38 results on '"Schwalb B"'
Search Results
2. Structure of a yeast closed complex with distorted DNA (core CCdist)
- Author
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Dienemann, C., primary, Schwalb, B., additional, Schilbach, S., additional, and Cramer, P., additional
- Published
- 2018
- Full Text
- View/download PDF
3. Structure of a yeast closed complex with distorted DNA (CCdist)
- Author
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Dienemann, C., primary, Schwalb, B., additional, Schilbach, S., additional, and Cramer, P., additional
- Published
- 2018
- Full Text
- View/download PDF
4. Periodic mRNA synthesis and degradation co-operate during cell cycle gene expression
- Author
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Eser, P., Demel, C., Maier, K. C., Schwalb, B., Pirkl, N., Martin, D. E., Cramer, P., and Tresch, A.
- Subjects
cell cycle ,gene regulation ,mRNA degradation ,mRNA turnover ,periodic transcription - Abstract
During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is achieved by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. Here, we used metabolic RNA labeling and comparative dynamic transcriptome analysis (cDTA) to derive mRNA synthesis and degradation rates every 5 min during three cell cycle periods of the yeast Saccharomyces cerevisiae. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation generally follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter. peerReviewed
- Published
- 2014
5. Dynamic transcriptome analysis (DTA)
- Author
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Schwalb, B.
- Subjects
FOS: Chemical sciences ,Dynamic transcriptome analysis, DTA, mRNA turnover, bioinformatics, synthesis and decay of mRNA - Abstract
So far, much attention has been paid to regulation of transcription. However, it has been realized that controlled mRNA decay is an equally important process. To understand the contributions of mRNA synthesis and mRNA degradation to gene regulation, we developed Dynamic Transcriptome Analysis (DTA). DTA allows to monitor these contributions for both processes and for all mRNAs in the cell without perturbation of the cellular system. DTA works by non-perturbing metabolic RNA labeling that supersedes conventional methods for mRNA turnover analysis. It is accomplished with dynamic kinetic modeling to derive the gene-specific synthesis and decay parameters. DTA reveals that most mRNA synthesis rates result in several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. In the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. In the following recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems. One of the technical obstacles of standard transcriptomics is the unknown normalization factor between samples, i.e. wild-type and mutant cells. Variations in RNA extraction efficiencies, amplification steps and scanner calibration introduce differences in the global intensity levels. The required normalization limits the precision of DTA. We have extended DTA to comparative DTA (cDTA), to eliminate this obstacle. cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. It therefore allows for direct comparison of RNA synthesis and decay rates between samples. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are five fold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4–Not complex causes decreased decay rates as expected, but also decreased synthesis rates. In this thesis, we provide a novel tool to estimate RNA synthesis and decay rates: a quantitative dynamic model to describe mRNA metabolism in growing cells to complement the biochemical protocol of DTA/cDTA. It can be applied to reveal rate changes for all kinds of perturbations, e.g. in knock-out or point mutation strains, in responses to stress stimuli or in small molecule interfering assays like treatments with miRNA or siRNA inhibitors. In doing so, we show that DTA is a valuable tool for miRNA target validation. The DTA/cDTA approach is in principle applicable to virtually every organism. The bioinformatic workflow of DTA/cDTA is implemented in the open source R/Bioconductor package DTA.
- Published
- 2012
- Full Text
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6. Cardiac tolerability of trimipramine in the elderly
- Author
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Malsch, C., primary, Jäger, K., additional, Schwalb, B., additional, and Fischer, W., additional
- Published
- 1996
- Full Text
- View/download PDF
7. O-3-4 - Cardiac tolerability of trimipramine in the elderly
- Author
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Malsch, C., Jäger, K., Schwalb, B., and Fischer, W.
