[Figure: see text].

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• elongace genetické transkripce * MeSH
• exprese genu MeSH
• interakční proteinové domény a motivy genetika   MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• molekulární modely MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• proteinové domény MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• transkripční elongační faktory chemie   metabolismus   MeSH
• transkripční faktory chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vnitřně neuspořádané proteiny chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

• MeSH
• buněčné linie MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• genový knockdown MeSH
• lidé MeSH
• myši knockoutované MeSH
• neurony chemie   metabolismus   MeSH
• posttranskripční úpravy RNA MeSH
• proteinové domény MeSH
• regulace genové exprese MeSH
• RNA-polymerasa II chemie   genetika   metabolismus   MeSH
• RNA * chemie   genetika   metabolismus   MeSH
• stabilita RNA MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• proteinové domény MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• RNA-helikasy chemie   metabolismus   MeSH
• RNA-polymerasa II chemie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminace genetické transkripce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To deal with the general problem of biomolecule specific binding analysis, we have applied the technique of difference spectra to the surface plasmon resonance (SPR)-enhanced total internal reflection ellipsometry measurement. We suggest a three-step treatment of the SPR background that can easily be integrated with the usual measurement routine. First, making use of the difference spectrum in ellipsometric angle Δ, single peak footprints of the topmost layer are obtained that facilitate its sensitive detection during film growth. Subsequently, circumventing the need for explicit knowledge of the substrate properties, the difference spectra peaks can be used for the end-point analysis of a binding. Finally, tracking the binding effectivity of the analyte we determine the injection speed and analyte concentration windows needed for successful monitoring of the film growth. We demonstrate our approach on a comprehensive two-stage binding experiment involving two biologically relevant molecules: the C-terminal domain (CTD) of RNA polymerase II and CTD-interacting domain of one of its transcription factors, the Rtt103 protein.

• MeSH
• povrchová plasmonová rezonance metody   MeSH
• proteinové domény MeSH
• RNA-polymerasa II chemie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   MeSH
• transkripční faktory chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• proteinkinasy metabolismus   MeSH
• proteolýza MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• transkripční faktory chemie   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• genetická transkripce MeSH
• methyltransferasy metabolismus   MeSH
• peptidylprolylisomerasa metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• prolin metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• RNA-polymerasa II * chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.

• MeSH
• adenosin chemie   metabolismus   MeSH
• autoradiografie MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• gonadotropiny metabolismus   MeSH
• imunohistochemie MeSH
• izotopové značení MeSH
• myši MeSH
• oocyty chemie   růst a vývoj   metabolismus   fyziologie   MeSH
• prasata MeSH
• RNA-polymerasa II chemie   genetika   metabolismus   MeSH
• umlčování genů * MeSH
• uridin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this article, we report the resonance assignment of CTD-interacting domain (CID) of pre-mRNA down-regulation (Nrd)1 bound to Ser5-phosphorylated CTD (pSer5) of RNA Polymerase II. The presented assignment of backbone and side-chain resonances of the Nrd1 CID proton, carbon and nitrogen nuclei will allow studies of the structure and interaction of CID with carboxy-terminal domain (CTD) of the RNA polymerase II.

• MeSH
• down regulace MeSH
• fosforylace MeSH
• izotopy chemie   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• peptidy chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• serin chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• fenotyp MeSH
• fúzní onkogenní proteiny fyziologie   chemie   MeSH
• lidé MeSH
• nádorová transformace buněk fyziologie   MeSH
• RNA-polymerasa II fyziologie   chemie   MeSH
• transkripční faktory fyziologie   MeSH
• vazba proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH