scholarly journals Dynamics of transcription elongation are finely tuned by dozens of regulatory factors

2021 ◽  
Author(s):  
Mary Couvillion ◽  
Kevin M Harlen ◽  
Kate C Lachance ◽  
Kristine Trotta ◽  
Erin Smtih ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Elongation ◽  
Antisense Transcription ◽  
Regulation Of Transcription ◽  
Pol Ii ◽  
Density Peaks ◽  
Large Numbers ◽  
Key Points ◽  
Opposing Effects

Understanding the complex network and dynamics that regulate transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of S. cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation dynamics. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Genes that are sensitive to disruptions in transcription elongation tend to couple changes in Pol II pausing and antisense transcription to transcription output. Our findings collectively show that the regulation of transcription elongation by a diverse array of factors affects gene expression levels and co-transcriptional processing by precisely balancing Pol II activity.

Linking transcription, RNA polymerase II elongation and alternative splicing

2020 ◽  
Vol 477 (16) ◽  
pp. 3091-3104 ◽  
Author(s):  
Luciana E. Giono ◽  
Alberto R. Kornblihtt
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Polymerase Ii ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Single Gene ◽  
Regulation Of Gene Expression ◽  
Regulation Of Transcription ◽  
Stepping Stones ◽  
Eukaryotic Transcription

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic — such as promoter structure — and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

scholarly journals A machine learning-based framework for modeling transcription elongation

2021 ◽  
Vol 118 (6) ◽  
pp. e2007450118
Author(s):  
Peiyuan Feng ◽  
An Xiao ◽  
Meng Fang ◽  
Fangping Wan ◽  
Shuya Li ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Gene Expression Regulation ◽  
Transcription Elongation ◽  
Transcriptional Elongation ◽  
Sequencing Data ◽  
Pol Ii ◽  
High Throughput Sequencing Data ◽  
Pol Ii Pausing ◽  
Elongation Process

RNA polymerase II (Pol II) generally pauses at certain positions along gene bodies, thereby interrupting the transcription elongation process, which is often coupled with various important biological functions, such as precursor mRNA splicing and gene expression regulation. Characterizing the transcriptional elongation dynamics can thus help us understand many essential biological processes in eukaryotic cells. However, experimentally measuring Pol II elongation rates is generally time and resource consuming. We developed PEPMAN (polymerase II elongation pausing modeling through attention-based deep neural network), a deep learning-based model that accurately predicts Pol II pausing sites based on the native elongating transcript sequencing (NET-seq) data. Through fully taking advantage of the attention mechanism, PEPMAN is able to decipher important sequence features underlying Pol II pausing. More importantly, we demonstrated that the analyses of the PEPMAN-predicted results around various types of alternative splicing sites can provide useful clues into understanding the cotranscriptional splicing events. In addition, associating the PEPMAN prediction results with different epigenetic features can help reveal important factors related to the transcription elongation process. All these results demonstrated that PEPMAN can provide a useful and effective tool for modeling transcription elongation and understanding the related biological factors from available high-throughput sequencing data.

AT7519, a Potent Multi-Targeted CDK Inhibitor, Is Active in CLL Patient Samples Independent of Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3161-3161
Author(s):  
Vicky Lock ◽  
Laurence Cooke ◽  
Murray Yule ◽  
Neil T Thompson ◽  
K. Della Croce ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Parp Cleavage ◽  
Regulation Of Transcription ◽  
Cytotoxic Effects ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Cyclin Dependent Kinases

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

scholarly journals PPM1G Binds 7SK RNA and Hexim1 To Block P-TEFb Assembly into the 7SK snRNP and Sustain Transcription Elongation

2015 ◽  
Vol 35 (22) ◽  
pp. 3810-3828 ◽  
Author(s):  
Swapna Aravind Gudipaty ◽  
Ryan P. McNamara ◽  
Emily L. Morton ◽  
Iván D'Orso
Keyword(s):  
Rna Polymerase Ii ◽  
Noncoding Rna ◽  
Kinase Inhibitor ◽  
Transcription Elongation ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
New Paradigm ◽  
Pol Ii ◽  
Small Nuclear Ribonucleoprotein ◽  
Rna Interaction

Transcription elongation programs are vital for the precise regulation of several biological processes. One key regulator of such programs is the P-TEFb kinase, which phosphorylates RNA polymerase II (Pol II) once released from the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. Although mechanisms of P-TEFb release from the snRNP are becoming clearer, how P-TEFb remains in the 7SK-unbound state to sustain transcription elongation programs remains unknown. Here we report that the PPM1G phosphatase (inducibly recruited by nuclear factor κB [NF-κB] to target promoters) directly binds 7SK RNA and the kinase inhibitor Hexim1 once P-TEFb has been released from the 7SK snRNP. This dual binding activity of PPM1G blocks P-TEFb reassembly onto the snRNP to sustain NF-κB-mediated Pol II transcription in response to DNA damage. Notably, the PPM1G-7SK RNA interaction is direct, kinetically follows the recruitment of PPM1G to promoters to activate NF-κB transcription, and is reversible, since the complex disassembles before resolution of the program. Strikingly, we found that the ataxia telangiectasia mutated (ATM) kinase regulates the interaction between PPM1G and the 7SK snRNP through site-specific PPM1G phosphorylation. The precise and temporally regulated interaction of a cellular enzyme and a noncoding RNA provides a new paradigm for simultaneously controlling the activation and maintenance of inducible transcription elongation programs.

scholarly journals RNA polymerase II pausing modulates hematopoietic stem cell emergence in zebrafish

Blood ◽  
2016 ◽  
Vol 128 (13) ◽  
pp. 1701-1710 ◽  
Author(s):  
Qiwen Yang ◽  
Xiuli Liu ◽  
Ting Zhou ◽  
Jennifer Cook ◽  
Kim Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hematopoietic Stem Cell ◽  
Zebrafish Embryos ◽  
Hematopoietic Stem ◽  
Pol Ii ◽  
Key Points ◽  
Ifn Γ ◽  
Pol Ii Pausing

Key Points Pol II pausing is required for HSC emergence in zebrafish embryos. TGFβ and IFN-γ signaling are oppositely regulated by Pol II pausing to regulate HSC emergence.

scholarly journals RPRD Proteins Control Transcription in Human Cells

2021 ◽  
Author(s):  
Kinga Winczura ◽  
Hurmuz Ceylan ◽  
Monika Sledziowska ◽  
Matt Jones ◽  
Holly Fagarasan ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Human Cells ◽  
Cellular Stress Response ◽  
Regulation Of Transcription ◽  
Biological Processes ◽  
Substantial Impact ◽  
Pol Ii ◽  
Number Of Factors ◽  
External Signals

The regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

scholarly journals Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology

2020 ◽  
Author(s):  
Yubo Cheng ◽  
Babita Gollen ◽  
Luke Saville ◽  
Christopher Isaac ◽  
Jogender Mehla ◽  
...  
Keyword(s):  
Stress Response ◽  
Rna Polymerase Ii ◽  
Amyloid Beta ◽  
Transcriptional Activation ◽  
Rna Processing ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Amyloid Toxicity ◽  
B2 Rna ◽  
Non Coding Rnas

ABSTRACTMore than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in the mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates transition to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi of an amyloid precursor protein knock-in mouse model and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD). This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity.

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