- Published
- 1996
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- View/download PDF
8. Evaluation of Cereblon-Directing Warheads for the Development of Orally Bioavailable PROTACs.
- Author
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Actis M, Cresser-Brown J, Caine EA, Steger M, Aggarwal A, Mayasundari A, Murphy N, Valinciute G, Li Y, Fu X, Yang L, Marsh G, Hughes G, Karadogan B, Wheeler D, Norley M, Spain F, Urh M, Roussel MF, Schwalb B, Daub H, Riching KM, Maple H, Nishiguchi G, and Rankovic Z
- Subjects
- Humans, Administration, Oral, Animals, Mice, Adaptor Proteins, Signal Transducing metabolism, Thalidomide pharmacokinetics, Thalidomide analogs & derivatives, Thalidomide pharmacology, Thalidomide administration & dosage, Structure-Activity Relationship, Cell Line, Tumor, Rats, Antineoplastic Agents pharmacology, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents chemistry, Antineoplastic Agents administration & dosage, Proteolysis Targeting Chimera, Ubiquitin-Protein Ligases metabolism, Biological Availability
- Abstract
PROTACs usually occupy physicochemical space outside the one defined by classical drug-like molecules, which often presents considerable challenges in their optimization and development for oral administration. We have previously reported phenyl glutarimide (PG)-based BET PROTAC SJ995973, with improved overall in vitro degradation and antiproliferative activities compared to its direct thalidomide-based analogue dBET1, but similarly poor in vivo pharmacokinetic profile. To further demonstrate the PG utility, we describe here optimization efforts that led to the discovery of an orally bioavailable BET-PROTAC SJ44236 ( 8 ), and results of a comprehensive in vitro/vivo comparative study with analogues containing alternative CRBN-directing warheads. Our study highlights the importance of considering warhead modifications when optimizing PROTACs for oral delivery.
- Published
- 2025
- Full Text
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9. Promoter-proximal RNA polymerase II termination regulates transcription during human cell type transition.
- Author
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Lysakovskaia K, Devadas A, Schwalb B, Lidschreiber M, and Cramer P
- Abstract
Metazoan gene transcription by RNA polymerase II (Pol II) is regulated in the promoter-proximal region. Pol II can undergo termination in the promoter-proximal region but whether this can contribute to transcription regulation in cells remains unclear. Here we extend our previous multiomics analysis to quantify changes in transcription kinetics during a human cell type transition event. We observe that upregulation of transcription involves an increase in initiation frequency and, at a set of genes, a decrease in promoter-proximal termination. In turn, downregulation of transcription involves a decrease in initiation frequency and an increase in promoter-proximal termination. Thus, promoter-proximal termination of Pol II contributes to the regulation of human gene transcription., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)
- Published
- 2025
- Full Text
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10. CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release.
- Author
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Velychko T, Mohammad E, Ferrer-Vicens I, Parfentev I, Werner M, Studniarek C, Schwalb B, Urlaub H, Murphy S, Cramer P, and Lidschreiber M
- Subjects
- Humans, Phosphorylation, Protein Kinase Inhibitors pharmacology, Mediator Complex metabolism, Mediator Complex genetics, HeLa Cells, Transcription Factor TFIIH metabolism, Transcription Factor TFIIH genetics, HEK293 Cells, Cyclin-Dependent Kinase-Activating Kinase, Cyclin-Dependent Kinases metabolism, Cyclin-Dependent Kinases genetics, RNA Polymerase II metabolism, RNA Polymerase II genetics, Promoter Regions, Genetic, Transcription Initiation, Genetic
- Abstract
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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11. MSL2 ensures biallelic gene expression in mammals.
- Author
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Sun Y, Wiese M, Hmadi R, Karayol R, Seyfferth J, Martinez Greene JA, Erdogdu NU, Deboutte W, Arrigoni L, Holz H, Renschler G, Hirsch N, Foertsch A, Basilicata MF, Stehle T, Shvedunova M, Bella C, Pessoa Rodrigues C, Schwalb B, Cramer P, Manke T, and Akhtar A
- Subjects
- Animals, Female, Male, Mice, DNA Methylation, Dosage Compensation, Genetic, Embryonic Development, Enhancer Elements, Genetic, Haploinsufficiency, Histones metabolism, Mice, Knockout, Promoter Regions, Genetic, Transcription Factors metabolism, Alleles, Gene Expression Regulation, Ubiquitin-Protein Ligases deficiency, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism
- Abstract
In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA
1,2 . This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders1,3 . Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter-enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease., (© 2023. The Author(s).)- Published
- 2023
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12. RNA transcription and degradation of Alu retrotransposons depends on sequence features and evolutionary history.
- Author
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Baar T, Dümcke S, Gressel S, Schwalb B, Dilthey A, Cramer P, and Tresch A
- Subjects
- Alu Elements genetics, Humans, Retroelements genetics, Transcription, Genetic, RNA genetics, RNA Polymerase II metabolism
- Abstract
Alu elements are one of the most successful groups of RNA retrotransposons and make up 11% of the human genome with over 1 million individual loci. They are linked to genetic defects, increases in sequence diversity, and influence transcriptional activity. Still, their RNA metabolism is poorly understood yet. It is even unclear whether Alu elements are mostly transcribed by RNA Polymerase II or III. We have conducted a transcription shutoff experiment by α-amanitin and metabolic RNA labeling by 4-thiouridine combined with RNA fragmentation (TT-seq) and RNA-seq to shed further light on the origin and life cycle of Alu transcripts. We find that Alu RNAs are more stable than previously thought and seem to originate in part from RNA Polymerase II activity, as previous reports suggest. Their expression however seems to be independent of the transcriptional activity of adjacent genes. Furthermore, we have developed a novel statistical test for detecting the expression of quantitative trait loci in Alu elements that relies on the de Bruijn graph representation of all Alu sequences. It controls for both statistical significance and biological relevance using a tuned k-mer representation, discovering influential sequence features missed by regular motif search. In addition, we discover several point mutations using a generalized linear model, and motifs of interest, which also match transcription factor-binding motifs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)
