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Ssu72 and Rtr1 Serine 5 Phosphates and Their Role in NNS and CPF Transcription TerminationVictorino, Jose Fabian 05 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Polyadenylation dependent transcription termination is dependent on the Cleavage
and Polyadenylation Factor complex (CPF) which is essential for the termination and
processing of mature RNA. Polyadenylation (PolyA) independent transcription
termination is carried out by the NNS (Nrd1-Nab3-Sen1) termination pathway, which
helps regulate termination and processing of non-coding RNA (ncRNA). The disruption
of these pathways can impact expression of nearby genes, both protein coding and noncoding.
Recruitment of termination pathway components is achieved through a domain
unique to the largest subunit of RNA Polymerase II (RNAPII) referred to as the Cterminal
domain (CTD), which contains a repeating heptad sequence, Y1S2P3T4S5P6S7,
and acts as a docking site for transcription regulatory proteins. Ssu72 is a serine 5
phosphatase and an essential member of the CPF complex. Rtr1 is also a serine 5
phosphatase, but its mechanism of action is less well characterized. Both Rtr1 and Ssu72
regulate transcription machinery recruitment through control of the phosphorylation
status of the CTD. My studies have focused on Rtr1 and Ssu72 mutants in yeast which
show evidence of transcription termination related phenotypes. Chromatin
immunoprecipitation of RNAPII followed by exonuclease treatment (ChIP-exo) studies
provide evidence of RNAPII transcription continuing through termination sites at ncRNA
genes as a result of a hyperactive Ssu72-L84F mutant, while an RTR1 knockout results in
increased premature RNAPII transcription termination. Northern blots and RNA
sequencing confirm premature transcription termination and decreased total RNA expression in the RTR1 knockout and increased length of ncRNA transcripts as well as
total RNA expression in the Ssu72-L84F mutant. Mass spectrometry analysis has
identified changes in the protein-protein interactions (PPI) within the CPF complex in the
Ssu72-L84F mutant and decreased PPIs between different transcription machinery in
RTR1 knockout cells. My results show that the CTD phosphatases Rtr1 and Ssu72 play
unique roles in the regulation of RNAPII termination in eukaryotes. / 2020-11-19
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EFFECTS OF INHIBITING CDK9 ON THE EXPRESSION OF PRIMARY RESPONSE GENESKeskin, Havva January 2011 (has links)
Flavopridol (FVP) is a well known pharmacological inhibitor of Cyclin Dependent Kinases (CDKs), with significant selectivity for Cyclin Dependent Kinase 9 (CDK9). Treatment of cells with FVP results in inhibition of transcription elongation. CDK9 is a serine/threonine kinase that associates with T-type cyclins. These complexes are designated transcription elongation factors (P-TEFb). P-TEFb controls transcription elongation by phosphorylating the carboxyl terminal domain (CTD) of RNA polymerase II (RNAPII) and negative elongation factors. Whether P-TEFb is required for the elongation of most genes transcribed by RNAPII or fraction of them is still debatable. The aim of my Thesis is to understand the early and late effects of FVP on primary response gene expression. Two different microarray analyses with RNA probes obtained from T98G and BJ-TERT cells were performed by Drs. Graña and Garriga to determine the effect of inhibiting CDK9 on global mRNA expression using a dominant negative mutant of CDK9 (dnCDK9) and FVP. These gene profiling experiments showed that FVP and dnCDK9 downregulate the expression of several genes. However, these studies also showed upregulation of a group of primary response genes (PRGs). The goal of this thesis was to bring some light into this unexpected phenomenon. I have found that several PRGs including FOS, JUNB, EGR1 and GADD45B, are rapidly and potently downregulated before they are upregulated upon FVP treatment in exponentially growing cells. In serum starved cells restimulated with serum, FVP also inhibits the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 are upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are apparently transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition. I also show that serum starvation does not affect the localization of RNAPII immediately downstream of the promoter of a PRG where RNAPII remains paused in the absence of mitogenic stimulation, suggesting that initiation is not rate limiting for transcription of at least certain PRGs in the absence of mitogens and remains dependent on transcription elongation. In sum, I have shown that certain PRG/IRGs are transcribed in the presence of FVP and their transcription might be independent of CDK9 suggesting a possible alternative mechanism of their transcription. I also determined that transcription initiation is not affected by serum starvation, as paused RNAPII appears to remain bound downstream of a PRG promoter in quiescent cells independently of the length of mitogenic starvation. / Molecular Biology and Genetics
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Structural and Kinetic Characterization of RNA Polymerase II C-Terminal Domain Phosphatase Ssu72 and Development of New Methods for NMR Studies of Large ProteinsWerner-Allen, Jonathan January 2011 (has links)
