Nascent Transcriptome Research Articles

Study of the RNA splicing kinetics via in vivo 5-EU labeling.

Splicing is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency, giving rise to alternative splicing products. At the same time, splice sites might be used at a variable rate. We used 5-ethynyl uridine labeling to sequence a nascent transcriptome of HeLa cells and deduced the rate of splicing for each donor and acceptor splice site. The following correlation analysis showed a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as a splicing rate decrease with a decreased complementarity of the donor splice site to U1 and acceptor sites to U2 snRNAs. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of the acceptor splicing site utilization, or the differences in splicing rate between long, short, and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the poly(A) site, which might be explained by the cooperativity of the splicing and polyadenylation. Additional analysis of splicing kinetics of SF3B4 knockdown cells suggested the impairment of a U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field, as they provide general splicing rate dependencies as well as help to justify the existence of slowly removed splice sites.

SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway

The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.

Post-transcriptional regulation shapes the transcriptome of quiescent budding yeast.

To facilitate long-term survival, cells must exit the cell cycle and enter quiescence, a reversible non-replicative state. Budding yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence is unknown. By investigating the nascent transcriptome, we identified over a thousand noncoding RNAs in quiescent and G1 yeast cells, and found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. Additionally, both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that, in quiescence, the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs. RNA sequencing through quiescent entry revealed two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, thereby facilitating efficient quiescence entry. Our work identified a previously unknown key biological role for the nuclear exosome-NNS pathway in mRNA regulation and uncovered a novel layer of gene-expression control in quiescence.

Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Dissecting key regulators of transcriptome kinetics through scalable single-cell RNA profiling of pooled CRISPR screens

We present a combinatorial indexing method, PerturbSci-Kinetics, for capturing whole transcriptomes, nascent transcriptomes and single guide RNA (sgRNA) identities across hundreds of genetic perturbations at the single-cell level. Profiling a pooled CRISPR screen targeting various biological processes, we show the gene expression regulation during RNA synthesis, processing and degradation, miRNA biogenesis and mitochondrial mRNA processing, systematically decoding the genome-wide regulatory network that underlies RNA temporal dynamics at scale.

P10.35.A CDK12/CDK13 INHIBITION DISRUPTS A TRANSCRIPTIONAL PROGRAM CRITICAL FOR GLIOBLASTOMA SURVIVAL

Abstract BACKGROUND Glioblastoma is the most prevalent and aggressive malignant tumor of the central nervous system. Aberrant transcriptional programs are key to development and maintenance of cancer cells, and these can be exploited to target a specific cancer. Thus, targeting the transcriptional addiction specific for gliomas could offer a large therapeutic potential. METHODS A panel of patient-derived primary glioma stem cell (GSC) lines and glioblastoma organoids (GBO) were used to investigate the effect of transcriptional cyclin-dependent kinase (tCDK) inhibitors using a range of survival assays. A CRISPR/CAS9-based competition assay was applied to study the effect of genetic ablation of individual tCDKs. CUT&RUN was utilized to generate the genome-wide maps of the total RNA polymerase II (RNAPII) and phosphorylated species, and SLAM-seq was used to identify changes in steady-state and nascent transcriptome. Cell cycle analyses were done using EdU incorporation to mark S-phase cells. RESULTS We show that pharmacological inhibition or genetic ablation of the tCDKs, CDK12 and CDK13, markedly reduces the in vitro proliferation of GSCs and of ex vivo glioblastoma organoids as well as migratory capacity of GSCs. Using a xenograft mouse model, we demonstrate that CDK12/13 inhibition reduces tumor growth in vivo, which compares favorably with the existing chemotherapeutic treatments. Mechanistically, inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII C-terminal Domain (CTD) phosphorylation, which in turn disrupts transcription and cell cycle progression in glioblastoma cells. CONCLUSION s CDK12/CDK13 inhibition may have a large therapeutic potential for glioblastoma treatment, both alone and in combination.

Nascent transcriptome reveals orchestration of zygotic genome activation in early embryogenesis

