Nascent RNA Transcripts Research Articles

Cockayne syndrome B protein is implicated in transcription and associated chromatin dynamics in homeostatic and genotoxic conditions.

The integrity of the actively transcribed genome against helix-distorting DNA lesions relies on a multilayered cellular response that enhances Transcription-Coupled Nucleotide Excision Repair (TC-NER). When defective, TC-NER is causatively associated with Cockayne-Syndrome (CS), a rare severe human progeroid disorder. Although the presence of unresolved transcription-blocking lesions is considered a driver of the aging process, the molecular features of the transcription-driven response to genotoxic stress in CS-B cells remain largely unknown. Here, an in-depth view of the transcriptional and associated chromatin dynamics that occur in CS-B cells illuminates the role of CSB therein. By employing high-throughput genome-wide approaches, we observed that absence of a functional CSB protein results in a delay in transcription progression, more positioned +1 nucleosomes, and less dynamic chromatin structure, compared to normal cells. We found that early after exposure to UV, CS-B cells released RNA polymerase II (RNAPII) from promoter-proximal pause sites into elongation. However, the magnitude of this response and the progression of RNAPII were reduced compared to normal counterparts. Notably, we detected increased post-UV retainment of unprocessed nascent RNA transcripts and chromatin-associated elongating RNAPII molecules. Contrary to the prevailing models, we found that transcription initiation is operational in CS-B fibroblasts early after UV and that chromatin accessibility showed a marginal increase. Our study provides robust evidence for the role of CSB in shaping the transcription and chromatin landscape both in homeostasis and in response to genotoxic insults, which is independent of its known role in TC-NER, and which may underlie major aspects of the CS phenotype.

8530 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells

Abstract Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None. Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses. Presentation: 6/3/2024

9273 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells

Abstract Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None. Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses. Presentation: 6/3/2024

Spectral neural approximations for models of transcriptional dynamics

The advent of high-throughput transcriptomics provides an opportunity to advance mechanistic understanding of transcriptional processes and their connections to cellular function at an unprecedented, genome-wide scale. These transcriptional systems, which involve discrete stochastic events, are naturally modeled using chemical master equations (CMEs), which can be solved for probability distributions to fit biophysical rates that govern system dynamics. While CME models have been used as standards in fluorescence transcriptomics for decades to analyze single-species RNA distributions, there are often no closed-form solutions to CMEs that model multiple species, such as nascent and mature RNA transcript counts. This has prevented the application of standard likelihood-based statistical methods for analyzing high-throughput, multi-species transcriptomic datasets using biophysical models. Inspired by recent work in machine learning to learn solutions to complex dynamical systems, we leverage neural networks and statistical understanding of system distributions to produce accurate approximations to a steady-state bivariate distribution for a model of the RNA life cycle that includes nascent and mature molecules. The steady-state distribution to this simple model has no closed-form solution and requires intensive numerical solving techniques: our approach reduces likelihood evaluation time by several orders of magnitude. We demonstrate two approaches, whereby solutions are approximated by 1) learning the weights of kernel distributions with constrained parameters or 2) learning both weights and scaling factors for parameters of kernel distributions. We show that our strategies, denoted by kernel weight regression and parameter-scaled kernel weight regression, respectively, enable broad exploration of parameter space and can be used in existing likelihood frameworks to infer transcriptional burst sizes, RNA splicing rates, and mRNA degradation rates from experimental transcriptomic data.

Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.

Regulation of nuclear transcription by mitochondrial RNA in endothelial cells.

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

SAT634 The Androgen Receptor Does Not Directly Regulate The Transcription Of DNA Damage Response Genes

Abstract Disclosure: S. Hasterok: None. T.G. Scott: None. D.G. Roller: None. A. Spencer: None. A.B. Dutta: None. K. Sathyan: None. D.E. Frigo: None. M.J. Guertin: None. D. Gioeli: None. The clinical success of combined androgen deprivation therapy (ADT) and radiation therapy (RT) in prostate cancer (PCa) created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in PCa cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at steady state in asynchronously growing PCa cells. Our data indicate that the clinical benefit of ADT and RT is not due to the direct regulation of DDR gene transcription by AR and that the clinical benefit of this therapeutic combination is instead likely due to both treatments independently reducing tumor cell fitness. Presentation: Saturday, June 17, 2023

Genome-wide measurement of RNA dissociation from chromatin classifies transcripts by their dynamics and reveals rapid dissociation of enhancer lncRNAs.

