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Phase separation of RNA-binding protein promotes polymerase binding and transcription

ABSTRACT: An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.

ORGANISM(S): Mus musculus

PROVIDER: GSE150399 | GEO | 2020/05/12

REPOSITORIES: GEO

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Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

Project description:The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.

2019-06-17 | GSE120656 | GEO

SGF29 nuclear condensates mediate cellular aging

Project description:Phase separation, a biophysical segregation of subcellular milieus referred as condensates, is known to regulate transcription but its impacts on physiological processes are less clear. Here, we asked how the candidate phase regulator SGF29, a component of the transcriptional coactivator complex SAGA, regulates transcriptional events in aging. We 36 demonstrate that SGF29 forms liquid-like nuclear condensates as human mesenchymal 37 progenitor cells (hMPCs) become senescent. Mechanistically, SGF29 partitions with the 38 transcription factor SP1, RNA polymerase II and the transcriptional coactivator MED4 to form transcriptionally active condensates. These then target specific genomic loci, such as the CDKN1A promoter, to drive senescence-related gene expression. The Arg 207 in the intrinsically disordered region is identified as the key amino acid residue for SGF29 to form phase separation and activate CDKN1A transcription. Our study links phase separation to aging regulation and reveals nuclear condensates as a functional unit shaping transcriptional landscapes in aging.

2023-02-24 | PXD040372 | iProX

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

Project description:RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits.

2022-08-09 | PXD035866 | iProX

Elongin A displays widespread association with actively transcribed genes and modulates enhancer RNA transcripiton.

Project description:Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes both to thousands of Pol II transcribed genes with a preference for sites of Pol II pausing and as well as a large number of active enhancers across the genome. EloA loss results in accumulation of transcripts at a subset of enhancers and their adjacent genes. However, EloA deletion results in only a modest decrease in the average elongation rate of Pol II globally. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interferes with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on eRNA synthesis and interacts with the rRNA producing/processing machinery in the nucleolus.

2020-12-10 | GSE151582 | GEO

RNA-binding protein WDR43 binds prevalently to open chromatin and promotes gene transcription

Project description:The intricate regulation of transcription underlies cellular differentiation and development. However, the general relevance of RNA-binding proteins (RBPs) in polymerase (Pol) II transcription is less understood. Here we revealed that WDR43, a novel chromatin-bound RBP, promotes transcription and is essential for self-renewal of embryonic stem cells (ESCs) and early embryonic development. WDR43 binds to promoter-associated non-coding and nascent RNAs, localizes at thousands of gene promoters and enhancers, and interacts with the Pol II transcription machinery. Recombinant WDR43 directly promotes transcription and the release of elongation factor P-TEFb in vitro. Depletion of WDR43 in ESCs leads to global defects in Pol II pause release and mRNA expression. These results demonstrate that WDR43 maintains high-level expression of pluripotency programs by modulating Pol II activity and transcription elongation. To extend these findings, we demonstrated widespread co-occupancy of open chromatin by multiple RBPs, suggesting an unforeseen role for RBPs in transcription control

2019-02-08 | GSE110358 | GEO

A role for phase separation to prevent RNA:DNA hybrids

Project description:The function of phase separation for many RNA-binding proteins is poorly understood, including FUS (FUSed in sarcoma). FUS binds RNA Pol II and controls phosphorylation of serine-2 (Ser2P). We uncovered a FUS activity involving no interactions with the polymerase. FUS could activate transcription of a non-eukaryote polymerase by preventing RNA invasion of DNA to form R-loops. This activity required binding to the nascent transcript and phase separation. We confirmed the activity in human cells using DRIP-seq, where R-loop sites formed were consistent with characteristic features of FUS activity, including transcription start sites where Ser2P signals accumulate after FUS knockdown. R-loop sites were enriched for sequences favored by the degenerate binding specificity of FUS. FUS also regulated R-loops near active retrotransposons and regions associated with DNA repeat expansion disorders. These findings offer a unified model that incorporates RNA-binding and phase separation to guard against instability from disruptive nucleic acid structures.

2022-06-25 | GSE206740 | GEO

Chromatin remodeling by antisense Pol II primes efficient Pol III transcription

Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is associated with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here that antisense RNA Polymerase II (Pol II) transcription is critical to prime and maintain nucleosome depletion at Pol III loci and allow efficient Pol III recruitment upon re-initiation of growth from stationary phase. Antisense Pol II is recruited by the Pcr1 transcription factor, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.

2022-12-24 | GSE193552 | GEO

Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modification on the C-terminal domain (CTD) of the largest subunit , generating a sophisticated PTM landscape for the spatiotemporal recruitment to transcriptional regulators. To delineate the protein interactomes recruited to Pol II at different stages of transcription, we in vitro reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 positions, the hallmark phosphorylation at the initation and productive elongation stages of transcription, respectively. Distinctive protein interactomes indicates different proteins are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) specifically binds to the Ser2 of the heptad. The loss of the interaction between CHERP and Pol II results in broad alternative splicing events. Our method points to a new method to distinguish the PTM codes that coordinate the transcription process.

2023-02-06 | MSV000091213 | MassIVE

Establishment of a novel in vitro transcription assay

Project description:RNA Polymerase II (Pol II) transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. By combing our in vitro transcription assays with other experiments (e.g. in vivo dynamic ChIP-seq assays), we identified PAF1 complex components as drivers for transcription recycling, revealing a new layer in controlling Pol II-dependent transcription. Our findings also point to RNA Pol II transcription recycling as a mechanism that functions aberrantly in disease states such as cancer, and indicate the potential to target factors such as PAF1 that drive RNA Pol II recycling in cancer.

2021-11-03 | PXD027143 | Pride

Establishment of a novel in vitro transcription assay

Project description:RNA Polymerase II (Pol II) transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. By combing our in vitro transcription assays with other experiments (e.g. in vivo dynamic ChIP-seq assays), we identified PAF1 complex components and phospho-MED1 as drivers for transcription recycling, revealing a new layer in controlling Pol II-dependent transcription. Our findings also point to RNA Pol II transcription recycling as a mechanism that functions aberrantly in disease states such as cancer, and indicate the potential to target factors such as PAF1 and phospho-MED1 that drive RNA Pol II recycling in cancer.

2021-11-03 | PXD017881 | Pride

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