Early embryo development requires maternal-to-zygotic transition, during which transcriptionally silent nuclei begin widespread gene expression during zygotic genome activation (ZGA).1-3 ZGA is vital for early cell fating and germ-layer specification,3,4 and ZGA timing is regulated by multiple mechanisms.1-5 However, controversies remain about whether these mechanisms are interrelated and vary among species6-10 and whether the timing of germ-layer-specific gene activation is temporally ordered.11,12 In some embryonic models, widespread ZGA onset is spatiotemporally graded,13,14 yet it is unclear whether the transcriptome follows this pattern. A major challenge in addressing these questions is to accurately measure the timing of each gene activation. Here, we metabolically label and identify the nascent transcriptome using 5-ethynyl uridine (5-EU) in Xenopus blastula embryos. We find that EU-RNA-seq outperforms total RNA-seq in detecting the ZGA transcriptome, which is dominated by transcription from maternal-zygotic genes, enabling improved ZGA timing determination. We uncover discrete spatiotemporal patterns for individual gene activation, a majority following a spatial pattern of ZGA that is correlated with a cell size gradient.14 We further reveal that transcription necessitates a period of developmental progression and that ZGA can be precociously induced by cycloheximide, potentially through elongation of interphase. Finally, most ectodermal genes are activated earlier than endodermal genes, suggesting a temporal orchestration of germ-layer-specific genes, potentially linked to the spatially graded pattern of ZGA. Together, our study provides fundamental new insights into the composition and dynamics of the ZGA transcriptome, mechanisms regulating ZGA timing, and its role in the onset of early cell fating.

Metabolic incorporation of electron-rich ribonucleosides enhances APEX-seq for profiling spatially restricted nascent transcriptome

The spatial arrangement of newly synthesized transcriptome in eukaryotic cells underlies various biological processes including cell proliferation and differentiation. In this study, we combine metabolic incorporation of electron-rich ribonucleosides (e.g., 6-thioguanosine and 4-thiouridine) with a peroxidase-mediated proximity-dependent RNA labeling technique (APEX-seq) to develop a sensitive method, termed MERR APEX-seq, for selectively profiling newly transcribed RNAs at specific subcellular locations in live cells. We demonstrate that MERR APEX-seq is 20-fold more efficient than APEX-seq and offers both high spatial specificity and high coverage in mitochondrial matrix. At the ER membrane, 91% of the transcripts captured by MERR APEX-seq encode for secretory pathway proteins, thus demonstrating the high spatial specificity of MERR APEX-seq in open subcellular compartments. Application of MERR APEX-seq to the nuclear lamina of human cells reveals a local transcriptome of 1,012 RNAs, many of which encode for nuclear proteins involved in histone modification, chromosomal structure maintenance, and RNA processing.

Super resolution microscopy reveals how elongating RNA polymerase II and nascent RNA interact with nucleosome clutches.

Transcription and genome architecture are interdependent, but it is still unclear how nucleosomes in the chromatin fiber interact with nascent RNA, and which is the relative nuclear distribution of these RNAs and elongating RNA polymerase II (RNAP II). Using super-resolution (SR) microscopy, we visualized the nascent transcriptome, in both nucleoplasm and nucleolus, with nanoscale resolution. We found that nascent RNAs organize in structures we termed RNA nanodomains, whose characteristics are independent of the number of transcripts produced over time. Dual-color SR imaging of nascent RNAs, together with elongating RNAP II and H2B, shows the physical relation between nucleosome clutches, RNAP II, and RNA nanodomains. The distance between nucleosome clutches and RNA nanodomains is larger than the distance measured between elongating RNAP II and RNA nanodomains. Elongating RNAP II stands between nascent RNAs and the small, transcriptionally active, nucleosome clutches. Moreover, RNA factories are small and largely formed by few RNAP II. Finally, we describe a novel approach to quantify the transcriptional activity at an individual gene locus. By measuring local nascent RNA accumulation upon transcriptional activation at single alleles, we confirm the measurements made at the global nuclear level.

