Nascent RNA Research Articles

Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation

Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation

Genome-wide kinetic profiling of pre-mRNA 3' end cleavage.

Cleavage and polyadenylation is necessary for the formation of mature mRNA molecules. The rate at which this process occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. Despite advances in high-throughput approaches for global kinetic profiling of RNA maturation, genome-wide 3' end cleavage rates have never been measured. Here, we describe a novel approach to estimate the rates of cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. We find that 3' end cleavage is fast on average, with half-lives under a minute, but highly variable across individual sites. Rapid cleavage is promoted by the presence of canonical sequence elements and an increased density of polyadenylation signals near a cleavage site. Finally, we find that cleavage rates are associated with the localization of RNA polymerase II at the end of a gene, and faster cleavage leads to quicker degradation of downstream readthrough RNA. Our findings shed light on the features important for efficient 3' end cleavage and the regulation of transcription termination.

Collisions of RNA polymerases behind the replication fork promote alternative RNA splicing in newly replicated chromatin

DNA replication produces a global disorganization of chromatin structure that takes hours to be restored. However, how these chromatin rearrangements affect the regulation of gene expression and the maintenance of cell identity is not clear. Here, we use ChOR-seq and ChrRNA-seq experiments to analyze RNA polymerase II (RNAPII) activity and nascent RNA synthesis during the first hours after chromatin replication in human cells. We observe that transcription elongation is rapidly reactivated in nascent chromatin but that RNAPII abundance and distribution are altered, producing heterogeneous changes in RNA synthesis. Moreover, this first wave of transcription results in RNAPII blockages behind the replication fork, leading to changes in alternative splicing. Altogether, our results deepen our understanding of how transcriptional programs are regulated during cell division and uncover molecular mechanisms that explain why chromatin replication is an important source of gene expression variability.

Targeting the cGAS-STING Pathway Inhibits Peripheral T-cell Lymphoma Progression and Enhances the Chemotherapeutic Efficacy.

Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. The efficacy of current first-line treatment is dismal, and novel agents are urgently needed to improve patient outcomes. A close association between the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway and tumor promotion exists, revealing prospective therapeutic targets. This study, investigates the role of the cGAS-STING pathway and its underlying mechanisms in PTCL progression. Single-cell RNA sequencing showes that the cGAS-STING pathway is highly expressed and closely associated with PTCL proliferation. cGAS inhibition suppresses tumor growth and impaires DNA damage repair. Moreover, Cdc2-like kinase 1 (CLK1) is critical for residual tumor cell survival after treatment with cGAS inhibitors, and CLK1 suppression enhances sensitivity to cGAS inhibitors. Single-cell dynamic transcriptomic analysis indicates reduced proliferation-associated nascent RNAs as the underlying mechanism. In first-line therapy, chemotherapy-triggered DNA damage activates the cGAS-STING pathway, and cGAS inhibitors can synergize with chemotherapeutic agents to kill tumors. The cGAS-STING pathway is oncogenic in PTCL, whereas targeting cGAS suppresses tumor growth, and CLK1 may be a sensitivity indicator for cGAS inhibitors. These findings provide a theoretical foundation for optimizing therapeutic strategies for PTCL, especially in patients with relapsed/refractory disease.

Structural visualization of transcription initiation in action.

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications.