<p>Ssu72 is a protein phosphatase that selectively targets phosphorylated serine residues at the 5th position (pS5) in the heptad repeats of the C-terminal domain (CTD) of RNA polymerase II, in order to regulate the CTD-mediated coupling between eukaryotic transcription and co-transcriptional events. The biological importance of Ssu72 is underscored by (1) the requirement of its activity for viability in yeast, and (2) the numerous phenotypes - affecting all three stages of the transcription cycle - that result from its mutation in yeast. Despite limited homology to the low molecular weight (LMW) subclass of protein tyrosine phosphatases (PTPs), several lines of evidence suggest that Ssu72 represents the founding member of a new class of enzymes, including its unique substrate specificity and an in vivo connection with the activity of proline isomerase Ess1.</p><p>The main focus of this thesis has been to structurally and kinetically characterize Ssu72, in order to define its relation to known enzyme families, to provide biochemical explanations for extant in vivo observations, and to allow future structure-guided investigations of its role in coordinating transcription with co-transcriptional events. To this end, we solved the structure of Ssu72 in complex with its pS5 CTD substrate, revealing an enzyme fold with unique structural features and a surprising substrate conformation with the pS5-P6 motif of the CTD adopting the cis configuration. Together with kinetic assays, the structure provides a new interpretation of the role of proline isomers in regulating the CTD phosphorylation state, with broad implications for CTD biology.</p><p>The second goal of this thesis has been to develop new methods for NMR studies of large proteins, which present unique challenges to conventional methods, including fast signal decay and severe signal degeneracy. The first of these new methods, the `just-in-time' HN(CA)CO, improves the sensitivity of a common backbone assignment experiment. The next two methods, the 4-D diagonal-suppressed TROSY-NOESY-TROSY and the 4-D time-shared NOESY, were designed for use with sparse sampling techniques that allow the acquisition of high-resolution, high-dimensionality datasets. These efforts culminate with global fold calculations for large proteins, including the 23 kDa Ssu72, with accurate and unambiguous automated assignment of NOE crosspeaks. We expect that the methods presented here will be particularly useful as the NMR community continues to push toward higher molecular weight targets.</p> / Dissertation
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Investigation of the role of essential proteins in gene silencing at the centromere of Schizosaccharomyces pombeDobbs, Edward January 2012 (has links)
The centromeres of eukaryotes have a region on which the kinetochore is assembled, flanked by heterochromatin which provides cohesion between the sister chromatids during cell division. When centromeric heterochromatin is lost chromosomes no longer segregate evenly into the daughter cells during cell division. In the fission yeast Schizosaccharomyces pombe (S. pombe) RNA interference (RNAi) is responsible for maintaining this heterochromatin. The pathway is part of a feedback loop whereby siRNAs generated from non-coding centromere transcripts are loaded into an Argonaute complex. The siRNAs guide the complex to the homologous centromere repeats in order to recruit Clr4 which modifies histone H3 with the heterochromatin mark H3K9me. A previous screen to find factors affecting centromere silencing isolated 13 loci termed centromere: suppressor of position-effect (csp) 1-13. Several csp mutants have been identified to be RNAi components. In this investigation the csp6 locus has been identified to be the Hsp70 gene ssa2+. It has been demonstrated that Argonaute proteins from plants and flies require Hsp70/90 chaperone activity for loading of siRNA. It therefore seems likely that Hsp70 may play a similar role in fission yeast. Genetic and biochemical techniques have been used in this study to investigate if the csp6 alleles are affecting siRNA loading in S. pombe. RNA Polymerase II (RNAPII) transcribes the pre-siRNA transcripts from the centromere repeats. csp3 was identified to be an allele of the RNAPII subunit rpb7+. rpb7-G150D was found to cause a silencing defect in the centromeric heterochromatin through a defect in transcription. Another RNAPII mutation, rpb2-m203, was found to have strong silencing defects caused by an unidentified non-transcriptional role in RNAi-mediated heterochromatin formation at the centromere. In order to gain more insight into the role of RNAPII in heterochromatin assembly I performed a screen in which the subunits rpb3 and rpb11 were subjected to random mutagenesis. Several mutants were isolated and characterisation of phenotypes regarding heterochromatin at the centromere has been carried out for nine of the mutants. As a result a novel phenomenon of RNAi-independent silencing at the centromere has been discovered.
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Investigations into the regulation of histone H2B monoubiquitination / Investigations into the regulation of histone H2B monoubiquitinationShchebet, Andrei 18 April 2011 (has links)
No description available.