Genome-Wide Analysis of the Regulatory Landscape in Different Pre-B-ALL Subtypes

BackgroundAcute pre-B leukemia results from a failure in cell differentiation. In normal development, cells commit to their lineage and differentiated phenotype state by the activation of transcription factors (TFs), or TF modules, in a series of decision points at which a choice is made between alternative lineage fates. Previously, we showed that the ETV6-RUNX1 gene translocation that occurs in 25% of pediatric leukemia patients silences regulatory regions containing RUNX binding sites (enhancers) at target gene loci implicated in lymphoid lineage differentiation (Teppo et al. 2016). However, the genome-wide changes in enhancer activities at binding sites of other TFs functioning in B-cell differentiation remain poorly understood. Moreover, the genome-wide identification of enhancers altered during leukemogenesis is incomplete.Material & MethodsTo measure transcripts generated both at enhancer and gene regions, global run-on sequencing (GRO-seq) technique was used to measure nascent RNAs (Heinäniemi et al. 2016) from human pre-B-ALL patient samples (N=35) and several cell lines representing different subtypes. For validation, additional samples were generated from cell line models with overexpression or silencing of leukemic TFs. To this collection of 151 nascent transcriptomes, additional 92 GRO-seq samples representing different cell lineages (Liivrand et al 2017) were used to compare the leukemic cells to primary cells and other cancers. Enhancer RNA (eRNA) levels were compared at TF binding motif centered enhancer regions. TF ChIP-seq data from leukemic cells and HSC were acquired from the NCBI Gene Expression Omnibus for validation of this approach.ResultsTo gain insight into the derailed regulatory landscape in leukemic cells, we identified eRNA regions both from normal and leukemic samples and performed statistical analysis distinguishing regulatory regions with differential activity between pre-B-ALL-subtypes or compared to normal CD34+ bone marrow precursors. Next, association of the significantly altered enhancers with gene transcripts was utilized to build regulatory maps for differentially expressed gene loci. For this purpose, we developed a transcript identification and quantification pipeline suitable for detecting altered expression at different coding and non-coding RNA loci from nascent transcriptomes (Liivrand et al 2017; unpublished). To globally evaluate altered TF activities across pre-B-ALL subtypes underlying the differences observed, we present here an unbiased approach based on eRNA quantification at TF motif-centered enhancers. The analysis of eRNA levels at binding sites of TFs with established role in leukemia was used as a benchmark: we could confirm low eRNA transcription at RUNX1 binding sites in the ETV6-RUNX1 subtype, elevated eRNA levels at HOXA9 and MEIS1 binding sites in the MLL rearranged subtype, while TCF3, BCL6 and PBX1 bound sites were most active in TCF3-PBX1 samples.ConclusionsOur results provide the first genome-wide transcriptional regulatory landscape of different pre-B-ALL subtypes. We identified novel subtype-specific active enhancers and annotated regulatory non-coding regions that can be utilized to evaluate the functional impact of genetic alterations outside gene regions. DisclosuresNo relevant conflicts of interest to declare.

Acute depletion of METTL3 implicates N 6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

RNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5′ splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5′ splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5′ splice site that is suppressed in the presence of m6A and upstream of a 5′ splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.

Decoding the function of an oncogenic transcription factor: finding the first responders

Transcription factors (TFs) are frequently altered in human diseases. Identifying the direct and immediate target genes of TFs is critical to understanding their role in pathophysiology. Stengel etal. (2020) applied chemogenetic and nascent transcriptome mapping technologies to define the core gene set regulated by AML1-ETO-an oncogenic TF fusion protein frequently found in acute myeloid leukemia (AML).

Metabolic Incorporation of Electron-Rich Ribonucleosides Enhanced APEX-Seq for Profiling Spatially Restricted Nascent Transcriptome

The spatial arrangement of newly synthesized transcriptome in eukaryotic cells underlies various biological processes including cell proliferation and differentiation. In this study, we combine metabolic incorporation of electron-rich ribonucleosides (e.g. 6-thioguanosine and 4-thiouridine) with peroxidase-mediated proximity-dependent RNA labeling technique (APEX-seq) to develop a new method, called MER APEX-seq, for selectively profiling newly transcribed RNAs at specific subcellular locations in live cells. We demonstrate that MER APEX-seq is 20-fold more efficient than APEX-seq and offers both high spatial specificity and high coverage in the mitochondrial matrix. Application of MER APEX-seq to the nuclear lamina of HEK293T cells reveals a local transcriptome of 1012 RNAs, many of which encode for nuclear proteins involved in histone modification, chromosomal structure maintenance, RNA splicing and processing.

RAI1 Regulates Activity-Dependent Nascent Transcription and Synaptic Scaling

Long-lasting forms of synaptic plasticity such as synaptic scaling are critically dependent on transcription. Activity-dependent transcriptional dynamics in neurons, however, remain incompletely characterized because most previous efforts relied on measurement of steady-state mRNAs. Here, we use nascent RNA sequencing to profile transcriptional dynamics of primary neuron cultures undergoing network activity shifts. We find pervasive transcriptional changes, in which ∼45% of expressed genes respond to network activity shifts. We further link retinoic acid-induced 1 (RAI1), the Smith-Magenis syndrome gene, to the transcriptional program driven by reduced network activity. Remarkable agreement among nascent transcriptomes, dynamic chromatin occupancy of RAI1, and electrophysiological properties of Rai1-deficient neurons demonstrates the essential roles of RAI1 in suppressing synaptic upscaling in the naive network, while promoting upscaling triggered by activity silencing. These results highlight the utility of bona fide transcription profiling to discover mechanisms of activity-dependent chromatin remodeling that underlie normal and pathological synaptic plasticity.

