subunit of RNA polymerase II (RNAPII), POLR2A, is extensively phosphorylated, and its phosphorylation is significantly reduced upon CDK1 inhibition during viral infection. Taken together, these data support a model in which CDK1 activity maintains a transcriptionally permissive cellular state required for efficient HSV-1 IE gene expression. Our data suggest that when CDK1 is pharmacologically inhibited, key transcriptional facilitators are dysregulated, impairing viral transcription and replication.

Platinum-based compounds and ultraviolet (UV) irradiation produce bulky DNA lesions that stall RNA polymerase II (RNAPII), activating transcription-coupled nucleotide excision repair (TC-NER), RNAPII degradation, and global transcriptional shutdown. However, the consequences of RNAPII bypassing such lesions remain unclear. We identified the acetyltransferase p300 as a key regulator of TC-NER-dependent RNAPII removal from damaged chromatin via a USP7-dependent mechanism. Loss of p300 permits RNAPII to bypass transcription-blocking lesions, sustaining transcription and full-length mRNA production despite DNA damage. This leads to continued translation, endoplasmic reticulum (ER) stress, and activation of the unfolded protein response (UPR), compromising cell viability. Notably, this stress response resensitizes tumors resistant to platinum-based chemotherapy. Our findings reveal a vulnerability in tumor cells that evade transcriptional shutdown and define a synthetic lethal interaction between p300 inhibition and platinum-induced DNA damage, offering a targeted strategy to overcome chemoresistance.

Fine-tuning DNA replication and transcription is crucial to prevent collisions between their machineries1. This is particularly important near promoters, where RNA polymerase II (RNAPII) initiates transcription and frequently arrests, forming R-loops2-4. Arrested RNAPII can obstruct DNA replication, which often initiates near promoters5,6. The mechanisms that rescue arrested RNAPII during elongation to avoid conflicts with co-directional replisomes remain unclear. Here, using genome-wide approaches and genetic screens, we identify CFAP20 as part of a protective pathway that salvages arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops near promoters, which leads to defects in replication timing and dynamics. These defects stem from accelerated replication-fork speeds that cause a secondary reduction in origin activity. Co-depletion of the Mediator complex or removal of R-loop-engaged RNAPII restores normal replication. Our findings suggest that transcription-dependent fork stalling in cis induces accelerated fork progression in trans, generating single-stranded DNA gaps. We propose that CFAP20 facilitates RNAPII elongation under high levels of Mediator-driven transcription, thereby preventing replisome collisions. This study provides a transcription-centred view of transcription-replication encounters, revealing how locally arrested transcription complexes propagate genome-wide replication phenotypes and defining CFAP20 as a key factor that safeguards genome stability.

Chronic lymphocytic leukemia is a B cell malignancy characterized by proliferation of B cells and is most prevalent in elderly populations that has poor prognosis in advanced stages. Bruton tyrosine kinase inhibitors such as ibrutinib and BCL-2 inhibitors such as venetoclax are considered one of the standard front-line treatments as shown by several clinical trials. However, several targeted and immunotherapies are emerging. Sonrotoclax is a BH3 mimetic BCL2i has been tested in combination with Zanubrutinib in phase I dose escalation/dose expansion study and resulted in a uMRD4 rates of 78% at a 320mg dose in the peripheral blood. No disease progression at a median follow-up of 10 months resulted with no atrial fibrillation and grade 3 infections at 8% of patients. Two clinical studies describe the construction of CAR T cell therapies for the treatment of CLL. CAR T cell therapies in two clinical studies describe constructions which are definite options, and combinations of BCL-2 and BTK inhibitors have been evaluated especially for double refractory disease. One study provided a protocol to recapitulate the TME of CLL to further precision medicine approaches for the disease with the aim of understanding resistance targeted therapies. Another agent, VIP152, is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy that phosphorylates RNA POLII by positive transcription elongation factor complex (P-TEFb). It is a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, producing dysregulated transcripts in CLL.

Stalling of elongating RNA polymerase II (RNAPII) at DNA lesions blocks transcription and triggers transcription-coupled repair (TCR). However, the mechanisms determining the fate of stalled RNAPII remain incompletely understood. Here, we develop a time-resolved assay to track RNAPII clearance and degradation at UV-induced lesions. We show that RNAPII ubiquitylation by CSB and the CRL4CSA ubiquitin ligase is essential, as loss of these proteins causes persistent RNAPII accumulation at damage sites. Downstream of CSB/CRL4CSA-mediated ubiquitylation, two distinct pathways mediate RNAPII removal. The primary rapid route relies on TFIIH, with its XPD helicase activity driving RNAPII dissociation after proper recruitment and positioning by ELOF1, UVSSA, and STK19. A secondary slow pathway is mediated by the ubiquitin-dependent segregase VCP, which compensates for impaired TFIIH function. While VCP contributes only minimally in TCR-proficient cells, inhibition of VCP in TFIIH-deficient contexts completely abrogates RNAPII clearance. Together, these findings establish a hierarchical program in which CSB/CRL4CSA-mediated ubiquitylation initiates RNAPII processing, TFIIH/XPD helicase activity provides the main clearance mechanism, and VCP-dependent extraction acts as a backup when TFIIH fails. This mechanistic framework explains how cells resolve DNA lesion-stalled RNAPII during normal and compromised TCR.

