RNA Polymerase II Research Articles

Structural insights into promoter-proximal pausing of RNA polymerase II at +1 nucleosome.

The metazoan transcription elongation complex (EC) of RNA polymerase II (RNAPII) generally stalls between the transcription start site and the first (+1) nucleosome. This promoter-proximal pausing involves negative elongation factor (NELF), 5,6-dichloro-1-β-d-ribobenzimidazole sensitivity-inducing factor (DSIF), and transcription elongation factor IIS (TFIIS) and is critical for subsequent productive transcription elongation. However, the detailed pausing mechanism and the involvement of the +1 nucleosome remain enigmatic. Here, we report cryo-electron microscopy structures of ECs stalled on nucleosomal DNA. In the absence of TFIIS, the EC is backtracked/arrested due to conflicts between NELF and the nucleosome. We identified two alternative binding modes of NELF, one of which reveals a critical contact with the downstream DNA through the conserved NELF-E basic helix. Upon binding with TFIIS, the EC progressed to the nucleosome to establish a paused EC with a partially unwrapped nucleosome. This paused EC strongly restricts EC progression further downstream. These structures illuminate the mechanism of RNAPII pausing/stalling at the +1 nucleosome.

Transcription elongation factor ELOF1 is required for efficient somatic hypermutation and class switch recombination.

Transcription elongation factor ELOF1 is required for efficient somatic hypermutation and class switch recombination.

Transcription-coupled AID deamination damage depends on ELOF1-associated RNA polymerase II.

Transcription-coupled AID deamination damage depends on ELOF1-associated RNA polymerase II.

Spt6-Spn1 interaction is required for RNA Polymerase II association and precise nucleosome positioning along transcribed genes.

Spt6-Spn1 interaction is required for RNA Polymerase II association and precise nucleosome positioning along transcribed genes.

Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

The histone chaperone and transcription elongation factor SPT6 is integral to RNA polymerase II (RNAPII) activity. SPT6 also plays a crucial role in regulating transcription termination, although the mechanisms involved are largely unknown. In an attempt to identify the pathways employed by SPT6 in this regulation, we found that, while SPT6 and its partner IWS1 interact and co-localize with RNAPII, their functions diverge significantly at gene termination sites. Depletion of SPT6, but not of IWS1, results in extensive readthrough transcription, indicating that SPT6 independently regulates transcription termination. Further analysis identified that the cleavage and polyadenylation factor PCF11 and the phosphatase regulatory protein PNUTS collaborate with SPT6 in this process. These findings suggest that SPT6 may facilitate transcription termination by recruiting PNUTS and PCF11 to RNAPII. Additionally, SPT6 and PNUTS jointly restrict promoter upstream transcripts (PROMPTs), whereas PCF11 presence is essential for their accumulation in the absence of SPT6 at hundreds of genes. Thus, SPT6, PCF11, and PNUTS have both distinct and overlapping functions in transcription termination. Our data highlight the pivotal role of SPT6 in ensuring proper transcription termination at the 5' and 3'-ends of genes.

Selection of stable reference genes in prunus persica fruit infected with monilinia laxa for normalisation of RT-qPCR gene expression data

Reverse transcription-quantitative PCR (RT-qPCR) is a powerful tool for quantifying gene expression. However, reference genes (RGs) for gene expression analysis in peach (Prunus persica) during interactions with Monilinia laxa, a major fungal pathogen that causes brown rot, have not been established. In this study, we analysed 12 candidate RGs in this pathosystem by analysing samples from 12 to 144 HAI. The stability of the RGs was evaluated using the ΔCq method and BestKeeper, NormFinder, and geNorm algorithms. Our results identified AKT3, RNA pol II (RPII) and SNARE (using geNorm), RPII, AKT3 and TEF2 (using NormFinder), AKT3, SNARE and RPII (using BestKeeper) and RPII, MUB6 and AKT3 (using the ΔCq method) as the most stable RGs for mRNA normalisation in this pathosystem across all tested samples. The geNorm algorithm was used to determine the optimal number of suitable RGs required for proper normalisation under these experimental conditions, indicating that the three RGs were sufficient for normalisation. Analysis of the results obtained using different algorithms showed that AKT3, RPII, and SNARE were the three most stable RGs. Furthermore, to confirm the validity of the reference genes, the expression levels of six genes of interest, involved in different metabolic pathways, were normalized in inoculated and uninoculated peach fruit. These findings provide a set of RGs for accurate RT-qPCR analysis in studies involving peach and M. laxa interactions, facilitating deeper insights into the molecular mechanisms underlying this important plant–pathogen relationship.

RNA Pol-II transcripts in nucleolar associated domains of cancer cell nucleoli

ABSTRACT We performed a comparative study of the non-ribosomal gene content of nucleoli from seven cancer cell lines, using identical methods of purification and analysis. We identified unique chromosomal domains associated with the nucleolus (NADs) and genes within these domains (NAGs). Four cell lines have relatively few NAGs, which appears mostly transcriptionally inactive, consistent with literature. The remaining three lines formed a separate group with nucleoli with unique features and NADS. They constitute larger number of common NAGs, marked by ATAC-seq and having accessible promoters, with histone markers for transcriptional activity and detectable RNA Pol II bound at their promoters. The transcripts of these genes are almost entirely exported from the nucleolus. These results indicate that RNA Pol II dependent transcription in NADs can vary widely in different cell types, presumably dependent on the cell’s developmental stage. Nucleolus-associated genes are likely to be distinguished marks reflecting the cell’s metabolism.

