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Probing ribosomal RNA structural rearrangements : a time lapse of ribosome assembly dynamicsBurlacu, Elena January 2016 (has links)
Ribosome synthesis is a very complex and energy consuming process in which pre-ribosomal RNA (pre-rRNA) processing and folding events, sequential binding of ribosomal proteins and the input of approximately 200 trans-acting ribosome assembly factors need to be tightly coordinated. In the yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus with the formation of a very large 90S-sized complex. This ~2.2MDa pre-ribosomal complex is subsequently processed into the 40S and 60S assembly intermediates (pre-40S and pre-60S), which subsequently mature largely independently. Although we have a fairly complete picture of the protein composition of these pre-ribosomes, still very little is known about the rRNA structural rearrangements that take place during the assembly of the 40S and 60S subunits and the role of the ribosome assembly factors in this process. To address this, the Granneman lab developed a method called ChemModSeq, which made it possible to generate nucleotide resolution maps of RNA flexibility in ribonucleoprotein complexes by combining SHAPE chemical probing, high-throughput sequencing and statistical modelling. By applying ChemModSeq to ribosome assembly intermediates, we were able to obtain nucleotide resolution insights into rRNA structural rearrangements during late (cytoplasmic) stages of 40S assembly and for the early (nucleolar) stages of 60S assembly. The results revealed structurally distinct cytoplasmic pre-40S particles in which rRNA restructuring events coincide with the hierarchical dissociation of assembly factors. These rearrangements are required to trigger stable incorporation of a number of ribosomal proteins and the completion of the head domain. Rps17, one of the ribosomal proteins that fully assembled into pre-40S complexes only at a later assembly stage, was further characterized. Surprisingly, my ChemModSeq analyses of nucleolar pre-60S complexes indicated that most of the rRNA folding steps take place at a very specific stage of maturation. One of the most striking observations was the stabilization of 5.8S pre-rRNA region, which coincided with the dissociation of the assembly factor Rrp5 and stable incorporation of a number of ribosomal proteins.
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Understanding tumour suppressive responses upon inhibition of ribosome maturationPantazi, Asimina January 2018 (has links)
Ribosome biogenesis is an essential biological process that is required for cell division and growth. Cancer cells alter their physiology in order to meet their excessive growth demands and therefore maintain abnormal metabolism and homeostasis. Under normal conditions, ribosome biogenesis is tightly regulated to maintain adequate ribosomal content of the cell. However, several oncogenes promote this process and elevated ribosome biogenesis is often found in cancer cells, where it can support the high biosynthetic demand of these cells. Hence, ribosome biogenesis is a process that might provide candidate targets for therapeutic intervention. The main aim of this research was to assess whether inhibition of late stage biogenesis of the 60S ribosomal subunit would result in tumour suppressive responses in normal and cancer cells. We focused upon two GTPases, EFL1 and LSG1, that catalyse the last two cytoplasmic reactions in the maturation of the 60S subunit. We observed that RNAi-based silencing of the GTPases in human lung fibroblasts triggered growth arrest and senescence, which was mediated by the p16 and p53 pathways. Inhibition of these pathways revealed that loss of p53 could bypass the senescence response. However, when cells were plated at low density, knockdown of LSG1 conferred a tumour suppressive response, even in the absence of p53. Knockdown of LSG1 in MCF-10A mammary epithelial cells that lack the p16 locus also induced a robust senescence response and this was also observed in transformed derivatives of MCF-10A cells. Preliminary data obtained in a 3D mammosphere culture model also revealed that inhibition of 60S maturation could elicit an antiproliferative response. Taken together, these data indicate that at least some cancer cells would be responsive to a therapy based upon inhibition of 60S subunit biogenesis. We further characterised the senescence response that was obtained through knockdown of LSG1 by performing gene expression analysis. This revealed a minimal Senescence-Associated Secretory Phenotype (SASP) that was restricted to members of the TGF-β family and lacked the canonical pro-inflammatory cytokines and chemokines that are found in the SASP of cells undergoing oncogene-induced senescence (OIS). Surprisingly, we also observed a dramatic increase in expression of multiple genes in the cholesterol biosynthesis pathway, although inhibition of this pathway indicated that cholesterol biosynthesis was not required for the senescence response. Further insight into the mechanisms of induction of the ribosomal stress senescence response was sought through pilot CRISPR screen and reverse phase protein array (RPPA) analyses. These revealed some interesting leads that will direct future studies.
