Transcription In Mammalian Cells Research Articles

Post-transcriptional splicing can occur in a slow-moving zone around the gene

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

H3K4me1 facilitates promoter-enhancer interactions and gene activation during embryonic stem cell differentiation

Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, we analyze dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of the mouse embryonic stem cells. We found that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers, induced upon neural differentiation.By contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion ofKMT2B, resulting in aggravated transcriptional defects. Our observations therefore implicate KMT2B in the catalysis of H3K4me1 at enhancers and provide additional support for an active role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells.

Programmable RNA base editing with photoactivatable CRISPR-Cas13

CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.

DNA Methylation Landscape of Peripheral T-Cell Lymphomas Not Otherwise Specified

DNA methylation is an epigenetic modification catalyzed by DNA methyltransferases that regulate gene transcription in mammalian cells. DNA methylation in gene promoters is associated with repressive histone marks and contributes to gene repression. This process is involved in various physiological processes, including development, X chromosome inactivation, genomic imprinting, differentiation, and hematopoiesis. The global, genome-wide deregulation of methylation patterns contributes to the pathogenesis of immune disorders, hematologic malignancies, and cancer. Because aberrations in DNA methylation can deregulate spatiotemporal gene expression in a cell type-specific manner, genome-wide changes in this epigenetic modification may contribute to tumor development. Specifically, the downregulation of tumor suppressors through promoter hypermethylation is a widely accepted mechanism in cancer progression. Due to its distinct patterns in cancer, DNA methylation can be used as a diagnostic and predictive biomarker. Peripheral T-cell lymphomas, Not Otherwise Specified (PTCL-NOS), are a group of heterogeneous T-cell malignancies with diverse cytogenic, molecular, and morphological phenotypes. Despite large-scale gene expression profiling, the molecular features-and DNA methylation in particular-of these rare but aggressive malignancies remain poorly understood. To better understand the molecular landscape of PTCL-NOS, we performed global methylation profiling via Whole Genome Bisulfite Sequencing on 10 lymphomas and compared those to patterns seen in normal T-cells and Anaplastic Large Cell Lymphoma (ALCL). Our analysis revealed large-scale deregulation of methylation in all tumors at levels of both differentially methylated cytosines and regions. The dominant molecular change was hypomethylation, which was, on average, 7 times more frequent than hypermethylation. Hypomethylation was not limited to repetitive elements but was also seen in all genomic elements, including promoters, enhancers, gene bodies, and exons. Out of 1,286 genes whose promoters were consistently hypomethylated in Peripheral T-cell lymphomas (PTCL), 39 genes were associated with an increase in expression, suggesting that the loss of methylation may have contributed to their deregulation. This hypomethylated and overexpressed signature contained genes that might be involved in tumor progression or maintenance, such as Rac GTPase activating protein 1 ( RACGAP1), regulator of chromosome condensation 1 ( RCC1), anillin actin-binding protein ( ANLN), and others. Out of 578 genes whose promoters were consistently hypermethylated in PTCL, the expression of 56 genes was decreased. These included genes with tumor suppressor function, such as splicing factor 3b subunit 1 ( SF3B1) and lymphoid enhancer-binding factor 1 ( LEF1). Our analysis also identified differential methylation between PTCL-NOS and ALCL in various genomic features. For example, 71 hypo- and 59 hypermethylated promoters were found only in ALCL but not PTCL-NOS. Conversely, 326 hypo- and 254 hypermethylated promoters were observed only in PTCL-NOS. Analysis of gene expression also revealed specific changes in both PTCL subtypes, manifested by the activation of different signaling pathways as identified by Ingenuity Pathway Analysis. PTCL-NOS was characterized by the suppression of the SUMOylation pathway, PTEN, and EIF2 Signaling, and the activation of NF-κB and Notch signaling. ALCLs were characterized by the suppression of the Role of CHK Proteins in Cell Cycle Control, Erythropoietin, ERBB signaling, the Coronavirus Pathogenesis pathway, IGF-1 signaling, and the activation of Glutathione-mediated Detoxification and Triacylglycerol Biosynthesis. Altogether, we identified recurrent specific methylation and expression changes that could serve as the basis for the development of tools facilitating the diagnosis and prognosis of PTCL-NOS. In addition, our data may contribute to the identification of epigenetically-regulated drivers of tumor initiation, progression, and maintenance.

A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons.

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.

Transcription factor UBF depletion in mouse cells results in downregulation of both downstream and upstream elements of the rRNA transcription network

Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing invivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling. shUBF cells display cell cycle G1 delay and reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells.

PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53

The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth.

Target Finder of Transcription Factor (TFoTF): a novel tool to predict transcription factor-targeted genes in cancer.

Transcription factors (TFs) are key players in the regulation of gene transcription in mammalian cells. Although high-throughput screening can be used to identify differentially expressed genes between comparable groups, the precision of the corresponding datasets is far from optimal. Here, we establish Target Finder of Transcription Factor (TFoTF), a method for the prediction of TF-targeted genes from genomic and cancer-related transcriptomic data. TFoTF can identify potential TF-targeted genes in large cancer datasets and efficiently estimate correlations between TFs and their targeted genes with a significant level of specificity, sensitivity, and precision. Overall, TFoTF is an easy-to-use tool that can be utilized to generate testable hypotheses in the context of cancer research projects.

Size- and Stereochemistry-Dependent Transcriptional Bypass of DNA Alkyl Phosphotriester Adducts in Mammalian Cells.

Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S P and R P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass.

A programmable system to methylate and demethylate N6-methyladenosine (m6A) on specific RNA transcripts in mammalian cells

RNA N6-methyladenosine (m6A) is the most abundant internal mRNA modification and forms part of an epitranscriptomic system that modulates RNA function. m6A is reversibly catalyzed by specific enzymes, and those modifications can be recognized by RNA-binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m6A participation in critical biological functions, this exploration has mainly been conducted through the global KO or knockdown of the writers, erasers, or readers of m6A. Consequently, there is a lack of information about the role of m6A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m6A. Here, we demonstrate a CRISPR/dCas13a-based RNA m6A editors, which can target RNAs using a single or multiple CRISPR RNA array to methylate or demethylate m6A in human 293T cells and mouse embryonic stem cells. We systematically assay its capabilities to enable the targeted rewriting of m6A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we use the system to specifically modulate m6A levels on the noncoding XIST (X-inactive specific transcript) to modulate X chromosome silencing and activation. The editors described here can be used to explore the roles of m6A in biological processes.

Structure and engineering of the minimal type VI CRISPR-Cas13bt3

SUMMARYType VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.

Next-Generation Sequencing-Based Analysis of the Effects of N1- and N6-Methyldeoxyadenosine Adducts on DNA Transcription.

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric N1- and N6-methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription. Our results revealed that 1mdA can induce multiple types of mutant transcripts and strongly inhibit DNA transcription, whereas 6mdA is a nonmutagenic DNA adduct that can exhibit a subtle but significant inhibitory effect on DNA transcription in vitro and in human cells. Moreover, our results demonstrated that the transcription-coupled nucleotide excision repair pathway is dispensable for the removal of 1mdA and 6mdA from the template DNA strand in human cells. These findings provided new important insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.

Sodium Ions as Regulators of Transcription in Mammalian Cells.

The maintenance of an uneven distribution of Na+ and K+ ions between the cytoplasm and extracellular medium is the basis for the functioning of any animal cell. Changes in the intracellular ratio of these cations occur in response to numerous stimuli and are important for the cell activity regulation. Numerous experimental data have shown that gene transcription in mammalian cells can be regulated by changes in the intracellular [Na+]i/[K+]i ratio. Here, we discuss possible mechanisms of such regulation in various cell types, with special attention to the [Ca2+]-independent signaling pathways that suggest the presence of an intracellular sensor of monovalent cations. As such sensor, we propose the secondary structures of nucleic acids called G-quadruplexes. They are widely represented in mammalian genomes and are often found in the promoters of genes encoding transcription factors.

SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells.

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone-fold domain (HFD)-containing proteins forming three histone-fold (HF) pairs, to which the double HFD-containing SUPT3H adds one HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that (i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H, (ii) SUPT3H is not essential for mESC survival, but required for their growth and self-renewal, and (iii) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.

Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor.