- Published
- 2022
- Full Text
- View/download PDF
13. Efficient RNA polymerase II pause release requires U2 snRNP function.
- Author
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Caizzi L, Monteiro-Martins S, Schwalb B, Lysakovskaia K, Schmitzova J, Sawicka A, Chen Y, Lidschreiber M, and Cramer P
- Subjects
- Animals, Drosophila Proteins genetics, Drosophila Proteins metabolism, Drosophila melanogaster enzymology, Drosophila melanogaster genetics, Feedback, Physiological, Gene Expression Regulation, HeLa Cells, Humans, K562 Cells, Positive Transcriptional Elongation Factor B genetics, Positive Transcriptional Elongation Factor B metabolism, Promoter Regions, Genetic, RNA Polymerase II genetics, RNA Precursors genetics, RNA Precursors metabolism, RNA Splicing, RNA, Messenger genetics, Ribonucleoprotein, U2 Small Nuclear genetics, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Spliceosomes genetics, Time Factors, RNA Polymerase II metabolism, RNA, Messenger biosynthesis, Ribonucleoprotein, U2 Small Nuclear metabolism, Spliceosomes enzymology, Transcription Elongation, Genetic
- Abstract
Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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- View/download PDF
14. Transcription activation depends on the length of the RNA polymerase II C-terminal domain.
- Author
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Sawicka A, Villamil G, Lidschreiber M, Darzacq X, Dugast-Darzacq C, Schwalb B, and Cramer P
- Subjects
- Enhancer Elements, Genetic, Gene Expression Profiling, Humans, MAP Kinase Signaling System, Promoter Regions, Genetic, Protein Domains, RNA Polymerase II genetics, RNA Polymerase II chemistry, RNA Polymerase II metabolism, Sequence Deletion, Transcriptional Activation
- Abstract
Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)
- Published
- 2021
- Full Text
- View/download PDF
15. Integrator is a genome-wide attenuator of non-productive transcription.
- Author
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Lykke-Andersen S, Žumer K, Molska EŠ, Rouvière JO, Wu G, Demel C, Schwalb B, Schmid M, Cramer P, and Jensen TH
- Subjects
- Cleavage And Polyadenylation Specificity Factor genetics, DNA-Binding Proteins genetics, HeLa Cells, Humans, Polyadenylation, Promoter Regions, Genetic, RNA Polymerase II genetics, RNA, Small Nuclear genetics, Cleavage And Polyadenylation Specificity Factor metabolism, DNA-Binding Proteins metabolism, RNA Polymerase II metabolism, RNA, Small Nuclear biosynthesis, Transcription Termination, Genetic
- Abstract
Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
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16. Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms.
- Author
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Maier KC, Gressel S, Cramer P, and Schwalb B
- Subjects
- Cell Line, Tumor, Humans, Machine Learning, Neural Networks, Computer, RNA Isoforms chemical synthesis, Uridine chemistry, Nanopore Sequencing methods, RNA Isoforms chemistry, RNA Stability
- Abstract
Eukaryotic genes often generate a variety of RNA isoforms that can lead to functionally distinct protein variants. The synthesis and stability of RNA isoforms is poorly characterized because current methods to quantify RNA metabolism use short-read sequencing and cannot detect RNA isoforms. Here we present nanopore sequencing-based isoform dynamics (nano-ID), a method that detects newly synthesized RNA isoforms and monitors isoform metabolism. Nano-ID combines metabolic RNA labeling, long-read nanopore sequencing of native RNA molecules, and machine learning. Nano-ID derives RNA stability estimates and evaluates stability determining factors such as RNA sequence, poly(A)-tail length, secondary structure, translation efficiency, and RNA-binding proteins. Application of nano-ID to the heat shock response in human cells reveals that many RNA isoforms change their stability. Nano-ID also shows that the metabolism of individual RNA isoforms differs strongly from that estimated for the combined RNA signal at a specific gene locus. Nano-ID enables studies of RNA metabolism at the level of single RNA molecules and isoforms in different cell states and conditions., (© 2020 Maier et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2020
- Full Text
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17. CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation.
