No description available.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein α (C/EBPα) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBPα. Additionally, the original clone found to interact with C/EBPα consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBPα mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBPα, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBPα-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBPα-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBPα. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBPα was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBPα, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBPα.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein α (C/EBPα) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBPα. Additionally, the original clone found to interact with C/EBPα consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBPα mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBPα, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBPα-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBPα-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBPα. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBPα was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBPα, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBPα.

No description available.

Nous montrons dans cette étude que dans les cellules déplétées pour la nucléoline, une plus faible accumulation de pré-ARNr est associée à une augmentation de marques d’hétérochromatine (H3K9me2) et une diminution de marques d’euchromatine (H4K12ac et H3K4me3) sur la chromatine des gènes ribosomiques. Des expériences de ChIP-seq montrent que la nucléoline est enrichie dans la région codante et promotrice de l’ADNr et est préférentiellement associée avec les gènes non méthylés des ARNr. La déplétion de la nucléoline entraîne une accumulation de l’ARN Pol I au début de l’ADNr et une diminution de UBF sur la région codante et promotrice. La nucléoline interfère avec la liaison de TTF-1 sur le promoteur-proximal T0, inhibant ainsi le recrutement de TIP5 du complexe NoRC, et établissant un état d’hétérochromatine répressive. Ces résultats révèlent l’importance de la nucléoline dans le maintien d’un état euchromatinien des ADNr et dans l’élongation de la transcription. Nous montrons aussi dans cette thèse que l’acétylation est une nouvelle modification post-traductionnelle de la nucléoline. Des études d’immunofluorescence utilisant l’anticorps anti nucléoline acétylée montrent que la nucléoline acétylée est exclue des nucléoles. De plus, par ChIP-seq nous n’avons jamais pu détecter d’association significative de la nucléoline acétylée sur la chromatine des ADNr. Aussi, nous n’avons détecté aucune activation de la transcription de Pol II sur des matrices de chromatine avec la nucléoline acétylée. Nous trouvons une distribution de la nucléoline acétylée majoritairement dans le nucléoplasme où elle co-localise parfaitement avec le facteur d’épissage SC35, et partiellement avec les structures marquées avec un anticorps dirigé contre Y12, mais ne co-localise pas avec des structures contenant la coïline, ce qui suggère que cette fraction de la nucléoline pourrait être impliquée dans la synthèse ou le métabolisme des pré-ARNm. / Here we have shown that, in nucleolin depleted cells, lower accumulation of pre-rRNA is associated with the increase in heterochromatin marks (H3K9me2) and decrease of the euchromatin histone marks (H4K12Ac and H3K4me3) in rDNA chromatin. ChIP-seq experiments show that nucleolin is enriched in the coding and promoter region of the rDNA and is preferentially associated with the unmethylated rRNA genes. Nucleolin knockdown results in the accumulation of RNAPI at the beginning of the rDNA and a decrease of UBF in the coding and promoter regions. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the NoRC subunit TIP5 and HDAC1 and establishing a repressive heterochromatin state. These results reveal the importance of nucleolin in the maintenance of the euchromatin state of rDNA and transcription elongation.In this thesis we have also shown that acetylation is a novel post-translational modification of nucleolin. Immuno-fluorescence studies using anti-acetylated nucleolin antibody illustrated that acetylated nucleolin is excluded from nucleoli and interestingly, neither could we detect any significant binding of ac-nucleolin on rDNA chromatin by doing ChIP-Seq, nor did we detect any activation of Pol II transcription with ac-nucleolin from DNA and chromatin templates. Moreover, we found acetylated nucleolin had a predominant nucleoplasmic distribution where it associates with the splicing factor SC35 and partially with the structures labeled with Y12 antibody, but not with coilin containing structures.