Long non-coding RNAs (lncRNAs) are involved in gene expression regulation in cis. Although enriched in the cell chromatin fraction, to what degree this defines their regulatory potential remains unclear. Furthermore, the factors underlying lncRNA chromatin tethering, as well as the molecular basis of efficient lncRNA chromatin dissociation and its impact on enhancer activity and target gene expression, remain to be resolved. Here, we developed chrTT-seq, which combines the pulse-chase metabolic labeling of nascent RNA with chromatin fractionation and transient transcriptome sequencing to follow nascent RNA transcripts from their transcription on chromatin to release and allows the quantification of dissociation dynamics. By incorporating genomic, transcriptomic, and epigenetic metrics, as well as RNA-binding protein propensities, in machine learning models, we identify features that define transcript groups of different chromatin dissociation dynamics. Notably, lncRNAs transcribed from enhancers display reduced chromatin retention, suggesting that, in addition to splicing, their chromatin dissociation may shape enhancer activity.

SpinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Epigenetic regulation of SARS-Cov-2 spike protein modulates cellular viability and nascent RNA transcription in neuronal cells

Epigenetic regulation of SARS-Cov-2 spike protein modulates cellular viability and nascent RNA transcription in neuronal cells

Single-cell detection of primary transcripts, their genomic loci and nuclear factors by 3D immuno-RNA/DNA FISH in T cells.

Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture ("3C"-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.

Butt-seq: a new method for facile profiling of transcription.

A wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, Butt-seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that Butt-seq is a simple and powerful technique to analyze transcription at a high level of resolution.

Abstract 251: Regulation Of Endotheliopathy By Chromatin-associated Long Non-coding RNA

Lining the critical interface between circulating blood and vascular wall, endothelial cells (ECs) play vital functions in health and disease. The optimal gene expression in ECs is essential to maintain endothelial homeostasis, and its dysregulation can lead to EC dysfunction or endotheliopathy, a common mechanism underlying many metabolic and cardiovascular diseases. Through chromatin-RNA contact profiling, we have identified several chromatin-associated long non-coding RNA (lncRNAs) that play essential roles in the regulation of endothelial function, including endothelial nitric oxide production, angiogenesis, endothelial-mesenchymal transition, and inflammatory activation. These include nuclear genome-transcribed lncRNAs, e.g. lncRNA-enhancing endothelial nitric oxide synthase (LEENE) and LINC00607 that promotes expression of fibronectin and plasminogen activator inhibitor-1. Our recent study has also revealed that mitochondria transcribed RNAs (mtRNA) are pervasively attached to nuclear chromatin and such mtRNA-association with chromatin is increased by pro-inflammatory, diabetes-mimicking stimuli. Suppression of a mitochondrial lncRNA in ECs attenuates nascent RNA transcription from the nuclear genome, including that of critical genes promoting pro-inflammatory activation. Finally, we show increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors. Together, these data highlight an emerging role of chromatin-associated lncRNAs in regulating endothelial biology.

MP01-05 REVEALING NEW PARADIGMS IN MALE INFERTILITY THROUGH THE LENS OF STRESS-INDUCED GRANULES

You have accessJournal of UrologyCME1 Apr 2023MP01-05 REVEALING NEW PARADIGMS IN MALE INFERTILITY THROUGH THE LENS OF STRESS-INDUCED GRANULES Joel E. Berends, Vijaya L. Dommeti, Jeffrey N. Dudley, Stephanie Carras, Catherine S. Nam, Tyler Robinson, Evan Keller, James M. Dupree, and Sethu Pitchiaya Joel E. BerendsJoel E. Berends More articles by this author , Vijaya L. DommetiVijaya L. Dommeti More articles by this author , Jeffrey N. DudleyJeffrey N. Dudley More articles by this author , Stephanie CarrasStephanie Carras More articles by this author , Catherine S. NamCatherine S. Nam More articles by this author , Tyler RobinsonTyler Robinson More articles by this author , Evan KellerEvan Keller More articles by this author , James M. DupreeJames M. Dupree More articles by this author , and Sethu PitchiayaSethu Pitchiaya More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Infertility affects one in six couples, with male factor contributing to ∼50% of cases, wherein 30% of these men have idiopathic infertility. Several male infertility etiologies are thought to exert heat stress and impact spermatogenesis. Upon heat shock, mammalian cells invoke evolutionarily conserved corrective measures that include induction of chaperone genes by heat shock factor (HSF), a transcription factor, and concentration of RNAs and proteins within membraneless, subcellular granules. Investigations on how HSFs mount response to stress and how these granules impact spermatogenesis are lacking. We aimed to characterize these stress response mechanisms in cultured cell and murine models. METHODS: We modeled various (e.g. heat, oxidative, hyperosmotic) stresses in human and mouse cell lines. Immunolabeling followed by super-resolution microscopy was used to visualize HSF granules. Metabolic RNA labeling coupled with Click Chemistry was used to visualize nascent RNA transcripts. Cleavage Under Targets and Tagmentation (CUT&Tag), an epigenomic technique, and Bru-Seq, a nascent transcriptomic technique, were used to probe HSF binding and newly formed RNA transcripts, respectively. Finally, we modeled acute (30 min) heat stress in mice and assessed testicular pathology by Johnson scoring. RESULTS: All stresses induced the formation of nuclear HSF granules and these granules colocalized with nascent RNA. Strikingly, CUT&Tag showed that HSF1 had stress-specific genomic occupancy, even at chaperone gene loci, although Bru-seq revealed the generic upregulation of all chaperone genes in all stresses. In addition, we discovered stress-specific transcriptional programs, that modulate cell fate. Finally, the testes of mice treated with 42°C heat stress had lower spermatogenesis activity than control mice. CONCLUSIONS: Epigenomic, nascent transcriptomic, and super-resolution microscopy data has revealed that HSFs drive stress-dependent epigenomic and transcriptomic programs by forming stress-induced granules. Our findings pave the way to understand mechanistic drivers of male infertility and can identify biomarkers that stratify and possibly explain idiopathic infertility. Source of Funding: This work was supported in part by the UCF PSRTA, Dornier MedTech, and the UM Urology Catalyst and Incubator Awards. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e3 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joel E. Berends More articles by this author Vijaya L. Dommeti More articles by this author Jeffrey N. Dudley More articles by this author Stephanie Carras More articles by this author Catherine S. Nam More articles by this author Tyler Robinson More articles by this author Evan Keller More articles by this author James M. Dupree More articles by this author Sethu Pitchiaya More articles by this author Expand All Advertisement PDF downloadLoading ...