- Author
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Tellier M, Zaborowska J, Caizzi L, Mohammad E, Velychko T, Schwalb B, Ferrer-Vicens I, Blears D, Nojima T, Cramer P, and Murphy S
- Subjects
- Chromatin metabolism, Cyclin-Dependent Kinases antagonists & inhibitors, Cyclin-Dependent Kinases genetics, Cyclin-Dependent Kinases metabolism, HEK293 Cells, Humans, Mutation, Phosphorylation, RNA biosynthesis, RNA Polymerase II chemistry, Sequence Analysis, RNA, Serine metabolism, Transcriptional Elongation Factors metabolism, Cyclin-Dependent Kinases physiology, RNA Polymerase II metabolism, Transcription Elongation, Genetic
- Abstract
Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
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18. Selective Mediator dependence of cell-type-specifying transcription.
- Author
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Jaeger MG, Schwalb B, Mackowiak SD, Velychko T, Hanzl A, Imrichova H, Brand M, Agerer B, Chorn S, Nabet B, Ferguson FM, Müller AC, Bergthaler A, Gray NS, Bradner JE, Bock C, Hnisz D, Cramer P, and Winter GE
- Subjects
- Animals, Cell Line, Tumor, Chromatin physiology, Drosophila, Gene Expression Profiling, Gene Knock-In Techniques, Humans, Mediator Complex genetics, Positive Transcriptional Elongation Factor B metabolism, RNA Polymerase II metabolism, Gene Expression Regulation, Mediator Complex physiology, Transcription, Genetic
- Abstract
The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.
- Published
- 2020
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19. The pause-initiation limit restricts transcription activation in human cells.
- Author
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Gressel S, Schwalb B, and Cramer P
- Subjects
- Cell Survival, Cyclin-Dependent Kinase 9 metabolism, Genome, HSP70 Heat-Shock Proteins genetics, Humans, K562 Cells, Kinetics, Positive Transcriptional Elongation Factor B metabolism, RNA Polymerase II metabolism, Gene Expression Regulation, Transcription, Genetic, Transcriptional Activation genetics, Transcriptional Activation physiology
- Abstract
Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region. Pausing Pol II limits the frequency of transcription initiation ('pause-initiation limit'), predicting that the pause duration must be decreased for transcriptional activation. To test this prediction, we conduct a genome-wide kinetic analysis of the heat shock response in human cells. We show that the pause-initiation limit restricts transcriptional activation at most genes. Gene activation generally requires the activity of the P-TEFb kinase CDK9, which decreases the duration of Pol II pausing and thereby enables an increase in the productive initiation frequency. The transcription of enhancer elements is generally not pause limited and can be activated without CDK9 activity. Our results define the kinetics of Pol II transcriptional regulation in human cells at all gene classes during a natural transcription response.
- Published
- 2019
- Full Text
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20. The Implication of Early Chromatin Changes in X Chromosome Inactivation.
- Author
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Żylicz JJ, Bousard A, Žumer K, Dossin F, Mohammad E, da Rocha ST, Schwalb B, Syx L, Dingli F, Loew D, Cramer P, and Heard E
- Subjects
- Acetylation, Animals, Chromatin genetics, Embryonic Stem Cells, Epigenomics methods, Female, Gene Silencing, Histone Deacetylases metabolism, Histones metabolism, Mice, Polycomb-Group Proteins metabolism, Protein Processing, Post-Translational, RNA, Long Noncoding metabolism, Transcription, Genetic, Ubiquitination, X Chromosome metabolism, Chromatin metabolism, X Chromosome Inactivation genetics, X Chromosome Inactivation physiology
- Abstract
During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
21. Promoter Distortion and Opening in the RNA Polymerase II Cleft.
- Author
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Dienemann C, Schwalb B, Schilbach S, and Cramer P
- Subjects
- Cryoelectron Microscopy, DNA Helicases genetics, DNA Helicases metabolism, DNA, Fungal metabolism, DNA, Fungal ultrastructure, Gene Expression Regulation, Fungal, Models, Molecular, Nucleic Acid Conformation, RNA Polymerase II metabolism, RNA Polymerase II ultrastructure, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Structure-Activity Relationship, Transcription Factor TFIIH genetics, Transcription Factor TFIIH metabolism, Transcription Initiation, Genetic, DNA, Fungal genetics, Promoter Regions, Genetic, RNA Polymerase II genetics, Saccharomyces cerevisiae genetics
- Abstract
Transcription initiation requires opening of promoter DNA in the RNA polymerase II (Pol II) pre-initiation complex (PIC), but it remains unclear how this is achieved. Here we report the cryo-electron microscopic (cryo-EM) structure of a yeast PIC that contains underwound, distorted promoter DNA in the closed Pol II cleft. The DNA duplex axis is offset at the upstream edge of the initially melted DNA region (IMR) where DNA opening begins. Unstable IMRs are found in a subset of yeast promoters that we show can still initiate transcription after depletion of the transcription factor (TF) IIH (TFIIH) translocase Ssl2 (XPB in human) from the nucleus in vivo. PIC-induced DNA distortions may thus prime the IMR for melting and may explain how unstable IMRs that are predicted in promoters of Pol I and Pol III can open spontaneously. These results suggest that DNA distortion in the polymerase cleft is a general mechanism that contributes to promoter opening., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
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22. CDK9-dependent RNA polymerase II pausing controls transcription initiation.