The GPN-loop GTPase Npa3 plays a critical role in RNA polymerase II (RNAPII) assembly and nuclear import. We employed here the npa3ΔC mutant, which supports normal RNAPII localization and function, to investigate potential links between Npa3 and target of rapamycin complex I (TORC1) signaling. The npa3ΔC cells exhibited increased sensitivity to rapamycin, a synthetic sickness interaction with tor1Δ, and a delayed growth recovery rate from rapamycin-induced G1 arrest. Co-expression analysis identified LTV1, a gene involved in TORC1 signaling and ribosome nuclear export, as one of the top genes co-expressed with NPA3. Furthermore, overexpression of eukaryotic translation initiation factor 1A (eIF1A, TIF11) or regulator of heterotrimeric G-protein signaling (RGS2) restored growth in npa3ΔC cells under rapamycin treatment. Interestingly, RGS2 also rescued growth under hygromycin B stress. Our findings suggest a genetic interplay between Npa3 and TORC1.

Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely debated. Here we investigated the relationship between cohesin and RNA polymerase II (RNAPII) using single-molecule mapping and live-cell imaging methods in human cells. Cohesin-mediated transcriptional loops were highly correlated with those of RNA polymerase II and followed the direction of gene transcription. Depleting RAD21, a subunit of cohesin, resulted in the loss of long-range (>100 kb) loops between distal (super-)enhancers and promoters of cell-type-specific downregulated genes. By contrast, short-range (<50 kb) loops were insensitive to RAD21 depletion and connected genes that are mostly constitutively expressed. This result explains why only a small fraction of genes are affected by the loss of long-range chromatin interactions in cohesin-depleted cells. Remarkably, RAD21 depletion appeared to upregulate genes that were involved in initiating DNA replication and disrupted DNA replication timing. Our results elucidate the multifaceted roles of cohesin in establishing transcriptional loops, preserving long-range chromatin interactions for cell-specific genes and maintaining timely DNA replication.

In eukaryotes, transcription elongation factors (TEFs) associate with RNA Polymerase II (RNAPII) to facilitate gene expression and couple transcription to co-transcriptional processes, including chromatin regulation and RNA processing. To further our understanding of TEF biology, we developed a domain-centric analysis pipeline to perform a broad survey of ten TEF orthologs -- Paf1, Ctr9, Cdc73, Rtf1, Leo1, Spt4, Spt5, Spt6, Spn1, and Elf1 -- across the Tree of Life and analyze their evolutionary patterns in a structural context. We report evidence for all ten TEFs being present in the last eukaryotic common ancestor, indicating that mechanisms of TEF-mediated transcription regulation are both ancient and conserved. However, some early-diverging eukaryotic clades exhibit signs of altered TEF domain composition. A comparative phylogenetic analysis highlighted conserved regions of TEFs that are detected in both metazoans and fungi and other regions that appear clade-specific, detected only in metazoans. These observations, together with additional insights generated from evolutionary rate covariation analysis, shed light on under-characterized aspects of TEFs, including domains for which functions have yet to be dissected.

In response to DNA damage, RPB1, the catalytic subunit of RNA Polymerase II (RNAPII), is degraded by the ubiquitin–proteasome system. Degradation models only consider transcriptionally engaged molecules, where a stalled RNAPII complex functions as a lesion-recognition factor, and its RPB1 subunit is proposed to be subsequently degraded to facilitate access of lesion-processing nucleotide excision repair (NER) factors. This transcription-coupled repair is complemented by the global genome repair (GG-NER) system, where lesions are recognized by the XPC and DDB2 factors. Here, we show that RPB1 degradation is controlled in trans by a pathway that depends on lesion processing by NER, irrespectively of whether the lesion is recognized by RNAPII itself or by XPC–DDB2. Incomplete repair due to absence of lesion-processing factors (XPA, XPB, XPD, XPF, or XPG) enhances RPB1 degradation, indicating that the signal controlling RPB1 abundance is started by lesion recognition and continues until DNA repair is completed. Consistent with an in trans mechanism, damage-induced RPB1 degradation is not restricted to active nor phosphorylated RPB1 molecules and depends on Cullin-RING ubiquitin ligases. These findings uncover a repair-dependent mechanism controlling RPB1 levels and provide a rationale for the control of gene expression under stress, where more damage implies more repair and less RPB1 levels, hence restricting RNAPII activity.