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The role of Rtr1 and Rrp6 in RNAPII in transcription terminationFox, Melanie Joy 31 August 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (RNAPII) is responsible for transcription of messenger RNA (mRNA) and many small non-coding RNAs. Progression through the RNAPII transcription cycle is orchestrated by combinatorial posttranslational modifications of the C-terminal domain (CTD) of the largest subunit of RNAPII, Rpb1, consisting of the repetitive sequence (Y1S2P3T4S5P6S7)n. Disruptions of proteins that control CTD phosphorylation, including the phosphatase Rtr1, cause defects in gene expression and transcription termination. There are two described RNAPII termination mechanisms. Most mRNAs are terminated by the polyadenylation-dependent cleavage and polyadenylation complex. Most short noncoding RNAs are terminated by the Nrd1 complex. Nrd1-dependent termination is coupled to RNA 3' end processing and/or degradation by Rrp6, a nuclear specific subunit of the exosome. The Rrp6-containing form a 3'-5' exonuclease complex that regulates diverse aspects of nuclear RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs). It remains unclear whether Rrp6 is directly involved in termination. We discovered that deletion of RRP6 promotes extension of multiple Nrd1-dependent transcripts resulting from improperly processed 3' RNA ends and faulty transcript termination at specific target genes. Defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of Nrd1 depletion from the nucleus. Our data indicate Rrp6 acts with Nrd1 globally to promote transcription termination in addition to RNA processing and/or degradation. Furthermore, we found that deletion of the CTD phosphatase Rtr1 shortens the distance of transcription before Nrd1-dependent termination of specific regulatory antisense transcripts (ASTs), increases Nrd1 occupancy at these sites, and increases the interaction between Nrd1 and RNAPII. The RTR1/RRP6 double deletion phenocopies an RRP6 deletion, indicating that the regulation of ASTs by Rtr1 requires Rrp6 activity and the Nrd1 termination pathway.
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Étude structure / fonction des sous-unités catalytiques de l'ARN polymérase IIDomecq, Céline January 2008 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.
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Genome-wide target identification of sequence-specific transcription factors through ChIP sequencingLee, Bum Kyu 17 November 2011 (has links)
The regulation of gene expression at the right time, place, and degree is crucial for many cellular processes such as proliferation and development. In addition, in order to maintain cellular life, cells must rapidly and appropriately respond to various environmental stimuli. Sequence-specific transcription factors (TFs) can recognize functional regulatory DNA elements in a sequence-specific manner so that they can regulate only a specific group of genes, a process which enables cells to cope with diverse internal and external stimuli. Human has approximately 1,400 sequence-specific TFs whose aberrant expression causes a wide range of detrimental consequences including developmental disorders, diseases, and cancers; therefore, it is pivotal to identify the binding sites of each sequence-specific TF in order to unravel its roles in and mechanisms of gene regulation.
Even though some TFs have been intensively studied, the majority of TFs still remain to be studied, particularly the tasks of identifying their genome-wide target genes and deciphering their biological roles in specific cellular contexts. Many questions remain unanswered: how many sites on the human genome a sequence-specific TF can bind; whether all TF-bound sites are functional; how a TF achieves binding specificity onto its targets; how and to what extent a TF is involved in gene regulation. Comprehensive identification of the binding sites of sequence-specific TFs and follow-up molecular studies including gene expression microarrays will provide close answers to these questions.
Chromatin Immunoprecipitation coupled with recently developed high-throughput sequencing (ChIP-seq) allows us to perform genome-scale unbiased identification of the binding sites of sequence-specific TFs. Here, to gain insight into gene regulatory functions of TFs as well as their influences on gene expression, we conducted, in diverse cell lines, genome-wide identification of the binding sites of several sequence-specific TFs (CTCF, E2F4, MYC, Pol II) that are involved in a wide range of biological functions, including cell proliferation, development, apoptosis, genome stability, and DNA repair. Analysis of ChIP-seq data provided not only comprehensive binding profiles of those TF across the genome in diverse cell lines, but also revealed tissue-specific binding of CTCF, MYC, and Pol II as well as combinatorial usage among these three factors. Analyses also showed that some CTCF binding sites were inherited from parents to children and regulated in an individual-specific as well as allele-specific manner. Finally, genome-wide target identification of several TFs will broaden our understanding of the gene regulatory roles of these sequence-specific TFs. / text
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Étude structure / fonction des sous-unités catalytiques de l'ARN polymérase IIDomecq, Céline January 2008 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal
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Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase IILacombe, Andrée-Anne 11 1900 (has links)
L’ARN polymérase II (ARNPII), l’enzyme responsable de la transcription des ARN messagers, procède au décodage du génome des organismes vivants. Cette fonction requiert l’action concertée de plusieurs protéines, les facteurs généraux de la transcription, par exemple, formant un réseau d’interactions protéine-protéine, plusieurs étant impliquées dans la régulation de l’ARNPII à différents niveaux. La régulation de la transcription a été largement étudiée durant les quatre dernières décennies. Néanmoins, nous en connaissons peu sur les mécanismes qui régulent l’ARNPII avant ou après la transcription.