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Identification de protéines associées à la RNA Hélicase DBP4 chez la levure saccharomyces cerevisiaeBa, Korka January 2007 (has links) (PDF)
La protéine nucléolaire Dbp4 est impliquée dans la biogenèse de l'ARN ribosomique (ARNr) 18S chez la levure Saccharomyces cerevisiae. Elle présente une interaction génétique avec le petit ARN nucléolaire (snoRNA) U14. Dbp4 est une RNA hélicase de la famille «DEAD-box» et elle est hautement conservée dans l'évolution. Son homologue chez l'humain (DDX10) serait impliqué dans certains types de cancers. La plupart des «DEAD-box » RNA hélicases sont essentielles à la croissance de la levure indiquant ainsi une absence de redondance de leurs fonctions sur leurs substrats spécifiques. De façon plus intéressante, il a été démontré que plusieurs hélicases seraient aussi impliqués dans la biogenèse des ribosomes. Les protéines «DEAD-box» sont caractérisées par la présence de neuf motifs conservés formant la région centrale catalytique de l'enzyme. Cette dernière est flanquée par deux extensions de composition variable aux extrémités N- et C-terminales. Ces extensions joueraient un rôle déterminant dans la reconnaissance du substrat et/ou la liaison de cofacteurs. Nos analyses bioinformatiques prédisent la présence d'un domaine d'interaction protéine-protéine appelé domaine «coiled-coil» (CC) à chacune des extrémités N-et C-terminale. Notre hypothèse est que Dbp4 serait associé à des protéines par l'intermédiaire de ces domaines CC. Afin d'identifier les partenaires protéiques potentiels de Dbp4, nous avons utilisé le système double hybride chez la levure en criblant une banque génomique de Saccharomyces cerevisiae. Parmi les clones positifs, nous avons identifié la présence des protéines Ifh1, Rrn5, Rps20, Rps2 et Msb2. Curieusement à l'exception de Msb2, toutes ces protéines sont impliquées dans la biogenèse des ribosomes et renferment des domaines CC comme Dbp4. Cela suggère que ces protéines sont des partenaires potentiels de Dbp4 pour la biogenèse des ribosomes. Pour confirmer ces résultats de double hybride, nous avons réalisé des tests in vivo par co-immunoprécipitation dont les résultats préliminaires n'ont pas été concluants. Des études complémentaires seront nécessaires afin d'éclaircir ces résultats. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Saccharomyces cerevisiae, Nucléole, Ribosomes, RNA hélicases, Dbp4.
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Examination of eukaryotic chaperonin-mediated nascent chain folding in the cytosol: a photocrosslinking approachEtchells, Stephanie Anne 15 November 2004 (has links)
TRiC (TCP-1 ring complex), a type II chaperonin, facilitates protein folding, and we previously showed that TRiC crosslinks to ribosome-bound actin and luciferase nascent chains. Here, it was found that actin and luciferase nascent chains were adjacent to more than one TRiC subunit at different stages of translation. Six and seven out of the eight TRiC subunits were photocrosslinked to the luciferase and actin nascent chains, respectively. Actin nascent chains with widely-spaced, site-specific probe locations were adjacent to the same three TRiC subunits (a, b and e) at different stages of translation. The exposure of other TRiC subunits to nascent chains varied with the length and identity of the nascent chain. In addition, the presence or absence of ATP influences the photocrosslinking yields. This suggests that ATP alters the conformation of the subunits and/or their affinity for the nascent chain. Photocrosslinking also revealed that TRiC is in close proximity to the exit site of the ribosomal tunnel, presumably to create a protected folding environment for the nascent chain.
Immunoprecipitations under native conditions revealed that prefoldin photocrosslinks to the actin nascent chain and that these prefoldin-containing photoadducts are coimmunoprecipitated with antibodies specific for the TRiC a subunit. This result suggests that prefoldin and TRiC bind simultaneously to the same actin nascent chain. Photocrosslinking studies with probes at position 68 in the actin nascent chain revealed that prefoldin binds to the nascent chain subsequently to TRiC binding.
An unknown protein with an apparent molecular mass of 105 kDa was shown to photocrosslink to the luciferase nascent chain in a length-dependent manner at specific probe locations close to the N-terminus of the nascent chain.
Thus, the nascent chain sees a variety of proteins in its immediate environment as it emerges from the ribosomal tunnel and undergoes its chaperonin-assisted folding.