- Author
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Gressel S, Schwalb B, Decker TM, Qin W, Leonhardt H, Eick D, and Cramer P
- Subjects
- B-Lymphocytes metabolism, Cell Line, Humans, Promoter Regions, Genetic, Protein Binding, Cyclin-Dependent Kinase 9 metabolism, RNA Polymerase II metabolism, Transcription Initiation, Genetic
- Abstract
Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.
- Published
- 2017
- Full Text
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23. Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex.
- Author
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Xu Y, Bernecky C, Lee CT, Maier KC, Schwalb B, Tegunov D, Plitzko JM, Urlaub H, and Cramer P
- Subjects
- Binding, Competitive, Cell Cycle Proteins chemistry, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cross-Linking Reagents chemistry, Cryoelectron Microscopy, Multiprotein Complexes metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, Protein Conformation, RNA Polymerase II genetics, RNA-Binding Proteins chemistry, RNA-Binding Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic, Transcriptional Elongation Factors genetics, Transcriptional Elongation Factors metabolism, Multiprotein Complexes chemistry, Nuclear Proteins chemistry, RNA Polymerase II chemistry, RNA Polymerase II metabolism, Saccharomyces cerevisiae Proteins chemistry, Transcriptional Elongation Factors chemistry
- Abstract
The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and Rtf1, and binds to the RNA polymerase II (Pol II) transcription elongation complex (EC). Here we report the reconstitution of Paf1C from Saccharomyces cerevisiae, and a structural analysis of Paf1C bound to a Pol II EC containing the elongation factor TFIIS. Cryo-electron microscopy and crosslinking data reveal that Paf1C is highly mobile and extends over the outer Pol II surface from the Rpb2 to the Rpb3 subunit. The Paf1-Leo1 heterodimer and Cdc73 form opposite ends of Paf1C, whereas Ctr9 bridges between them. Consistent with the structural observations, the initiation factor TFIIF impairs Paf1C binding to Pol II, whereas the elongation factor TFIIS enhances it. We further show that Paf1C is globally required for normal mRNA transcription in yeast. These results provide a three-dimensional framework for further analysis of Paf1C function in transcription through chromatin.
- Published
- 2017
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24. Genome-wide Analysis of RNA Polymerase II Termination at Protein-Coding Genes.
- Author
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Baejen C, Andreani J, Torkler P, Battaglia S, Schwalb B, Lidschreiber M, Maier KC, Boltendahl A, Rus P, Esslinger S, Söding J, and Cramer P
- Subjects
- Binding Sites, Chromosomal Proteins, Non-Histone genetics, Chromosomal Proteins, Non-Histone metabolism, DNA, Fungal genetics, Exoribonucleases genetics, Exoribonucleases metabolism, Models, Genetic, Protein Binding, RNA Polymerase II genetics, RNA Precursors genetics, RNA, Fungal genetics, RNA, Messenger genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Transcriptional Elongation Factors genetics, Transcriptional Elongation Factors metabolism, mRNA Cleavage and Polyadenylation Factors genetics, mRNA Cleavage and Polyadenylation Factors metabolism, DNA, Fungal metabolism, RNA 3' End Processing, RNA Polymerase II metabolism, RNA Precursors biosynthesis, RNA, Fungal biosynthesis, RNA, Messenger biosynthesis, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