Dans la première partie de cette thèse, nous poursuivons la caractérisation du réseau d’interactions de l’ARNPII dans la fraction soluble de la cellule humaine, travail qui a débuté précédemment dans notre laboratoire. Ce réseau, développé à partir de la méthode de la purification d’affinité en tandem couplée à la spectrométrie de masse (AP-MS) et à des méthodes d’analyses bioinformatiques, nous amène une foule d’informations concernant la régulation de l’ARNPII avant et après son interaction avec la chromatine. Nous y identifions des protéines qui pourraient participer à l’assemblage de l’ARNPII telles des chaperonnes et les protéines du complexe R2TP/prefoldin-like ainsi que des protéines impliquées dans le transport nucléocytoplasmique. Au centre de ce réseau se trouvent RPAP4, une GTPase qui semble se positionner à l’interface entre ces protéines régulatrices et l’ARNPII. Nous avons donc entamé l’étude la fonction de RPAP4, ce qui nous a menés à la conclusion que RPAP4 est essentielle à l’import nucléaire de l’ARNPII au noyau, où elle exerce sa fonction. Nous avons également montré que les motifs G et GPN sont essentiels à la fonction de RPAP4. Le traitement des cellules avec le bénomyl nous montre aussi que la fonction de RPAP4 et l’import nucléaire de l’ARNPII requièrent l’action des microtubules.
La deuxième partie de la thèse s’intéresse à une autre protéine positionnée au centre du réseau, RPAP2. Cette dernière partage plusieurs interactions avec RPAP4. Elle est aussi essentielle à la localisation nucléaire de l’ARNPII et interagit directement avec celle-ci. RPAP4 et RPAP2 étant toutes deux des protéines cytoplasmiques qui font la navette entre le noyau et le cytoplasme, nous présentons des évidences que RPAP4 est impliquée dans l’export nucléaire de RPAP2 pour permettre à celle-ci d’être disponible dans le cytoplasme pour l’import de l’ARNPII dans le noyau.
Dans la troisième partie de la thèse, nous étudions plus en profondeur les modifications post-traductionnelles de RPAP4, ce qui nous aide à mieux comprendre sa propre régulation et sa fonction auprès de l’ARNPII. RPAP4 est phosphorylée en mitose par la MAP kinase ERK5. Cette phosphorylation favorise l’interaction entre RPAP4 et RPAP2, ce qui empêche RPAP2 d’interagir avec l’ARNPII pendant la mitose, prévenant du même coup, son interaction avec la chromatine pendant cette phase du cycle cellulaire où la transcription est presque inexistante. / RNA polymerase II, the enzyme responsible for transcription of messenger RNA, decodes the genome of living organisms. This function requires the concerted action of several proteins, including transcription factors, which form a protein-protein interaction network. Many of them are implicated in the regulation of RNAPII transcription. Although regulation of transcription has been largely studied during the last four decades, little is known about mechanisms that regulate RNAPII prior and after the transcription reaction.
In the first part of this thesis, we continue the characterization of the RNAPII interaction network of RNAPII in the soluble fraction of the human cell. This network, developed using tandem affinity purification method coupled with mass spectrometry (AP-MS) and bioinformatic analysis, provides a wealth of information about RNAPII regulation prior and after its interaction with chromatin for transcription. We identified proteins that can be involved in RNAPII assembly, including chaperones and the cochaperone complex R2TP prefoldin-like, and proteins involved in nucleocytoplasmic shuttling. RPAP4 is a GTPase that occupies a central position in this network being at the interface between these regulatory proteins and RNAPII. We therefore started to study the function of RPAP4, which lead us to conclude that RPAP4 is essential for RNAPII nuclear import. We also report that G domains and the GPN motif are essential for RPAP4 function. Treatment of the cells with benomyl suggests that microtubules are required for RPAP4 function and RNAPII nuclear import.
The second part concerns another protein found in the network that is also centrally positioned in the network, called RPAP2. RPAP2 shares many interactions with RPAP4. This protein is also essential for the nuclear import of RNAPII as it interacts directly with it. RPAP4 and RPAP2 being cytoplasmic proteins that shuttle between the cytoplasm and the nucleus, we show evidences that RPAP4 is implicated in RPAP2 nuclear export to make it available for RNAPII nuclear import.
In the third part, we study RPAP4 post-translational modifications, which help us to understand its own regulation and its function with RNAPII. RPAP4 is phosphorylated in mitosis by the MAP kinase ERK5. This phosphorylation promotes the interaction between RPAP4 and RPAP2. It prevents RPAP2 and RNAPII interaction and RNAPII chromatin localization in mitosis where transcription is mostly nonexistent.
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