Profiling the Binding Activities of Peptides and Inhibitors to the U2 Auxiliary Factor Homology Motif (UHM) Domains.

RNA splicing is a biological process to generate mature mRNA (mRNA) by removing introns and annexing exons in the nascent RNA transcript and is executed by a multiprotein complex called spliceosome. To aid RNA splicing, a class of splicing factors use an atypical RNA recognition domain (UHM) to bind with U2AF ligand motifs (ULMs) in proteins to form modules that recognize splice sites and splicing regulatory elements on mRNA. Mutations of UHM containing splicing factors have been found frequently in myeloid neoplasms. To profile the selectivity of UHMs for inhibitor development, we established binding assays to measure the binding activities between UHM domains and ULM peptides and a set of small-molecule inhibitors. Additionally, we computationally analyzed the targeting potential of the UHM domains by small-molecule inhibitors. Our study provided the binding assessment of UHM domains to diverse ligands that may guide development of selective UHM domain inhibitors in the future.

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3’-end processing machineries.

Cytokine-induced transcriptional memory is evident in the kinetics of transcriptional bursts.

During the onset of cytokine-induced transcriptional memory, chromatin-associated proteins, modifications of DNA, and histones act in concert to control gene expression and maintain transcriptional states over several generations. This process is well-exemplified by the transient and amplified expression of pro-inflammatory GBP gene products, which have been shown to exhibit cytokine-induced transcriptional memory under the control of cohesin. However, the effects of transcriptional memory on the transcriptional kinetics of GBP genes remain largely unexplored. Here, we characterize the first-order transcriptional kinetics of GBP5 by modeling ensemble snapshots of nascent and mature RNA transcript counts acquired using fluorescence in-situ hybridization. Our model rigorously infers kinetic parameters for a multi-state model of promoter activation, transcription, and mature RNA nuclear export during cytokine induction, and illustrates the kinetic effects of cytokine-induced transcriptional memory. To perform robust parameter inference and prevent overfitting, we utilize a Sequential Monte Carlo (SMC) algorithm to compute the full posterior distribution on the first-order rate constants. Bayesian model selection reveals modifications to the GBP5 transcriptional state space in HeLa cells primed with interferon-gamma.

Rotation manipulation of single-molecule magnetic trapping and gene transcription regulation dynamics