RNA polymerase II at histone genes predicts outcome in human cancer.

Genome-wide hypertranscription is common in human cancer and predicts poor prognosis. To understand how hypertranscription might drive cancer, we applied our formalin-fixed paraffin-embedded (FFPE)-cleavage under targeted accessible chromatin method for mapping RNA polymerase II (RNAPII) genome-wide in FFPE sections. We demonstrate global RNAPII elevations in mouse gliomas and assorted human tumors in small clinical samples and discover regional elevations corresponding to de novo HER2 amplifications punctuated by likely selective sweeps. RNAPII occupancy at S-phase-dependent histone genes correlated with WHO grade in meningiomas, accurately predicted rapid recurrence, and corresponded to whole-arm chromosome losses. Elevated RNAPII at histone genes in meningiomas and diverse breast cancers is consistent with histone production being rate-limiting for S-phase progression and histone gene hypertranscription driving overproliferation and aneuploidy in cancer, with general implications for precision oncology.

A cluster of RNA Polymerase II molecules is stably associated with an active gene.

In eukaryotic nuclei, transcription is associated with discrete foci of RNA Polymerase II (RNAPII) molecules. How these clusters interact 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 are reduced upon transcription elongation. We show that single clusters are stably associated with active gene loci during transcription and that cluster intensities are strongly correlated with transcriptional output. Our data suggest that prior to ZGA, RNAPII clusters prime genes for activation, whereas after ZGA, clusters are composed mostly of elongating molecules at individual genes.

Dynamic control of RNA-DNA hybrid formation orchestrates DNA2 activation at stalled forks by RNAPII and DDX39A.

Dynamic control of RNA-DNA hybrid formation orchestrates DNA2 activation at stalled forks by RNAPII and DDX39A.

Assembly of the Xrn2/Rat1-Rai1-Rtt103 termination complexes in mesophilic and thermophilic organisms.

The 5'-3' exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from Saccharomyces cerevisiae and Chaetomium thermophilum. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Restrictor slows early transcription elongation to render RNA polymerase II susceptible to termination at non-coding RNA loci.

The eukaryotic genome is broadly transcribed by RNA polymerase II (RNAPII) to produce protein-coding messenger RNAs (mRNAs) and a repertoire of non-coding RNAs (ncRNAs). Whereas RNAPII is very processive during mRNA transcription, it terminates rapidly during synthesis of many ncRNAs, particularly those that arise opportunistically from accessible chromatin at gene promoters or enhancers. The divergent fates of mRNA versus ncRNA species raise many questions about how RNAPII and associated machineries discriminate functional from spurious transcription. The Restrictor complex, comprised of the RNA binding protein ZC3H4 and RNAPII-interacting protein WDR82, has been implicated in restraining the expression of ncRNAs. However, the determinants of Restrictor targeting and the mechanism of transcription suppression remain unclear. Here, we investigate Restrictor using unbiased sequence screens, and rapid protein degradation followed by nascent RNA sequencing. We find that Restrictor promiscuously suppresses early elongation by RNAPII, but this activity is blocked at most mRNAs by the presence of a 5' splice site. Consequently, Restrictor is a critical determinant of transcription directionality at divergent promoters and prevents transcriptional interference. Finally, our data indicate that rather than directly terminating RNAPII, Restrictor acts by reducing the rate of transcription elongation, rendering RNAPII susceptible to early termination by other machineries.

Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters.

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs and Mediator contain intrinsically-disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of RNAPII transcription at hundreds of promoters simultaneously. We show rapid RNAPII activation is IDR-dependent, without condensate formation. For example, the MED1-IDR can functionally replace a native TF, activating RNAPII with similar (not identical) kinetics; however, MED1-IDR squelches transcription as a condensate, but activates as a single-protein. TFs and Mediator cooperatively activate RNAPII bursting and re-initiation and surprisingly, Mediator can drive TF-promoter recruitment, without TF-DNA binding. Collectively, RIFT addressed questions largely intractable with cell-based methods, yielding mechanistic insights about IDRs, condensates, enhancer-promoter communication, and RNAPII bursting that complement live-cell imaging data.