RNA molecules are not merely a combination of four bases of A, C, G, and U. Chemical modifications occur in almost all RNA species and play diverse roles in gene expression regulation. The abundant cellular RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), are known to have the highest density of RNA modifications, which exert critical functions in rRNA and tRNA biogenesis, stability, and subsequent translation. In recent years, modifications on low-abundance RNA species in mammalian cells, such as messenger RNA (mRNA), regulatory noncoding RNA (ncRNA), and chromatin-associated RNA (caRNA), have been shown to contain multiple different chemical modifications with functional significance. As the most abundant mRNA modification in mammals, N6-methyladenosine (m6A) affects nearly every stage of mRNA processing and metabolism, with the antibody-based m6A-MeRIP-seq (methylated RNA immunoprecipitation sequencing) followed by high-throughput sequencing widely employed in mapping m6A distribution transcriptome-wide in diverse biological systems. In addition to m6A, other chemical modifications such as pseudouridine (Ψ), 2'-O-methylation (Nm), 5-methylcytidine (m5C), internal N7-methylguanosine (m7G), N1-methyladenosine (m1A), N4-acetylcytidine (ac4C), etc. also exist in polyA-tailed RNA in mammalian cells, requiring effective mapping approaches for whole-transcriptome profiling of these non-m6A mRNA modifications. Like m6A, the antibody-based enrichment followed by sequencing has been the primary method to study distributions of these modifications. Methods to more quantitatively map these modifications would dramatically improve our understanding of distributions and modification density of these chemical marks on RNA, thereby bettering informing functional implications. In this Account, aimed at both single-base resolution and modification fraction quantification, we summarize our recent advances in developing a series of chemistry- or biochemistry-based methods to quantitatively map RNA modifications, including m6A, Ψ, m5C, m1A, 2'-O-methylation (Nm), and internal m7G, in mammalian mRNA at base resolution. These new methods, including m6A-SAC-seq, eTAM-seq, BID-seq, UBS-seq, DAMM-seq, m1A-quant-seq, Nm-Mut-seq, and m7G-quant-seq, promise to conduct base-resolution mapping of most major mRNA modifications with low RNA input and uncover dynamic changes in modification stoichiometry during biological and physiological processes, facilitating future investigations on these RNA modifications in regulating cellular gene expression and as potential biomarkers for clinical diagnosis and prognosis. These quantitative sequencing methods allow the mapping of most mRNA modifications with limited input sample requirements. The same modifications on diverse RNA species, such as caRNA, ncRNA, nuclear nascent RNA, mitochondrial RNA, cell-free RNA (cfRNA), etc., could be sequenced using the same methods.

Quantifying the impact of initial RNA primer length on nucleotide addition by RNA polymerase I

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence.

Abstract LB_A20: Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors

Abstract Background: Drug resistance is a major obstacle to successful treatment of cancer and leukemia. The role of RNA epigenetics in cancer drug resistance is largely unknown. Our previous studies demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018). The Cancer Genome Atlas Program (TCGA) data demonstrate that elevated expression levels of NSUN1 and NSUN2 are correlated with significantly shorter patients’ survival in almost all cancer subtypes (https://www.proteinatlas.org/ENSG00000111641-NOP2/pathology). However, due to the lack of NSUN1 and NSUN2 proteins’ crystal structures, no NSUN1/2-targeting therapeutics have been developed yet. Methods: 1. Argonne artificial intelligence (AI)/supercomputer design of NSUN1/2-targeting small-molecule compound libraries, simulation of compound-protein interactions and analysis of structure activity relationship (SAR). 2. Novel RNA epigenetic technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). 3. Functional drug screening, H&E histology and immunohistochemistry (IHC). 4. Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results:1. Identification of the ligand-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of NSUN1/2-targeting small-molecule libraries by AI/supercomputing. 2. Identification of several NSUN1/2 inhibitors that effectively inhibit growth of the venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. 3. Demonstration of the lead inhibitors execute their effects through disruption of NSUN1/2-mediated (ACS) and translational complexes (TLC). 4. Characterization of the lead inhibitors’ binding modules with AI/supercomputing SAR analysis. 5. In vivo validation of the efficacies of the identified lead inhibitors using syngeneic AML mouse models. Conclusion:By leveraging the power of the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in oncogenic bioprocesses and drug (venetoclax) resistance, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancer subtypes. Citation Format: Jason X Cheng, Shaun Wood, Derrick Tang, Linda Degenstein, Andrzej Joachimiak, Rick Stevens. Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A20.