Metabolic Labeling of RNAs Uncovers Hidden Features and Dynamics of the Arabidopsis Transcriptome

Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants. Here, we demonstrate that RNA labeling with the modified, nontoxic uridine analog 5-ethynyl uridine (5-EU) in Arabidopsis (Arabidopsis thaliana) seedlings provides insight into plant transcriptome dynamics. Pulse labeling with 5-EU revealed nascent and unstable RNAs, RNA processing intermediates generated by splicing, and chloroplast RNAs. Pulse-chase experiments with 5-EU allowed us to determine RNA stabilities without the need for chemical transcription inhibitors such as actinomycin and cordycepin. Inhibitor-free, genome-wide analysis of polyadenylated RNA stability via 5-EU pulse-chase experiments revealed RNAs with shorter half-lives than those reported after chemical inhibition of transcription. In summary, our results indicate that the Arabidopsis nascent transcriptome contains unstable RNAs and RNA processing intermediates and suggest that polyadenylated RNAs have low stability in plants. Our technique lays the foundation for easy, affordable, nascent transcriptome analysis and inhibitor-free analysis of RNA stability in plants.

Imaging nascent transcription in wholemount vertebrate embryos to characterize zygotic genome activation.

A major event in early embryo development is the awakening of the embryonic genome, a process of large-scale transcriptional induction termed zygotic genome activation (ZGA). To understand how ZGA is controlled temporally and spatially, tools are required to image and quantify nascent transcription in wholemount embryos. In this chapter, we describe a metabolic labeling approach that leverages 5-ethynyl uridine (5-EU) incorporation into newly transcribed RNAs. Subsequently, click chemistry is used to conjugate these nascent transcripts to fluorophores for wholemount confocal imaging or biotin for RNA sequencing. Such an approach facilitates direct visualization of the global transcriptional state of each cell during early embryogenesis and provides a spatial map of gene expression activity. We describe this procedure for imaging nascent transcription in a vertebrate embryo Xenopus laevis, and use it as our model the onset of large-scale ZGA. Unlike cell culture systems in which 5-EU can be added to the media, metabolic labeling in Xenopus embryos requires microinjection in one-cell or two-cell stage embryos. This method is a powerful tool to quantify the nascent transcriptome at a single-cell level and to dissect mechanisms that control ZGA. We propose that this methodology can be applied broadly in other embryonic systems, and demonstrate the feasibility using zebrafish cleavage stage embryos. Finally, we demonstrate how to sequence the nascent transcriptome via 5-EU incorporation and separation of zygotic vs maternal RNAs. Altogether, our generalizable methodology will facilitate new insights into gene regulation and spatial patterning of ZGA during early embryogenesis.

Expanding the Scope of RNA Metabolic Labeling with Vinyl Nucleosides and Inverse Electron-Demand Diels-Alder Chemistry.

Optimized and stringent chemical methods to profile nascent RNA expression are still in demand. Herein, we expand the toolkit for metabolic labeling of RNA through application of inverse electron demand Diels-Alder (IEDDA) chemistry. Structural examination of metabolic enzymes guided the design and synthesis of vinyl-modified nucleosides, which we systematically tested for their ability to be installed through cellular machinery. Further, we tested these nucleosides against a panel of tetrazines to identify those which are able to react with a terminal alkene, but are stable enough for selective conjugation. The selected pairings then facilitated RNA functionalization with biotin and fluorophores. We found that this chemistry not only is amenable to preserving RNA integrity but also endows the ability to both tag and image RNA in cells. These key findings represent a significant advancement in methods to profile the nascent transcriptome using chemical approaches.

Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH

Visualization of the transcriptome and the nuclear organization in situ in individual cell is the holy grail of single cell analysis. Here, we demonstrate a multiplexed single molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hours in mouse embryonic stem cells as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extensive reprogramming of the nascent transcriptome during iPSC to hepatocyte differentiation

Hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) provide a renewable source of cells for drug discovery, disease modelling and cell-based therapies. Here, by using GRO-Seq we provide the first genome-wide analysis of the nascent RNAs in iPSCs, HLCs and primary hepatocytes to extend our understanding of the transcriptional changes occurring during hepatic differentiation process. We demonstrate that a large fraction of hepatocyte-specific genes are regulated at transcriptional level and identify hundreds of differentially expressed non-coding RNAs (ncRNAs), including primary miRNAs (pri-miRNAs) and long non-coding RNAs (lncRNAs). Differentiation induced alternative transcription start site (TSS) usage between the cell types as evidenced for miR-221/222 and miR-3613/15a/16-1 clusters. We demonstrate that lncRNAs and coding genes are tightly co-expressed and could thus be co-regulated. Finally, we identified sets of transcriptional regulators that might drive transcriptional changes during hepatocyte differentiation. These included RARG, E2F1, SP1 and FOXH1, which were associated with the down-regulated transcripts, and hepatocyte-specific TFs such as FOXA1, FOXA2, HNF1B, HNF4A and CEBPA, as well as RXR, PPAR, AP-1, JUNB, JUND and BATF, which were associated with up-regulated transcripts. In summary, this study clarifies the role of regulatory ncRNAs and TFs in differentiation of HLCs from iPSCs.

Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH

Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.