The Arabidopsis floral repressor locus FLC is epigenetically silenced during winter cold to align flowering with spring. During weeks of cold exposure, FLC transcription is progressively reduced both by transcriptional repression mediated by FLC antisense transcription and epigenetic silencing implemented through a Polycomb-mediated epigenetic switch. In the warm, FLC is transcriptionally repressed by coordinated changes in transcription initiation and RNA PolII speed in a mechanism involving proximal termination. Whether similar mechanisms contribute to the cold-induced FLC transcriptional repression is unknown. Here, we combine mathematical modelling and transcription profiling to investigate FLC transcriptional changes during the cold. We find different dynamics of spliced and unspliced transcripts during cold exposure with only a small change in PolII speed. We also show that, unlike short-term cold, long-term cold temperatures drive an increase in splicing rates while simultaneously reducing productive transcription at FLC. This process is influenced by antisense COOLAIR transcription but does not rely on proximal COOLAIR termination. Cold-induced transcriptional repression of FLC thus involves a decoupling of changes in productive transcription initiation from PolII speed and rates of co-transcriptional splicing, a different mechanism from that repressing FLC in the warm.

Noncoding RNAs have increasingly recognized roles in critical molecular mechanisms of disease. However, the noncoding genome of Drosophila melanogaster, one of the most powerful disease model organisms, has been understudied. Here, we present FLYNC—FLY noncoding RNA discovery and classification—a novel explainable boosting machine model that accurately predicts the probability of a newly identified RNA transcript being a long noncoding RNA (lncRNA). Integrated into an end-to-end bioinformatics pipeline capable of processing single cell or bulk RNA sequencing data, FLYNC outputs potential new noncoding RNA genes. FLYNC leverages large-scale genomic and transcriptomic datasets to identify patterns and features that distinguish noncoding genes from protein-coding genes, thereby facilitating lncRNA prediction. We demonstrate the application of FLYNC to publicly available Drosophila adult head bulk transcriptome and single-cell transcriptomic data from Drosophila neural stem cell lineages and identify several novel tissue- and cell-specific lncRNAs. We have further experimentally validated the existence of a set of FLYNC predicted lncRNAs by RT-PCR and RNA PolII binding. Overall, our findings demonstrate that FLYNC serves as a robust tool for identifying lncRNAs in D. melanogaster, transcending current limitations in ncRNA identification and harnessing the potential of machine learning.

Reversible cytoplasmic foci of RNA polymerase II subunits serve as proteostatic hubs orchestrating transcriptional reprogramming.

CTCF (CCCTC-binding factor) is crucial for organizing mammalian genomes into domains and structural loops, yet its role in enhancer–promoter interactions remains unclear. Here, we demonstrate that 3D enhancer architecture undergoes marked reorganization upon CTCF depletion in activated CD4+ T cells. Despite this, active transcription, particularly driven by STAT5-bound super-enhancers, maintains enhancer loops independently of CTCF. Interestingly, robust enhancer–promoter interactions are associated with the release of RNA polymerase II (RNAPII) pausing and require CTCF-dependent 3D genome organization to shape immune-related gene expression patterns in CD4+ T cells. Notably, CTCF depletion reprograms the transcriptional response of CD4+ T cells to JAK inhibitors by rewiring the STAT5 enhancer network rather than altering the upstream JAK/STAT signaling cascade. This study emphasizes the role of 3D enhancer architecture orchestrated by CTCF and active transcription in directing precise cell identity gene expression through RNAPII pause-release in CD4+ T cells.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds testosterone mediating gene expression for sexual, somatic, and behavioral functions and is involved in various conditions, including androgen insensitivity syndrome and prostate cancer. Our previous work revealed the actin-dependent formation of transcriptional hubs consisting of the AR, the mammalian formin disheveled-associated activator of morphogenesis 2 (DAAM2) and active RNA Polymerase II (RNA Pol-II). Of note, highly dynamic nuclear F-actin polymerization by DAAM2, directly at the AR is essential for androgen signaling. To better understand actin-driven AR transcriptional activity, we turned our interest to the unconventional myosin VI, which was previously proposed to be involved in RNA Pol-II transcription. Indeed, dihydrotestosterone-dependent mass spectrometry of immunoprecipitated eGFP–myosin VI identified the AR as a prominent associator. Consistent with this, structured illumination microscopy in prostate cancer cells revealed signal-dependent nuclear enrichment of myosin VI, which localized in close proximity to AR as well as RNA Pol-II clusters and the actin nucleator DAAM2. Using live-cell structured illumination microscopy imaging, we directly visualized a ligand-dependent dynamic association between AR, myosin VI, and nuclear actin, revealing their spatially coordinated reorganization at AR clusters. Pharmacological inhibition of actin polymerization or inhibition of the myosin VI motor domain disrupted the formation of AR-related transcriptional clusters. Furthermore, reporter gene analysis and proliferation assays supported a critical role for myosin VI in AR signaling. Our findings thus uncover myosin VI as an essential regulator for the spatial organization of androgen-dependent transcription.