Abstract C128: PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells

Abstract PT-112 is a novel immunogenic small molecule under Phase 2 development in metastatic castration-resistant prostate cancer and thoracic malignancies. PT-112 has been shown to release damage-associated molecular patterns, essential immune signaling components of immunogenic cell death (ICD), and to elicit anticancer immune responses. In addition, PT-112 causes mitochondrial and endoplasmic reticulum (ER) stress in vitro. Here, we sought to elucidate early molecular effects of PT-112 that culminate in cancer cell death. We assessed nucleolar protein phenotypes in human prostate cancer cells treated with PT-112 using antibody labeling, followed by confocal microscopy. We also conducted nascent RNA sequencing in human cancer cells (non-small cell lung, prostate, and renal cell carcinoma) following 1- and 6-hour PT-112 treatment. We then performed an analysis using Enrichr across multiple databases to identify biological pathways linked to clusters of differentially expressed genes (p ≤ 0.001) in PT-112-treated cells. Pathways affected by PT-112 (p ≤ 0.05) were sorted by statistical significance. In prostate cancer cells, PT-112 caused changes in nucleolar protein labeling. The most pronounced effect, observed after 8 or more hours of treatment, was rapid NPM1 relocation from nucleoli to nucleoplasm, a hallmark of nucleolar stress and disruption of ribosomal biogenesis (RiBi). PT-112 also induced gene expression changes in the treated cancer cell lines, resulting in statistically significant repression of pathways related to RiBi, ribosomal RNA (rRNA) processing and translation. Notably, a pattern across these pathways was the reduced expression of many ribosomal genes, consistent with RiBi inhibition. Additional analysis identified oncogenic pathways such as Myc and NF-κB significantly inhibited by PT-112. The degree of pathway suppression increased with treatment time. These results demonstrate that PT-112 rapidly causes nucleolar stress and RiBi inhibition, suggesting these are central to PT-112-induced cancer cell death. This mechanism could also explain the selective killing of cancer cells by PT-112, given that aberrant nucleolar size and number as well as ribosomal activity in cancer cells make them more sensitive to these drug effects. The broad consequences of impaired ribosome production and downstream protein translation may explain PT-112’s anticancer and immunogenic effects. Additionally, these disruptions have been reported to cause an increase in free ribosomal proteins and changes in nucleolar protein localization, which have been shown to induce ER stress and shuttle pro-apoptotic proteins to the mitochondria. These additional consequences of RiBi inhibition and nucleolar stress may contribute to or drive previously observed PT-112-induced cancer organelle stress and ICD. While further validation of these findings is needed, this work deepens our understanding of PT-112’s mechanism of action. Present findings, together with demonstrated clinical activity of PT-112, position it as a promising small molecule cancer immunotherapy. Citation Format: Christina Y. Yim, Maria T. Congenie, Hannah L. Johnson, Maureen G. Mancini, Fabio Stossi, Michael A. Mancini, Joey Azofeifa, Matthew R. Price, Johan Baeck, Tyler D. Ames. PT-112, a novel immunogenic cell death inducer, causes ribosomal biogenesis inhibition and organelle stress in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C128.

PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.

Gene regulation is dependent on the production of mRNAs and a repertoire of non-coding RNAs by RNA polymerase II (RNAPII). Precision run-on sequencing (PRO-seq) maps the position of engaged RNAPII complexes at single-nucleotide resolution and can reveal direct targets of regulation, locations of enhancers, and transcription mechanisms that are difficult or impossible to measure by analysis of total cellular RNA. Briefly, this method first involves permeabilizing cells with mild detergents to remove intracellular NTPs and halt transcription. Transcription is then resumed in the presence of biotin-NTPs and sarkosyl to allow transcriptional incorporation of a single biotinylated NTP by RNAPII. The biotin moiety is then bound to streptavidin beads to stringently enrich for nascent RNAs. Sequencing libraries are then generated such that the first base read corresponds to the 3' end of the nascent transcript. Here, we describe our current protocol for generating PRO-seq libraries from metazoan cells, including adaptations of previously published protocols to incorporate unique molecular identifiers, reduce ligation bias, and improve library yields. Additional commentary describes quality control and processing of PRO-seq data and references for more advanced downstream analysis such as gene and enhancer identification. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell permeabilization for PRO-seq Basic Protocol 2: Construction of PRO-seq libraries Support Protocol: Adenylation of 3' adapter.

Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from an RNA polymerase. Here, we describe a concise, high-resolution cotranscriptional RNA chemical probing procedure called variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL). We demonstrate the accuracy and resolution of TECprobe-VL by replicating and extending previous analyses of ZTP and fluoride riboswitch folding and mapping the folding pathway of a ppGpp-sensing riboswitch. In each system, we show that TECprobe-VL identifies coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-VL as an accessible method for mapping cotranscriptional RNA folding pathways.

Overcoming Venetoclax Resistance in Leukemia with AI-Designed RNA Epigenetic Inhibitors

Background : Venetoclax resistance is a major obstacle to successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Its underlying mechanisms are multifaceted and have been linked to monocytic differentiation, reprograming mitochondrial metabolism, and increased expression of BRD4 and other BCL2-like proteins, etc. Our previous studies demonstrated that specific RNA cytosine methyltransferase, namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018) ( Figure 1A). The Cancer Genome Atlas Program (TCGA) data demonstrated that elevated expression of NSUN1 and NSUN2 is correlated with significantly shorter patients' survival in almost all cancer subtypes. However, due to the lack of the crystal structures of NSUN1 and NSUN2 proteins, no NSUN1/2-targeting therapeutics have been developed yet. Methods: AI/supercomputing design of NSUN1/2-targeting drug libraries and analysis of structure activity relationship (SAR). Newly developed functional technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). NSUN1/2-targeting functional drug screening (NSUN1/2-FDS) assay and drug growth inhibition (MTT) assay. H&E histology and immunohistochemistry (IHC). Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results: Identification of several ligan-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of small-molecule libraries to targe these ligand-binding surfaces/modules with AI/supercomputing structural prediction and ligand-receptor interaction simulation. Identification of several effective small-molecule inhibitors of NSUN1 and NSUN2 by functional screening of the AI-designed drug libraries in venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. Defining the binding modules of the identified NSUN1/2 inhibitors and selecting lead inhibitors through supercomputer/AI-directed SAR analysis. Elucidation of the action mechanisms of NSUN1/2 lead inhibitors thorough disruption of NSUN1/2-mediated ACS in the nucleus and translational complexes (TLC) in the ribosomes and mitochondria ( Figure 1A). In vivo validation of the efficacies of the identified lead inhibitors using a syngeneic AML mouse model. As shown in Figure 1B, NSUN1-17, one of the lead inhibitors, efficiently eliminated the AML cells/blasts from the blood vessels and parenchyma of the lungs of the syngeneic AML mice. In addition, our siRNA knock-down experiments demonstrated that downregulation of NSUN1/2 expression re-sensitizes venetoclax-resistant monocytic leukemia cells to a low dose of venetoclax. Our NIH/3T3 soft-agarose assay confirmed the oncogenic transformation capability of NSUN1 and NSUN2. We have also successfully established the homozygous ( Nsun2-/-) mouse model with markedly reduced body weight, but a normal lifespan, compared to the heterozygous mice ( Nsun2+/-). Conclusion: By leveraging the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in maintaining oncogenic transcription and translation as well as venetoclax resistance in AML, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancers.

