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1.
Methods Mol Biol ; 2351: 41-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382183

RESUMO

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.


Assuntos
Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Transcrição Genética , Animais , Células Cultivadas , Cromatina/genética , Biologia Computacional/métodos , DNA , Biblioteca Gênica , Humanos , Reação em Cadeia da Polimerase , RNA , RNA Polimerase II/metabolismo , Software
2.
Mol Cell ; 81(17): 3589-3603.e13, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34324863

RESUMO

Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3'-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3'-RNA processing factors at the 5'-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5'-elongation control and 3'-RNA processing.

3.
Nucleic Acids Res ; 49(8): 4402-4420, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33788942

RESUMO

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression.


Assuntos
Sequência Conservada , RNA Polimerase II/metabolismo , RNA-Seq/métodos , Transcrição Genética , Algoritmos , Sequência de Bases , DNA/química , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , RNA Polimerase II/química
4.
EMBO J ; 39(7): e101548, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32107786

RESUMO

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , RNA Polimerase II/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Fúngico/metabolismo , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética
5.
Proc Natl Acad Sci U S A ; 114(42): 11133-11138, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29073019

RESUMO

RNA polymerase II contains a long C-terminal domain (CTD) that regulates interactions at the site of transcription. The CTD architecture remains poorly understood due to its low sequence complexity, dynamic phosphorylation patterns, and structural variability. We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3'-end RNA-processing and transcription termination factor. Rtt103 forms homodimers via its long coiled-coil domain and associates densely on the repetitive sequence of the phosphorylated CTD via its N-terminal CTD-interacting domain. The CTD-Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins.


Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química
6.
EMBO Rep ; 18(6): 906-913, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28468956

RESUMO

Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.


Assuntos
RNA Polimerase II/química , Proteínas de Saccharomyces cerevisiae/química , Treonina/química , Fatores de Transcrição/química , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteólise , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Treonina/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Tirosina/metabolismo
7.
Wiley Interdiscip Rev RNA ; 4(1): 1-16, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23042580

RESUMO

RNA polymerase II (RNA pol II) is not only the fundamental enzyme for gene expression but also the central coordinator of co-transcriptional processing. RNA pol II associates with a large number of enzymes and protein/RNA-binding factors through its C-terminal domain (CTD) that consists of tandem repeats of the heptapeptide consensus Y(1)S(2)P(3) T(4)S(5)P(6)S(7). The CTD is posttranslationally modified, yielding specific patterns (often called the CTD code) that are recognized by appropriate factors in coordination with the transcription cycle. Serine phosphorylations are currently the best characterized elements of the CTD code; however, the roles of the proline isomerization and other modifications of the CTD remain poorly understood. The dynamic remodeling of the CTD modifications by kinases, phosphatases, isomerases, and other enzymes introduce changes in the CTD structure and dynamics. These changes serve as structural switches that spatially and temporally regulate the binding of processing factors. Recent structural studies of the CTD bound to various proteins have revealed the basic rules that govern the recognition of these switches and shed light on the roles of these protein factors in the assemblies of the processing machineries.


Assuntos
Processamento de Proteína Pós-Traducional , RNA Polimerase II , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Metiltransferases/metabolismo , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Fosfoproteínas Fosfatases , Prolina/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Transcrição Genética
8.
PLoS One ; 7(3): e33482, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22432028

RESUMO

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo.


Assuntos
DNA Polimerase I/química , DNA Polimerase I/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Biocatálise , Exonucleases/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Ligação Proteica , Alinhamento de Sequência , Deleção de Sequência , Relação Estrutura-Atividade
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