25. TT-seq captures enhancer landscapes immediately after T-cell stimulation.
- Author
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Michel M, Demel C, Zacher B, Schwalb B, Krebs S, Blum H, Gagneur J, and Cramer P
- Subjects
- Base Pairing, Enhancer Elements, Genetic, Gene Expression Regulation drug effects, Humans, Jurkat Cells, RNA analysis, Transcription, Genetic drug effects, Transcriptional Activation, Gene Expression Profiling methods, Ionomycin pharmacology, Sequence Analysis, RNA methods, T-Lymphocytes drug effects, Tetradecanoylphorbol Acetate pharmacology
- Abstract
To monitor transcriptional regulation in human cells, rapid changes in enhancer and promoter activity must be captured with high sensitivity and temporal resolution. Here, we show that the recently established protocol TT-seq ("transient transcriptome sequencing") can monitor rapid changes in transcription from enhancers and promoters during the immediate response of T cells to ionomycin and phorbol 12-myristate 13-acetate (PMA). TT-seq maps eRNAs and mRNAs every 5 min after T-cell stimulation with high sensitivity and identifies many new primary response genes. TT-seq reveals that the synthesis of 1,601 eRNAs and 650 mRNAs changes significantly within only 15 min after stimulation, when standard RNA-seq does not detect differentially expressed genes. Transcription of enhancers that are primed for activation by nucleosome depletion can occur immediately and simultaneously with transcription of target gene promoters. Our results indicate that enhancer transcription is a good proxy for enhancer regulatory activity in target gene activation, and establish TT-seq as a tool for monitoring the dynamics of enhancer landscapes and transcription programs during cellular responses and differentiation., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2017
- Full Text
- View/download PDF
26. Accurate Promoter and Enhancer Identification in 127 ENCODE and Roadmap Epigenomics Cell Types and Tissues by GenoSTAN.
- Author
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Zacher B, Michel M, Schwalb B, Cramer P, Tresch A, and Gagneur J
- Subjects
- Algorithms, Chromatin metabolism, Computational Biology methods, Histones metabolism, Humans, Regulatory Elements, Transcriptional genetics, Enhancer Elements, Genetic genetics, Epigenomics methods, Promoter Regions, Genetic genetics
- Abstract
Accurate maps of promoters and enhancers are required for understanding transcriptional regulation. Promoters and enhancers are usually mapped by integration of chromatin assays charting histone modifications, DNA accessibility, and transcription factor binding. However, current algorithms are limited by unrealistic data distribution assumptions. Here we propose GenoSTAN (Genomic STate ANnotation), a hidden Markov model overcoming these limitations. We map promoters and enhancers for 127 cell types and tissues from the ENCODE and Roadmap Epigenomics projects, today's largest compendium of chromatin assays. Extensive benchmarks demonstrate that GenoSTAN generally identifies promoters and enhancers with significantly higher accuracy than previous methods. Moreover, GenoSTAN-derived promoters and enhancers showed significantly higher enrichment of complex trait-associated genetic variants than current annotations. Altogether, GenoSTAN provides an easy-to-use tool to define promoters and enhancers in any system, and our annotation of human transcriptional cis-regulatory elements constitutes a rich resource for future research in biology and medicine., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
- Full Text
- View/download PDF
27. TT-seq maps the human transient transcriptome.
- Author
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Schwalb B, Michel M, Zacher B, Frühauf K, Demel C, Tresch A, Gagneur J, and Cramer P
- Subjects
- Base Pairing, Gene Expression Profiling, Humans, Polyadenylation, Promoter Regions, Genetic, RNA, Long Noncoding genetics, DNA-Directed RNA Polymerases metabolism, RNA, Messenger genetics, Terminator Regions, Genetic, Transcription Termination, Genetic, Transcriptome
- Abstract
Pervasive transcription of the genome produces both stable and transient RNAs. We developed transient transcriptome sequencing (TT-seq), a protocol that uniformly maps the entire range of RNA-producing units and estimates rates of RNA synthesis and degradation. Application of TT-seq to human K562 cells recovers stable messenger RNAs and long intergenic noncoding RNAs and additionally maps transient enhancer, antisense, and promoter-associated RNAs. TT-seq analysis shows that enhancer RNAs are short-lived and lack U1 motifs and secondary structure. TT-seq also maps transient RNA downstream of polyadenylation sites and uncovers sites of transcription termination; we found, on average, four transcription termination sites, distributed in a window with a median width of ~3300 base pairs. Termination sites coincide with a DNA motif associated with pausing of RNA polymerase before its release from the genome., (Copyright © 2016, American Association for the Advancement of Science.)
- Published
- 2016
- Full Text
- View/download PDF
28. Fine mapping of genome activation in bovine embryos by RNA sequencing.
- Author
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Graf A, Krebs S, Zakhartchenko V, Schwalb B, Blum H, and Wolf E
- Subjects
- Animals, Base Sequence, Cattle metabolism, Chromosome Mapping, Gene Expression Profiling, Gene Ontology, Genomics methods, Molecular Sequence Data, Sequence Analysis, RNA, Cattle genetics, Embryo, Mammalian metabolism, Gene Expression Regulation, Developmental genetics, Genome genetics, Transcriptional Activation genetics
- Abstract
During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10(3) different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.