Targeting the Cgas-Sting Pathway Inhibits the Progression and Enhances the Chemotherapy Efficacy of Peripheral T-Cell Lymphoma

Objective Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. Owing to its complex clinicopathological characteristics and diagnostic difficulties, the underlying molecular mechanism leading to the high invasiveness and rapid progression remains significantly unidentified. The efficacy of current first-line treatment is dismal, novel agents and therapeutic strategies are urgently needed to improve the prognosis of PTCL patients, especially for relapsed/refractory (RR) ones. Recent studies have indicated a close association between the cGAS-STING pathway and tumor promotion, which may provide new therapeutic targets. Herein, we investigated the role of the cGAS-STING pathway and its underlying mechanism in PTCL progression. Methods Firstly, we conducted single-cell RNA sequencing (scRNA-seq) to examine cellular identity and investigate the expression heterogeneity of cGAS-STING pathway. The cGAS-STING pathway score (CSPS) was calculated based on the expression of seven marker genes of this pathway. Immunofluorescence was performed to detect cGAS and STING expression in PTCL tissue microarray containing 35 PTCL samples and 8 normal lymph node biopsies. To assess the tumor-killing activity of cGAS-STING pathway inhibitors, here we tested five drugs including cGAS inhibitors G150 and G140, STING inhibitor H151, TANK binding kinase-1 (TBK1) inhibitor TBK1/Ikkε-IN-5 and signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic. Protein mass spectrometry was used to evaluate the protein changes in PTCL cell lines after G150 treatment. Next, we established a xenograft model to test the anti-tumor effect of G150 and TG003 (an inhibitor of CLK1) single-agent or combination therapy in vivo. Furthermore, we applied single-cell dynamic RNA sequencing to investigate the mechanism on the synergistic effect of TG003 and G150 in inhibition of cell proliferation. CCK8 and flow cytometric analysis were performed to detected the cellular viability and the apoptotic rate with the drug combination compared with single-drug treatment. Results scRNA-seq and showed the cGAS-STING pathway was overexpressed in PTCL with high heterogeneity and could be used as a prognostic marke (Fig. A-D). cGAS expression was positively correlated with DNA repair. cGAS inhibition effectively suppressed tumor growth via cGAS-STING-RELB-STAT3 signaling pathway and impairing DNA damage repair in PTCL. G150 treatment led to a concentration-dependent DNA damage (Fig. E-G). Moreover, single-cell dynamic RNA sequencing revealed that TG003 functions through inducing the reduction of proliferation-related nascent RNAs on the basis of G150 treatment (Fig. H,I). CLK1 played a critical role in the survival of residual tumor cells after treatment with cGAS inhibitors, and CLK1 suppression could enhance the sensitivity to cGAS inhibitors (Fig. J). In first-line therapy, chemotherapy-induced DNA damage activated the cGAS-STING pathway, which may ultimately lead to relapse or progression. cGAS inhibitors could synergize with chemotherapeutic agents in tumor killing (Fig. K-N). Fig. O summarized the oncogenic role of cGAS-STING pathway and the interaction between Tfh cells and B cells in AITL. Conclusion Herein, we demonstrated for the first time that the activated cGAS-STING pathway plays an oncogenic role in PTCL progression, in part by enhancing DNA repair and proliferation, which may also trigger chemotherapy resistance and ultimate relapse. CLK1 may serve as an indicator for the sensitivity to cGAS inhibitors. This may partly unveil the deficiency of first-line therapy and lay foundation for clinical application of cGAS-STING pathway-associated inhibitors.

Transcriptional responses of cancer cells to heat shock-inducing stimuli involve amplification of robust HSF1 binding

Responses of cells to stimuli are increasingly discovered to involve the binding of sequence-specific transcription factors outside of known target genes. We wanted to determine to what extent the genome-wide binding and function of a transcription factor are shaped by the cell type versus the stimulus. To do so, we induced the Heat Shock Response pathway in two different cancer cell lines with two different stimuli and related the binding of its master regulator HSF1 to nascent RNA and chromatin accessibility. Here, we show that HSF1 binding patterns retain their identity between basal conditions and under different magnitudes of activation, so that common HSF1 binding is globally associated with distinct transcription outcomes. HSF1-induced increase in DNA accessibility was modest in scale, but occurred predominantly at remote genomic sites. Apart from regulating transcription at existing elements including promoters and enhancers, HSF1 binding amplified during responses to stimuli may engage inactive chromatin.