<p>\abstract</p><p>Transcription and translation describe the flow of genetic information from DNA to mRNA to protein. Recent studies show that at a single cell level, these processes are stochastic, which results in the variation of the number of mRNA and proteins even under identical environmental conditions. Because the number of mRNA and protein in each single cell are actually very small, these variations can be crucial for cellular function in diverse contexts, such as development, stress response, immunological and nervous system function. Most studies examine the origin and effects of stochastic gene expression using computer simulations. My goal is to develop a theoretical framework to study activity-dependent gene expression using simplified models that capture essential features. </p><p>I have examined the dynamics of stochastic gene regulation in three contexts. First, I examine how fluctuations in promoter accessibility lead to "bursty" transcription, during which genes are turned "on" or "off" stochastically. I describe a mathematical formalism to represent bursty gene expression in a coarse-grained manner as a Markov process and derive a master equation for the time evolution of the probability distribution of the number of mRNA molecules. This allows us to examine how transcript number responds to time varying stimuli. This model forms a basic building block for understanding the signal transmission and noise of the transcription process to time varying inputs as would be sensed by cells in dynamic environments. In addition to synthesis, gene expression is subject to additional modes of regulation. One such mechanism that controls transcript numbers is by microRNAs (miRNAs), which pair with target mRNAs to repress protein production following transcription. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. I explore the functional roles of feedback regulation by miRNAs and show that protein fluctuations strongly depend on the mode of miRNA-mediated repression. I discuss the functional implications of protein fluctuations arising from miRNA-mediated repression on gene regulatory networks. Finally, I examine the impact of fluctuations on alternative splicing, which is a major source for proteomic complexity in higher eukaryotes. Although the proteins regulating alternative splicing have been extensively studied, little is known about how noise arising from the stochastic nature of alternative splicing contributes to the entire gene expression process. I explore the functional roles and noise properties of alternative splicing, focusing on the case of exon skipping and intron retention. I show that while the overall counts of the mRNAs of the two isoforms are independent and Poisson distributed, diffusion and binding of the splicing factors contributes to the variance in the abundance of the isoforms. </p><p>Noise in gene expression may be of particular relevance in the nervous system. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify neuronal excitability and connectivity, ultimately influencing higher order brain function. Finally, I examine the implications of our studies for activity dependent gene expression in the nervous system.</p> / Dissertation

No description available.

No description available.

Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.