- Published
- 2014
- Full Text
- View/download PDF
29. Periodic mRNA synthesis and degradation co-operate during cell cycle gene expression.
- Author
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Eser P, Demel C, Maier KC, Schwalb B, Pirkl N, Martin DE, Cramer P, and Tresch A
- Subjects
- Cell Cycle genetics, Gene Expression Regulation, Fungal, Genome, Fungal, Promoter Regions, Genetic, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Transcription Factors biosynthesis, Transcription Factors genetics, Transcription, Genetic, Gene Expression Profiling, Genes, cdc, RNA Stability genetics, RNA, Messenger biosynthesis
- Abstract
During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is achieved by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. Here, we used metabolic RNA labeling and comparative dynamic transcriptome analysis (cDTA) to derive mRNA synthesis and degradation rates every 5 min during three cell cycle periods of the yeast Saccharomyces cerevisiae. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation generally follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter.
- Published
- 2014
- Full Text
- View/download PDF
30. Transcriptome surveillance by selective termination of noncoding RNA synthesis.
- Author
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Schulz D, Schwalb B, Kiesel A, Baejen C, Torkler P, Gagneur J, Soeding J, and Cramer P
- Subjects
- Down-Regulation, Nuclear Proteins metabolism, Promoter Regions, Genetic, RNA, Antisense metabolism, Saccharomyces cerevisiae genetics, RNA, Fungal genetics, RNA, Untranslated genetics, RNA-Binding Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription Termination, Genetic, Transcriptome
- Abstract
Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription in the yeast S. cerevisiae is globally restricted by early termination that relies on the essential RNA-binding factor Nrd1. Depletion of Nrd1 from the nucleus results in 1,526 Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at most promoters and antisense NUTs in most 3' regions of genes. Nrd1 and its partner Nab3 preferentially bind RNA motifs that are depleted in mRNAs and enriched in ncRNAs and some mRNAs whose synthesis is controlled by transcription attenuation. These results define a global mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and to suppress antisense transcription., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
31. Global analysis of eukaryotic mRNA degradation reveals Xrn1-dependent buffering of transcript levels.
- Author
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Sun M, Schwalb B, Pirkl N, Maier KC, Schenk A, Failmezger H, Tresch A, and Cramer P
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Cluster Analysis, Exoribonucleases genetics, Gene Expression Regulation, Fungal, Kinetics, Models, Genetic, Mutation, N-Glycosyl Hydrolases metabolism, RNA, Fungal biosynthesis, RNA, Messenger biosynthesis, Repressor Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Substrate Specificity, Exoribonucleases metabolism, RNA Stability, RNA, Fungal metabolism, RNA, Messenger metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative dynamic transcriptome analysis (cDTA) that uses nonperturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
32. Drosophila miR-277 controls branched-chain amino acid catabolism and affects lifespan.
- Author
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Esslinger SM, Schwalb B, Helfer S, Michalik KM, Witte H, Maier KC, Martin D, Michalke B, Tresch A, Cramer P, and Förstemann K
- Subjects
- Aging, Animals, Animals, Genetically Modified, Cells, Cultured, Drosophila melanogaster genetics, Drosophila melanogaster physiology, Gene Expression Regulation, High-Throughput Nucleotide Sequencing, Insulin metabolism, Longevity, Sequence Analysis, RNA, Amino Acids, Branched-Chain metabolism, Drosophila melanogaster metabolism, MicroRNAs genetics, MicroRNAs metabolism, TOR Serine-Threonine Kinases metabolism
- Abstract
Development, growth and adult survival are coordinated with available metabolic resources, ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21-23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life in Drosophila melanogaster and determined that miR-277 is downregulated during adult life. Molecular analysis revealed that this miRNA controls branched-chain amino acid (BCAA) catabolism and as a result it can modulate the activity of the TOR kinase, a central growth regulator, in cultured cells. Metabolite analysis in cultured cells as well as flies suggests that the mechanistic basis may be an accumulation of branched-chain α-keto-acids (BCKA), rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g., translational fidelity. Constitutive miR-277 expression shortens lifespan and is synthetically lethal with reduced insulin signaling, indicating that metabolic control underlies this phenotype. Transgenic inhibition with a miRNA sponge construct also shortens lifespan, in particular on protein-rich food. Thus, optimal metabolic adaptation appears to require tuning of cellular BCAA catabolism by miR-277.
- Published
- 2013
- Full Text
- View/download PDF
33. Mediator phosphorylation prevents stress response transcription during non-stress conditions.
- Author
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Miller C, Matic I, Maier KC, Schwalb B, Roether S, Strässer K, Tresch A, Mann M, and Cramer P
- Subjects
- Amino Acid Motifs, Mediator Complex chemistry, Mediator Complex genetics, Phosphorylation, RNA Polymerase II genetics, RNA Polymerase II metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Gene Expression Regulation, Fungal, Mediator Complex metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic
- Abstract
The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.
- Published
- 2012
- Full Text
- View/download PDF
34. Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation.