Transcriptional regulation arises from the dynamic and combinatorial actions of multiple regulatory factors on genomic DNA. Although many epigenomic regulators have been identified, the precise order in which these factors accumulate at individual gene loci to activate transcription remains unclear. Here we show a single-cell data integration framework that infers the binding order of multiple chromatin factors at single-cell resolution. Central to this framework is sci-mtChIL-seq, a scalable single-cell method that simultaneously profiles genome-wide binding of RNA polymerase II (RNAPII) and diverse epigenomic regulators. By defining transcriptional states through RNAPII occupancy and integrating multiple sci-mtChIL-seq datasets, we systematically link the combinatorial patterns of transcription factor binding, histone modifications and chromatin remodeling. This framework reveals the temporal coordination among chromatin factors during transcriptional activation, providing a powerful approach to uncover context-dependent epigenomic dynamics and the principles of gene regulation in complex cellular systems.

Transcription-coupled nucleotide excision repair (TC-NER or TCR) is initiated when the ATPase Cockayne syndrome protein B (CSB) recognizes a DNA lesion stalled RNA polymerase II (RNAPII) and forms a stable complex. Here, we report that poly(ADP-ribose) polymerase-1 (PARP1), that plays a key role in the lesion recognition step of global genomic NER, also facilitates the earliest step of TCR. PARP1, which is associated with RNAPII during normal transcription, interacts with and stabilizes CSB on the lesion-stalled RNAPII. CSB stimulates PARP1’s activity to form PAR, and in turn CSB is PARylated mainly at its N-terminal PAR-binding motif (PBM) to promote its stabilization with RNAPII, whereas its minor PARylation at the C-terminal domain suppresses its ATPase function, thus limiting the window of time for ATP-dependent lesion recognition by CSB. The loss of PARP1, treatment with inhibitors of PARP or poly(ADP-ribose) glycohydrolase (PARG) to prevent PAR synthesis or its catabolism to generate free PAR or engineering N-terminal PARylation-resistant CSB decrease the efficiency of cells for TCR. PARP1 mutant Caenorhabditis elegans larvae exhibit a pronounced TCR-deficient phenotype. Our findings uncover an evolutionarily conserved role of PARP1 and PAR metabolism in the initiation of TCR.

The final step of oocyte growth, which reorganizes chromatin from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) configuration is essential for embryonic development after meiotic maturation and fertilization. The underlying mechanisms remain unknown. We identify RNA polymerase II (RNAPII) degradation as the key driver of this process. Inhibitors that trigger RNAPII degradation, but not nucleoside-based transcription inhibitors, induce NSN-to-SN transition in oocytes. By establishing miniTrim-Away for nuclear proteins and using segregase and proteasome inhibitors, we demonstrate that RNAPII degradation is necessary and sufficient for NSN-to-SN transition. Further experiments reveal that RNAPII degradation results in a global collapsing force and a local attractive force required for the transition to SN configuration. Finally, embryos derived from NSN oocytes have aberrant RNAPII levels and localization, and are defective in maternal-to-zygotic transition. Our study elucidates the mechanistic framework of oocyte chromatin reorganization and presents a strategy for inducing fully grown oocyte nuclei.

Transcription mediated by RNA polymerase II (RNAPII) involves multiple stages, including initiation, promoter-proximal pausing for capping, elongation, and termination. The C-terminal domain of RNAPII (CTD) contains repetitions of the heptad consensus sequence, such as Y1S2P3T4S5P6S7 with some variety, and the phosphorylated positions in the heptad sequences are altered according to the transcriptional stages. The interaction between several regulatory protein factors and the phosphorylated heptad sequence plays an important role in the accurate progress of transcription. A subset of these regulatory proteins possesses a CTD-interacting domain (CID) that specifically recognizes the phosphorylated CTD and mediates stage-specific transcriptional control. Among them, SCAF8 (RBM16), which also contains a CID, plays a key role in accurate transcriptional termination in conjunction with its paralog SCAF4. Despite their importance, the precise molecular mechanisms through which SCAF8 and SCAF4 coordinate transcriptional termination via their CID domains remain poorly understood. In this study, we report the 1H, 15N, and 13C NMR resonance assignments and solution structure of the human SCAF8 CID domain. The structure exhibits an α1-α2-α3-α4-α5-α6-α7-α8 helical topology, consistent with the previously determined crystal structure. These assignments provide a valuable foundation for understanding how SCAF8 interacts with the RNAPII CTD and contributes to transcriptional elongation and termination.