DNA transcription is regulated by a range of diverse mechanisms and primarily by transcription factors that recruit the RNA polymerase complex to the promoter region on the DNA. Protein binding to DNA at nearby or distant sites can synergistically affect this process in a variety of ways, but mainly through direct interactions between DNA-binding proteins. Here we show that a Transcription Activator-Like Effector (TALE), which lacks an activation domain, can enhance transcription in mammalian cells when it binds in the vicinity of and without direct interaction with several different dimeric or monomeric transcription factors. This effect was observed for several TALEs regardless of the recognition sequences and their DNA-bound orientation. TALEs can exert an effect over the distance of tens of nucleotides and it also potentiated KRAB-mediated repression. The augmentation of transcriptional regulation of another transcription factor is characteristic of TALEs, as it was not observed for dCas9/gRNA, zinc finger, or Gal4 DNA-binding domains. We propose that this mechanism involves an allosteric effect exerted on DNA structure or dynamics. This mechanism could be used to modulate transcription but may also play a role in the natural context of TALEs.

Non-coding 7S RNA inhibits transcription via mitochondrial RNA polymerase dimerization

The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both invitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.

Emerging roles of BET proteins in transcription and co-transcriptional RNA processing.

Transcription by RNA polymerase II (Pol II) gives rise to all nuclear protein-coding and a large set of non-coding RNAs, and is strictly regulated and coordinated with RNA processing. Bromodomain and extraterminal (BET) family proteins including BRD2, BRD3, and BRD4 have been implicated in the regulation of Pol II transcription in mammalian cells. However, only recent technological advances have allowed the analysis of direct functions of individual BET proteins with high precision in cells. These studies shed new light on the molecular mechanisms of transcription control by BET proteins challenging previous longstanding views. The most studied BET protein, BRD4, emerges as a master regulator of transcription elongation with roles also in coupling nascent transcription with RNA processing. In contrast, BRD2 is globally required for the formation of transcriptional boundaries to restrict enhancer activity to nearby genes. Although these recent findings suggest non-redundant functions of BRD4 and BRD2 in Pol II transcription, more research is needed for further clarification. Little is known about the roles of BRD3. Here, we illuminate experimental work that has initially linked BET proteins to Pol II transcription in mammalian cells, outline main methodological breakthroughs that have strongly advanced the understanding of BET protein functions, and discuss emerging roles of individual BET proteins in transcription and transcription-coupled RNA processing. Finally, we propose an updated model for the function of BRD4 in transcription and co-transcriptional RNA maturation. This article is categorized under: RNA Processing > 3' End Processing RNA Processing > Splicing Regulation/Alternative Splicing.

Starting the engine of the powerhouse: mitochondrial transcription and beyond.

Mitochondria are central hubs for cellular metabolism, coordinating a variety of metabolic reactions crucial for human health. Mitochondria provide most of the cellular energy via their oxidative phosphorylation (OXPHOS) system, which requires the coordinated expression of genes encoded by both the nuclear (nDNA) and mitochondrial genomes (mtDNA). Transcription of mtDNA is not only essential for the biogenesis of the OXPHOS system, but also generates RNA primers necessary to initiate mtDNA replication. Like the prokaryotic system, mitochondria have no membrane-based compartmentalization to separate the different steps of mtDNA maintenance and expression and depend entirely on nDNA-encoded factors imported into the organelle. Our understanding of mitochondrial transcription in mammalian cells has largely progressed, but the mechanisms regulating mtDNA gene expression are still poorly understood despite their profound importance for human disease. Here, we review mechanisms of mitochondrial gene expression with a focus on the recent findings in the field of mammalian mtDNA transcription and disease phenotypes caused by defects in proteins involved in this process.

Distinguishing between models of mammalian gene expression: telegraph-like models versus mechanistic models.

Two-state models (telegraph-like models) have a successful history of predicting distributions of cellular and nascent mRNA numbers that can well fit experimental data. These models exclude key rate limiting steps, and hence it is unclear why they are able to accurately predict the number distributions. To answer this question, here we compare these models to a novel stochastic mechanistic model of transcription in mammalian cells that presents a unified description of transcriptional factor, polymerase and mature mRNA dynamics. We show that there is a large region of parameter space where the first, second and third moments of the distributions of the waiting times between two consecutively produced transcripts (nascent or mature) of two-state and mechanistic models exactly match. In this region: (i) one can uniquely express the two-state model parameters in terms of those of the mechanistic model, (ii) the models are practically indistinguishable by comparison of their transcript numbers distributions, and (iii) they are distinguishable from the shape of their waiting time distributions. Our results clarify the relationship between different gene expression models and identify a means to select between them from experimental data.