- Author
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Sun M, Schwalb B, Schulz D, Pirkl N, Etzold S, Larivière L, Maier KC, Seizl M, Tresch A, and Cramer P
- Subjects
- Cell Nucleus genetics, Cell Nucleus metabolism, Feedback, Physiological, Gene Expression Regulation, Fungal, Genome, Fungal, Point Mutation, RNA, Fungal genetics, RNA, Messenger genetics, Ribonucleases genetics, Ribonucleases metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Transcription, Genetic, Transcriptome, Gene Expression Profiling methods, RNA Stability, RNA, Fungal metabolism, RNA, Messenger biosynthesis, Saccharomyces cerevisiae genetics
- Abstract
To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.
- Published
- 2012
- Full Text
- View/download PDF
35. Measurement of genome-wide RNA synthesis and decay rates with Dynamic Transcriptome Analysis (DTA).
- Author
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Schwalb B, Schulz D, Sun M, Zacher B, Dümcke S, Martin DE, Cramer P, and Tresch A
- Subjects
- Cell Cycle, Oligonucleotide Array Sequence Analysis, RNA Stability, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Schizosaccharomyces cytology, Schizosaccharomyces genetics, Transcriptome, Gene Expression Profiling methods, Genome-Wide Association Study, Software
- Abstract
Standard transcriptomics measures total cellular RNA levels. Our understanding of gene regulation would be greatly improved if we could measure RNA synthesis and decay rates on a genome-wide level. To that end, the Dynamic Transcriptome Analysis (DTA) method has been developed. DTA combines metabolic RNA labeling with standard transcriptomics to measure RNA synthesis and decay rates in a precise and non-perturbing manner. Here, we present the open source R/Bioconductor software package DTA. It implements all required bioinformatics steps that allow the accurate absolute quantification and comparison of RNA turnover.
- Published
- 2012
- Full Text
- View/download PDF
36. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast.
- Author
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Miller C, Schwalb B, Maier K, Schulz D, Dümcke S, Zacher B, Mayer A, Sydow J, Marcinowski L, Dölken L, Martin DE, Tresch A, and Cramer P
- Subjects
- Gene Expression Regulation, Fungal, Genome, Fungal, Half-Life, Logistic Models, Oligonucleotide Array Sequence Analysis, RNA, Fungal biosynthesis, RNA, Fungal genetics, RNA, Messenger genetics, Stress, Physiological, Transcription Factors metabolism, Transcription, Genetic, Gene Expression Profiling methods, RNA Stability, RNA, Messenger biosynthesis, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA-derived mRNA synthesis rates identified 16 stress-specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.
- Published
- 2011
- Full Text
- View/download PDF
37. [Observations on use of modern hypnotics. More rapid sleep onset, more frequent uninterrupted sleep and sleep duration].
- Author
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Wiegand MH, Roszinsky-Köcher G, Schwalb B, Fischer W, and Eckert MW
- Subjects
- Azabicyclo Compounds, Family Practice, Humans, Treatment Outcome, Hypnotics and Sedatives therapeutic use, Piperazines therapeutic use, Sleep Initiation and Maintenance Disorders drug therapy, Sleep Stages drug effects
- Published
- 2001
38. [Effectiveness of zopiclone in disorders of initiating and maintaining sleep. Results of a drug monitoring study in 811 general practices].
- Author
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Wiegand MH, Roszinsky-Köcher G, Schwalb B, Fischer W, and Eckert MW
- Subjects
- Adult, Aged, Azabicyclo Compounds, Data Interpretation, Statistical, Drug Monitoring, Family Practice, Female, Humans, Hypnotics and Sedatives administration & dosage, Hypnotics and Sedatives adverse effects, Male, Middle Aged, Piperazines administration & dosage, Piperazines adverse effects, Sleep Initiation and Maintenance Disorders diagnosis, Time Factors, Hypnotics and Sedatives therapeutic use, Piperazines therapeutic use, Sleep Initiation and Maintenance Disorders drug therapy
- Abstract
Background and Method: In a drug monitoring study with 811 participating general practitioners, safety and tolerability of zopiclone were studied in 2416 patients with disorders of initiating and maintaining sleep., Results: In general, zopiclone was efficient in all forms of insomnia; the subjective sleep duration was prolonged for 2 hours on average. Patients without somatic complaints or comorbidity showed the greatest benefit. Daytime well-being and vigilance were in general not impaired. Drug related adverse events occurred rarely; the great majority of the participating physicians rated the treatment with zopiclone as efficient and acceptable., Conclusion: In this drug monitoring study, zopiclone proved an efficient hypnotic for the treatment of disorders of initiating and maintaining sleep.
- Published
- 2001
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