Nascent ribosomal RNA act as surfactant that suppresses growth of fibrillar centers in nucleolus

Liquid-liquid phase separation (LLPS) has been thought to be the biophysical principle governing the assembly of the multiphase structures of nucleoli, the site of ribosomal biogenesis. Condensates assembled through LLPS increase their sizes to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their sizes do not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the FC growth, we here construct a minimal theoretical model by taking into account nascent pre-rRNAs tethered to FC surfaces by RNA polymerase I. The prediction of this theory was supported by our experiments that quantitatively measure the dependence of the size of FCs on the transcription level. This work sheds light on the role of nascent RNAs in controlling the size of nuclear bodies.

Exosome complex components 1 and 2 are vital for early mammalian development

Exosome Complex Components 1 and 2 (EXOSC1 and 2) are two proteins in the RNA Exosome complex whose main function is 5’ → 3’ RNA degradation and processing. The RNA exosome complex is comprised of nine subunits that form two separate components: the S1/KH cap and the PH-core. EXOSC1 and 2 are both part of the S1/KH cap and are involved in binding nascent RNA. As part of a systemic characterization of early lethal alleles produced by the Knockout Mouse Project, we have examined Exosc1 and Exosc2 homozygous null (mutant) embryos to determine developmental and molecular phenotypes of embryos lacking their functions. Our studies reveal that Exosc1 null embryos implant and form an egg cylinder but are developmentally delayed and fail to initiate gastrulation by embryonic day 7.5. In contrast, Exosc2 null embryos are lethal during peri-implantation stages, and while they do form a morphologically normal blastocyst at E3.5, they cannot be recovered at post-implantation stages. We show the absence of stage-specific developmental and altered lineage-specification in both Exosc1 and Exosc2 mutant embryos and conclude that these genes are essential for the successful progression through early mammalian development.

ERNA co-expression network uncovers TF dependency and convergent cooperativity

Enhancer RNAs (eRNAs) are non-coding RNAs produced by transcriptional enhancers that are highly correlated with their activity. Using a capped nascent RNA sequencing (PRO-cap) dataset in human lymphoblastoid cell lines across 67 individuals, we identified inter-individual variation in the expression of over 80 thousand transcribed transcriptional regulatory elements (tTREs), in both enhancers and promoters. Co-expression analysis of eRNAs from tTREs across individuals revealed how enhancers are associated with each other and with promoters. Mid- to long-range co-expression showed a distance-dependent decay that was modified by TF occupancy. In particular, we found a class of “bivalent” TFs, including Cohesin, that both facilitate and isolate the interaction between enhancers and/or promoters, depending on their topology. At short distances, we observed strand-specific correlations between nearby eRNAs in both convergent and divergent orientations. Our results support a cooperative model of convergent eRNAs, consistent with eRNAs facilitating adjacent enhancers rather than interfering with each other. Therefore, our approach to infer functional interactions from co-expression analyses provided novel insights into the principles of enhancer interactions as a function of distance, orientation, and binding landscapes of TFs.

PRO-IP-seq tracks molecular modifications of engaged Pol II complexes at nucleotide resolution

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we develop Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq), which double-selects nascent RNAs and transcription complexes, and track phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncover precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide (+1 nt), through early (+18 to +30 nt) and late (+31 to +60 nt) promoter-proximal pause, and into productive elongation. Pol II CTD is predominantly unphosphorylated from initiation until the early pause-region, whereas serine-2- and serine-5-phosphorylations are preferentially deposited in the later pause-region. Upon pause-release, serine-7-phosphorylation rapidly increases and dominates over the region where Pol II assembles elongation factors and accelerates to its full elongational speed. Interestingly, tracking CTD modifications upon heat-induced transcriptional reprogramming demonstrates that Pol II with phosphorylated CTD remains paused on thousands of heat-repressed genes. These results uncover dynamic Pol II regulation at rate-limiting steps of transcription and provide a nucleotide-resolution technique for tracking composition of engaged transcription complexes.