An oncoprotein CREPT functions as a co-factor in MYC-driven transformation and tumor growth

Understanding the mechanisms behind MYC-driven oncogenic transformation could pave the way for identifying novel drug targets. This study explored the role of CREPT in MYC-induced malignancy by generating MYC-transformed mouse embryonic fibroblasts (MEFs) with conditional CREPT deletion. Our results demonstrated that the loss of CREPT significantly impaired MYC-induced colony formation and cell proliferation, indicating that CREPT is essential for the malignant transformation of MEFs. Reintroducing CREPT in CREPT-deficient cells restored malignant properties. Furthermore, CREPT overexpression alone enhanced colony formation upon MYC induction but was insufficient to induce transformation without MYC, suggesting a cooperative interaction between CREPT and MYC in malignant transformation. CREPT deletion resulted in delayed cell cycle progression during the G2/M and S phases. CREPT enhanced the expression of MYC target genes by directly interacting with MYC through the CID domain of CREPT and the PEST domain of MYC. Arginine 34 of CREPT was identified as a critical residue for the interaction with MYC, and its mutation lost the ability of CREPT to promote MYC-driven colony formation and tumor growth in colorectal cancer models. Additionally, CREPT facilitated the recruitment of RNA Polymerase II (RNAPII) to MYC-binding promoters, promoting transcriptional initiation of MYC-targeted genes. Our study also revealed a strong correlation between CREPT and MYC expression in various human cancers, particularly in colorectal cancer, where their interaction appears to play a significant role in tumorigenesis. These findings suggest that the CREPT-MYC interaction is crucial for the progression of MYC-driven cancers and presents a potential target for therapeutic intervention.

Inter-chromosomal transcription hubs shape the 3D genome architecture of African trypanosomes

AbstractThe eukaryotic nucleus exhibits a highly organized 3D genome architecture, with RNA transcription and processing confined to specific nuclear structures. While intra-chromosomal interactions, such as promoter-enhancer dynamics, are well-studied, the role of inter-chromosomal interactions remains poorly understood. Investigating these interactions in mammalian cells is challenging due to large genome sizes and the need for deep sequencing. Additionally, transcription-dependent 3D topologies in mixed cell populations further complicate analyses. To address these challenges, we used high-resolution DNA-DNA contact mapping (Micro-C) in Trypanosoma brucei, a parasite with continuous RNA polymerase II (RNAPII) transcription and polycistronic transcription units (PTUs). With approximately 300 transcription start sites (TSSs), this genome organization simplifies data interpretation. To minimize scaffolding artifacts, we also generated a highly contiguous phased genome assembly using ultra-long sequencing reads. Our Micro-C analysis revealed an intricate 3D genome organization. While the T. brucei genome displays features resembling chromosome territories, its chromosomes are arranged around polymerase-specific transcription hubs. RNAPI-transcribed genes cluster, as expected from their localization to the nucleolus. However, we also found that RNAPII TSSs form distinct inter-chromosomal transcription hubs with other RNAPII TSSs. These findings highlight the evolutionary significance of inter-chromosomal transcription hubs and provide new insights into genome organization in T. brucei.

Transcription-coupled repair - mechanisms of action, regulation, and associated human disorders.

The transcription-coupled repair (TCR) pathway resolves transcription-blocking DNA lesions to maintain cellular function and prevent transcriptional arrest. Stalled RNA polymerase II (RNAPII) triggers repair mechanisms, including RNAPII ubiquitination, which recruit UVSSA and TFIIH. Defects in TCR-associated genes cause disorders like Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and recently defined AMeDS. TCR safeguards transcription, linking its failure to neurodegeneration and disease phenotypes.

Phenotypic screens identify SCAF1 as critical activator of RNAPII elongation and global transcription.

Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used asmall interferingRNA(siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits,SCAF1 (SR-related C-terminal domain-associated factor 1), using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery is synthetically lethal.

Mechanisms of synergistic Mediator recruitment in RNA polymerase II transcription activation revealed by single-molecule fluorescence.

Transcription activators trigger transcript production by RNA Polymerase II (RNApII) via the Mediator coactivator complex. Here the dynamics of activator, Mediator, and RNApII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNApII to the template required activator and an upstream activator sequence (UAS), but not a core promoter. While Mediator and RNApII sometimes bind as a pre-formed complex, more commonly Mediator binds first and subsequently recruits RNApII to form a preinitiation complex precursor (pre-PIC) tethered to activators on the UAS. Interestingly, Mediator occupancy has a highly non-linear response to activator concentration, and fluorescence intensity measurements show Mediator preferentially associates with templates having at least two activators bound. Statistical mechanical modeling suggests this "synergy" is not due to cooperative binding between activators, but instead occurs when multiple DNA-bound activator molecules simultaneously interact with a single Mediator.

RNA Polymerase II hypertranscription at histone genes in cancer FFPE samples.

Genome-wide hypertranscription is common in human cancer and predicts poor prognosis. To understand how hypertranscription might drive cancer, we applied our FFPE-CUTAC method for mapping RNA Polymerase II (RNAPII) genome-wide in formalin-fixed paraffin-embedded (FFPE) sections. We demonstrate global RNAPII elevations in mouse gliomas and assorted human tumors in small clinical samples and discover regional elevations corresponding to de novo HER2 amplifications punctuated by likely selective sweeps. RNAPII occupancy at replication-coupled histone genes correlated with WHO grade in meningiomas, accurately predicted rapid recurrence, and corresponded to whole-arm chromosome losses. Elevated RNAPII at histone genes in meningiomas and diverse breast cancers is consistent with histone production being rate-limiting for S-phase progression and histone gene hypertranscription driving overproliferation and aneuploidy in cancer, with general implications for precision oncology.