Macromolecular assemblies such as the ribosome, spliceosome, polymerases are imperative for cellular functions. The current understanding of these important machineries and many other assemblies at the molecular level is poor. The lack of structural data for many macromolecular assemblies further causes a bottleneck in understanding the cellular processes and the various disease manifestations. Hence, it is essential to characterize the structures and molecular architectures of these macromolecular assemblies. Though the number of 3-D structures for individual proteins structures or domains in the Protein Data Bank (PDB) is growing, the number of structures deposited for macromolecular assemblies is relatively poor. Hence, apart from the use of experimental techniques for characterizing macromolecular assembly structures, the use of computational techniques would help in supplementing the growth of macromolecular assembly structures. This thesis deals with the use of integrative approaches where computational methods are combined with experimental data to model and understand the mechanistic features of macromolecular assemblies with a special focus on a sub-complex of the spliceosome machinery. Chapter 1 of this thesis provides an introduction to protein-protein interactions and macromolecular assemblies. Further, the modelling of macromolecular assemblies using integrative methods are discussed, with a subsequent introduction to the spliceosome machinery. In chapter 2, modelling studies were performed on the proteins involved in the general amino acid control mechanism, which is triggered in yeast under amino acid starvation conditions. The proteins involved in the study were Gcn1, a ribosome binding protein and the RWD-domain containing proteins Gcn2, Yih1, Gir2 and Mtc5. From laboratory experiments it is known that in order for Gcn2 activation, an eIF2α kinase, its RWD-domain has to bind to Gcn1 and the residue Arg-2259 is important for this interaction. As the 3-D structure for the Gcn1 region containing Arg-2259 is not currently available, its 3-D structure was inferred using fold recognition and comparative modelling techniques. Further, in order to understand the Gcn2 RWD domain-Gcn1 molecular interaction, a complex structure was inferred by using a restrained protein-protein docking procedure. As the proteins, Yih1 and Gir2 are known to bind to Gcn1 using their RWD-domains, first the structures of the RWD-domain containing proteins including Mtc5 were inferred using a Gcn2 RWD domain NMR structure. Additionally, the Gcn1-Gcn2 complex was used to build a set of complexes to explain the binding of other RWD domain containing proteins Yih1, Gir2 and Mtc5. The important molecular interactions were obtained on analysing the interacting residues in these complexes. Thus, the Gcn1-Gcn2 interaction at the molecular level has been proposed for the first time. Future experiments guided by the protein-protein complex models and the proposed set of mutations should provide an understanding about the critical molecular interactions involved in the general amino acid control mechanism. Chapter 3 describes an integrative approach that was used to decipher a pseudo-atomic model of the closed form of human SF3b complex. SF3b is a multi-protein complex containing seven components – p14, SF3b49, SF3b155, SF3b145, SF3b130, SF3b14b and SF3b10. It recognizes the branch point adenosine in the pre-mRNA as part of U2 snRNP or U11/U12 di-snRNP in the spliceosome. Although, the cryo-EM map for human SF3b complex has been available for more than a decade, the structure and relative spatial arrangement of all components in the complex are not yet known. The integrative modelling approach used here involved utilizing structural data in the form of available X-ray and NMR structures, fold recognition and comparative modelling as well as currently available experimental datasets, along with the available cryo-EM density map to provide a model with high structural coverage. Hence, the molecular architecture of closed form human SF3b complex was derived that can now provide insights into the functioning of SF3b in splicing. This might also help the future high resolution structure determination efforts of the entire human spliceosome machinery In chapter 4, the molecular architecture of the closed form of SF3b complex obtained from the use of integrative modelling approach (Chapter 3) is extensively discussed. The structure-function relationships for some of the SF3b components based on the pseudo-atomic model has also been provided. In addition, the extreme flexibility associated with some of the SF3b components based on dynamics analysis has also been examined. Further, using an existing U11/U12 di-snRNP cryo-EM map and the closed form SF3b complex pseudo-atomic model, an open form of the SF3b complex was modelled and the component structures were fit into it. Hence, it was found that the transition between closed and open forms is primarily caused by a flap containing the HEAT repeat protein, SF3b155. This Protein is also known to harbour cancer causing mutations and has the potential to affect the Closed to open transition as well as SF3b complex structure and stability. Thus, this provides a framework for the future understanding of the closed to open transition in SF3b functioning within the spliceosome. Chapter 5 builds upon the integrative modelling approach (Chapter 3) that proposed the molecular architecture of the closed form of human SF3b complex and an open form of SF3b that was derived due to a flap opening of the closed form and which might help in accommodating RNA and other trans-acting factors within the U11/U12 di-snRNP (Chapter 4). In the current chapter, the SF3b open form and its interaction with the RNA elements is studied. The 5' end of U12 snRNA and its interaction with pre-mRNA in branch point duplex was modelled guided by the open form of SF3b that provided the necessary structural constraints and the RNA model is topologically consistent with the existing biochemical data. Further, utilizing the SF3b opens form-RNA model and the existing experimental knowledge, an extensive discussion has been provided on how the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation as well as its potential implications for branch point adenosine recognition fidelity. Moreover, the reasons for SF3b to be defined as a “fuzzy” complex - a complex with highly flexible folded regions along with intrinsically disordered regions is also discussed. Hence, the current work adds to the excellent developments made previously and deepens the understanding of the structure-function relationship of the human SF3b complex in the context of the spliceosome machinery. In chapter 6, a methodology has been proposed for the use of evolutionary conservation of protein-protein interfacial residues in multiple protein cryo-EM density based fitting of the protein components in the low-resolution density maps of multi-protein assemblies. First, the methodology was tested on a dataset of simulated density maps generated at four different resolutions -10, 15, 20 and 25 Å. On utilizing the evolutionary conservation scores obtained from multiple sequence alignments to score the fitted complexes, it was found that there was a decrease in the conservation scores when compared to that of the crystal structures, which were used to generate the simulated density maps. Further, the assessment of the multiple protein density fitting technique to align the actual protein-protein interface residues correctly using a performance metric called F-measure showed there was a decrease in performance as the resolutions became poorer. Hence, based on evolutionary conservations scores as well as F-measure the decrease in conservation scores or performance was found to be mainly due to the errors associated with the fitting process. Subsequently, a refinement methodology was designed involving the use of conservation scores, which improved the accuracy of the fitted models and the same, was observed in an experimental cryo-EM density test case of RyR1-FKBP12 complex. Hence, the conservation information acts as an effective filter to distinguish the incorrectly fitted structures and improves the accuracy of the fitting of the protein structures in the density maps. Thus, one can incorporate the conserved surface residues information in the current density fitting tools to reduce ambiguity and improve the accuracy of the macromolecular assembly structures determined using cryo-EM. In the concluding chapter 7, the learnings on the structural and mechanistic features of protein assemblies obtained from the use of computational techniques and integration of experimental datasets is discussed. In chapter 2, the modelling of a binary macromolecular complex such as the Gcn1-Gcn2 complex was performed using computational structure prediction strategies to understand the molecular basis of its interaction. Due to the potential inaccuracies which can exist in computational modelling, the chapters 3 to 5 dealt with the use of integrative approaches, primarily guided by the cryo-EM map, in order to decipher the molecular architecture of the human SF3b complex in the closed and open forms as well as its contribution for branch point adenosine recognition. Based on the extensive experience gained in modelling of assemblies using cryo-EM data in the previous chapters, a new method has been proposed on the use of evolutionary conservation information to improve the accuracy of cryo-EM density based fitting. Hence, these studies have provided strategies for modelling macromolecular assemblies as well as a deeper understanding of its mechanistic features.