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From knobs to a central pseudoknot : understanding 40S ribosomal subunit biogenesis through Bud23Sardana, Richa 26 August 2015 (has links)
Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text
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Organization of mitochondrial gene expression in yeast : Specific features of organellar protein synthesisKehrein, Kirsten January 2014 (has links)
Mitochondria contain their own genetic system, encoding key subunits of the oxidative phosphorylation system. These subunits are expressed by an organelle-specific gene expression machinery. This work revealed a number of fundamental aspects of mitochondrial gene expression and provides evidence that this process is organized in a unique and organelle-specific manner which likely evolved to optimize protein synthesis and assembly in mitochondria. Most importantly, improving the experimental handling of ribosomes we could show that mitochondrial ribosomes are organized in large assemblies that we termed MIOREX complexes. Ribosomes present in these complexes organize gene expression by recruiting multiple factors required for post-transcriptional steps. In addition, we could reveal mechanisms by which ribosome-interactor complexes modulate and coordinate the expression and assembly of the respiratory chain subunits. For example we showed that the Cbp3-Cbp6 complex binds to the ribosome in proximity to the tunnel exit to coordinate synthesis and assembly of cytochrome b. This location perfectly positions Cbp3-Cbp6 for direct binding to newly synthesized cytochrome b and permits Cbp3-Cbp6 to establish a feedback loop that allows modulation of cytochrome b synthesis in response to assembly efficiency. Likewise the interaction of the membrane-anchor proteins Mba1 and Mdm38 with the tunnel exit region enables them to participate in the translation of the two intron-encoding genes COX1 and COB in addition to their role in membrane insertion. In summary, work presented in this thesis shows that mitochondrial gene expression is a highly organized and regulated process. The concepts and technical innovations will facilitate the elucidation of many additional and important aspects and therefore contribute to the general understanding of how proteins are synthesized in mitochondria. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>
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The role of bystin in embryo implantation and in ribosomal biogenesisFUKUDA, Michiko N, MIYOSHI, Masaya, NADANO, Daita January 2007 (has links)
No description available.
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Modulation and Ligand Selectivity of Mammalian Odorant ReceptorsJiang, Yue January 2015 (has links)
<p>In mammals, the perception of smell starts with the activation of odorant receptors (ORs) by volatile molecules in the environment. Mammalian genomes typically encode large numbers of ORs, with approximately 400 intact ORs in human and more than 1000 in mouse. Central to the question of how olfactory stimuli are represented at the peripheral level is defining the ligand selectivity and activity regulation of ORs.</p><p>Processing of chemosensory signals in the brain is dynamically regulated in part by an animal’s physiological state. The Matsunami lab previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. In chapter 2, I demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.</p><p>Understanding odor coding requires comprehensive mapping between odorant receptors and corresponding odorants. In chapter 3, I present a high-throughput in vivo method to identify repertoires of odorant receptors activated by odorants, using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor-stimulated mice followed by RNA-Seq. This approach screens endogenously expressed odorant receptors against an odorant in one set of experiments, using awake and freely behaving mice. In combination with validations in a heterologous system, we identify sets of odorant receptors for two odorants, acetophenone and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), encompassing 69 receptor-odorant pairs. I also identified shared amino acid residues specific to the acetophenone or TMT receptors, and developed a model to predict receptor activation. This study provides a means to understand the combinatorial coding of odors in vivo.</p> / Dissertation
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Studies of Aminoglycoside AntibioticsZhu, Hongkun 30 September 2016 (has links)
No description available.
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Nascent Peptides That Induce Translational ArrestWoolstenhulme, Christopher J 01 March 2014 (has links) (PDF)
Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. Certain proteins are capable of stalling the ribosome during their own synthesis. Stalling events are used by both prokaryotic and eukaryotic cells to regulate gene expression. Characterization of natural stalling peptides shows that only a few strategically placed amino acids are needed to inactivate the ribosome. These motifs share little sequence similarity suggesting that there are more stalling motifs yet to be discovered. Here we use two genetic selections in E. coli to discover novel stalling peptides and detail their subsequent characterization. Kinetic studies show that some of these nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, such as polyproline sequences, peptidyl transfer to a subset of aminoacyl-tRNAs is inhibited. Translation factor EF-P alleviates pausing of the polyproline motifs, but has little or no effect on other stalling sequences. The EF-P ortholog eIF5A also alleviates pausing of polyproline sequences in yeast. Our studies show that short peptides sequences are capable of stalling the ribosome during elongation and termination through different mechanisms. These sequences are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.
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