Gene transcription regulation is a key step for gene expression in all organisms and responsible for the transmission of genetic information and genome integrity. As one of the most important mechanisms in gene transcription, an RNA polymerase (RNAP) specifically interacts with and unwinds genome DNA to form a transcription bubble where a nascent RNA transcript is polymerized, taking one of the unwound DNA strands as its template. The RNAP translocates along the DNA to transcribe the whole gene by carrying the transcription bubble. In such a way, an RNAP completes its biological task of gene expression by physically acting as a molecular machinery. Thus, an RNAP molecule can be considered as a research object for physicists who are willing to uncover the mechanisms of life processes in a physical view. To achieve this, single-molecule method has been invented and used widely. As one of these methods, single-molecule magnetic trapping manipulates biological molecules by applying extension force or torque to the magnetic beads tethered through biological molecule to pre-coated glass surfaces by manipulating the position or rotation of a pair of magnets. A linear DNA molecule can be manipulated in such a way to generate plectonemes, i.e. DNA supercoils, under an extension force of 0.3 pN (1 pN = 10<sup>–12</sup> N), possessing the feature that the number of unwound base pairs of a supercoiled DNA can be observed by the changes in the number of supercoils reflected by the DNA extension changes. Thus, the DNA unwound by RNAP, i.e. the transcription bubble, during transcription can be observed in this way. By monitoring the kinetics of the transcription bubble in real time, this method thus allows single-molecule detection with single-base resolution and a high-throughput data collection fashion in the kinetic studies of transcription. Owing to the advantages of the manipulation of DNA supercoils with single-molecule magnetic trapping, one can mimic the mechanistic feature of DNAs in vivo and characterize the kinetics of transcription under such conditions. This method can also be combined with single-molecule fluorescence method which can be applied to studying the mechanism of transcription regulation while monitoring the behaviors of fluorescently labeled biological molecules that interact with functional RNAP molecules, providing examples for studying the mechanisms of transcription regulations in more complex systems.

Genome-wide Mapping of 5'-monophosphorylated Ends of Mammalian Nascent RNA Transcripts.

In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5'-monophosphorylated (5'-PO4) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5'-PO4 ends can be a target of further processing or be subjected to 5'-3' exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5'-PO4 ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5'-PO4 RNA ends with the 3'-hydroxyl group of a 5' adaptor (5'-PO4 Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5'-PO4 groups precisely. This protocol successfully detects the 5'monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5'-3' exonuclease Xrn2, 5'-PO4 Bru-Seq maps the 5' splice sites of debranched introns and mRNA and tRNA 3' end processing sites cleaved by CPSF73 and RNaseZ, respectively. Key features • Metabolic labeling for brief periods with bromouridine focuses the analysis of 5'-PO4 RNA ends on the population of nascent transcripts that are actively transcribed. • Detects 5'-PO4 RNA ends on nascent transcripts produced by all RNA polymerases. • Detects 5'-PO4 RNA ends at single nucleotide resolution.

Increased Expression of the RBPMS Splice Variants Inhibits Cell Proliferation in Ovarian Cancer Cells.

RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of family proteins that bind to nascent RNA transcripts and regulate their splicing, localization, and stability. Evidence indicates that RBPMS controls the activity of transcription factors associated with cell growth and proliferation, including AP-1 and Smads. Three major RBPMS protein splice variants (RBPMSA, RBPMSB, and RBPMSC) have been described in the literature. We previously reported that reduced RBPMS levels decreased the sensitivity of ovarian cancer cells to cisplatin treatment. However, little is known about the biological role of the RBPMS splice variants in ovarian cancer cells. We performed RT-PCR and Western blots and observed that both RBPMSA and RBPMSC are reduced at the mRNA and protein levels in cisplatin resistant as compared with cisplatin sensitive ovarian cancer cells. The mRNA and protein levels of RBPMSB were not detectable in any of the ovarian cancer cells tested. To better understand the biological role of each RBPMSA and RBPMSC, we transfected these two splice variants in the A2780CP20 and OVCAR3CIS cisplatin resistant ovarian cancer cells and performed cell proliferation, cell migration, and invasion assays. Compared with control clones, a significant reduction in the number of colonies, colony size, cell migration, and invasion was observed with RBPMSA and RBPMSC overexpressed cells. Moreover, A2780CP20-RBPMSA and A2780CP20-RBPMSC clones showed reduced senescence-associated β-galactosidase (β-Gal)-levels when compared with control clones. A2780CP20-RBPMSA clones were more sensitive to cisplatin treatment as compared with A2780CP20-RBPMSC clones. The A2780CP20-RBPMSA and A2780CP20-RBPMSC clones subcutaneously injected into athymic nude mice formed smaller tumors as compared with A2780CP20-EV control group. Additionally, immunohistochemical analysis showed lower proliferation (Ki67) and angiogenesis (CD31) staining in tissue sections of A2780CP20-RBPMSA and A2780CP20-RBPMSC tumors compared with controls. RNAseq studies revealed many common RNA transcripts altered in A2780CP20-RBPMSA and A2780CP20-RBPMSC clones. Unique RNA transcripts deregulated by each RBPMS variant were also observed. Kaplan-Meier (KM) plotter database information identified clinically relevant RBPMSA and RBPMSC downstream effectors. These studies suggest that increased levels of RBPMSA and RBPMSC reduce cell proliferation in ovarian cancer cells. However, only RBPMSA expression levels were associated with the sensitivity of ovarian cancer cells to cisplatin treatment.