The Integrator Complex Is Essential for Erythropoiesis

Erythropoiesis is a finely orchestrated process that involves the generation of a ~2.5 million red blood cells per second to maintain homeostasis and prevent anemia. During terminal maturation, erythroid precursors upregulate erythroid-specific genes while silencing non-erythroid genes in the setting of a cell that is rapidly dividing, and nucleus that is condensing in preparation for enucleation. Our group has shown that regulation of RNAPII activity is an essential determinant of erythroid cell function. During terminal erythroid maturation, RNA polymerase II (RNAPII) levels decline dramatically, becoming a scarce resource allocated to erythroid genes, such as alpha and beta globin (Murphy Blood 2021). The mechanism(s) by which RNAPII is removed from genes unnecessary to erythroid differentiation and shunted to produce mRNA essential for red blood cell function are unknown. The Integrator Complex (INT) is a modular and multi-subunit machinery that binds to paused RNAPII and promotes promoter-proximal termination. INT is highly expressed in maturing erythroid cells, although little is known about the function of INT during erythropoiesis. We hypothesize that INT facilitates promoter-proximal termination at non-erythroid genes to maintain RNAPII availability during terminal erythroid maturation. The balance between kinase and phosphatase activity is a key regulator of RNAPII activity. Phosphorylation of the c-terminal domain of RNAPII by pTEFb facilitates RNAPII pause release, and promotes active transcription. Alternatively, subunit 8 of INT (INTS8) can dephosphorylate the c-terminal domain of RNAPII through association with protein phosphatase 2a (PP2A) (Huang Mol Cell 2020), enforcing RNAPII pausing and antagonizing pTEFb activity. Integrator subunit 11 then cleaves the nascent RNA causing promoter-proximal transcriptional termination (Wu PNAS 2017). To gain insights into the function of INT in erythropoiesis, we utilized the RNP method of Crispr/Cas 9 genome editing (Gundry Cell Reports 2016) to delete INTS8 from CD34+ HSPCs, and then subjected them to erythroid differentiation and colony forming assays. Disruption of INTS8 significantly impaired erythroid proliferation and viability. It also delayed erythroid maturation, evident by delayed GPA expression, less mature morphology, and lower rates of benzidine positivity. Moreover, INTS8 disruption led to a decline in erythroid colony-forming ability and decreased expression of alpha and beta globin. Together these data indicate that INT function is essential for erythropoiesis. Integrator subunit 1 (INTS1) serves as a molecular scaffold for the complex, and can be used as an indicator of INT levels and occupancy. Western blotting demonstrated that INTS1 protein increased after CD36 selection and remained high throughout terminal erythroid maturation. To delineate the genomic targets of INT we performed CUT&RUN for INTS1 on day 7 and day 9 of erythroid culture following CD36 selection (as described in Gautier, Cell Reports 2016), corresponding to the basophilic and polychromatic stages of erythroid maturation. Approximately 50% of sites of INT occupancy were located at transcription start sites at both time points. Motif analyses of sites of INTS1 occupancy were enriched for transcription factors involved in signaling, including STAT5 (p1e-189) and SMAD2 (p1e-170), and INTS1 was present at several known STAT5 target genes including MYC and CCNB1. Interestingly, ~74% of genes with stably paused RNAPII, as indicated by a pausing index (PI) >4, were occupied by INTS1. Further, the level of INTS1 occupancy was significantly higher at genes with a PI >4 than genes with PI <4. The genomic occupancy of INTS increased from day 7 to day 9. Transcription start sites that gained INTS1 occupancy during maturation lost RNPII, suggesting a role for INT in promoting RNAPII eviction at these loci. Collectively, our findings identify a critical role for INT in governing erythroid maturation and gene expression, and provide novel insights into the transcriptional regulation of erythropoiesis.

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription.

Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage, and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the SPT5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of noncoding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from, Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.