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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Function/structure relationship study of trichosanthin, a Chinese medicinal protein, and its interaction with acidic ribosomal protein, PO. / CUHK electronic theses & dissertations collection

January 2006 (has links)
Previous research showed that the C-terminal tail of TCS can be deleted to generate a mini-TCS (C7-TCS) with antigenicity. The second topic of my study is to resolve the role of the C-terminal of TCS. Structure of C7-TCS showed that deletion of the C-terminal tail destabilizes the protein structure and makes Trp192 more solvent exposed. The relationship between the C-terminal tail and Trp192 was determined by mutating Trp192 to Phe in wild-type TCS and C7-TCS, generating W192F-TCS and W192F-C7-TCS. The crystal structure of C7-TCS, [W192F]-TCS and [W192F]-C7-TCS were determined and compared. Trp192 was identified as an important residue in stabilizing the conformation of TCS. Besides, the accumulative effect of Trp192 and the C-terminal tail is significant on the ribosome-inactivating activity. By comparing the structures, it was found that, the hydrogen bond formed by amino acids 240 and 35 seems to be essential for the structure and amino acid 240 should be a critical residue for the connection of the N-terminal and C-terminal domains in trichosanthin. / Ribosome-inactivating activity is the most important activity of TCS and RIPs. Therefore, the third topic of my study is to find the important of interaction between TCS and ribosomal proteins. Two ribosomal proteins, P0 and P1, have been identified previously to interact with TCS. By yeast two-hybrid screening, three cut of ten charge residues in TCS were identified to be the interaction sites between TCS and ribosomal protein P0. The interaction region was located on the surface of TCS near the entrance to the active pocket. The interaction with P0 was shown to be carried out by electrostatic interaction between the positively charge residues of TCS. However, the mutation of all the concerned residues in TCS gave only a mild reduction in inhibiting the protein synthesis of an in vitro reticulocyte translation system, showing that the interaction between TCS and P0 only plays a minor role in the ribosomal inactivating activity of TCS. / The first topic of my research is to find the role of Glu-85. The structure of [E85Q]-TCS and AMP complex was obtained. It is deduced that there are two sites for substrate binding in TCS, one is for recognition and another ion hydrolysis. The structure also indicated that protonation of substrate adenine is carried out by a water molecule in the active pocket of TCS during its N-glycosidase action. / Trichosanthin (TCS) is a Chinese medicinal protein isolated froth the root tuber of Trichosanthes kirilowi Maximowicz. It is a 27kDa protein with multiple pharmacological properties, including abortifacient, anti-tumor and anti-human immunodeficiency virus (HIV). It is believed that the pharmacological properties of TCS are related to ribosome-inactivation, by breaking, the specific glycosidic bond of adenine 4324 from the 28S rRNA. / Too Hiu Mei. / "February 2006." / Advisers: Pang-Chui Shaw; Kam-Bo Wong. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6213. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 164-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
22

Structure determination of ribosomal proteins and development of new methods in biomolecular NMR

Helgstrand, Magnus January 2001 (has links)
This thesis concerns different areas of biomolecular nuclearmagnetic resonance spectroscopy (NMR). In the first part of thethesis a new formalism for simulations of NMR pulse sequencesis introduced. The formalism is derived both from classicalmechanics and quantum mechanics and is presented forhomonuclear and heteronuclear spin systems. The formalism hasalso been adapted to systems in chemical exchange. Simulationsof pulse sequences should be more straightforward using the newformalism. In the second part of the thesis the NMR solution structuresof two ribosomal proteins are described. The ribosome isresponsible for protein production in all living cells and tounderstand the mechanism of the ribosome it is important toknow the three dimensional structure. In this thesis thestructures of S16 and S19, two of the proteins in the smallribosomal subunit, are presented. S16 is a mixed α /βprotein with a five-stranded parallel-antiparallel β-sheetand two α -helices. S19 is s mixed α/β proteinwith a three-stranded parallel-antiparallel β -sheet, oneα -helix and a short 310-helix. In the third part of the thesis a program for semiautomaticassignment of NMR-spectra is presented. Assigning resonances inthe NMR spectrum is a labor-intensive process, which can takelong time. In semiautomatic assignment a computer program aidsthe user in finding assignments but leaves all decisions to theuser, thus speeding up the process. The program described inthis thesis is a new version of ANSIG, called Ansig forWindows. The program runs on PCs under Windows and has severaltools for semiautomatic assignment. <b>Keywords:</b>nuclear magnetic resonance, structuredetermination, ribosomal proteins, NMR simulations, NMR theory,NMR assignment software, semiautomatic assignment
23

Ribosomal Proteins in Diamond-Blackfan Anemia : Insights into Failure of Ribosome Function

Badhai, Jitendra January 2009 (has links)
Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a defect in red blood cell production. The disease is associated with growth retardation, malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. In a cellular model for DBA, siRNA knock-down of RPS19 results in a relative decrease of other ribosomal (r) proteins belonging to the small subunit (RPS20, RPS21, RPS24) when compared to r-proteins from the large ribosomal subunit (RPL3, RPL9, RPL30, RPL38). RPS19 mutant cells from DBA patients show a similar and coordinated down-regulation of small subunit proteins. The mRNA levels of the small subunit r-proteins remain relatively unchanged. We also show that RPS19 has an extensive number of transcriptional start sites resulting in mRNAs of variable 5’UTR length. The short variants are translated more efficiently. Structural sequence variations in the 5’UTR of RPS19 found in DBA patients show a 20%-30% reduced translational activity when compared to normal transcripts. Primary fibroblast from DBA patients with truncating mutations in RPS19 or RPS24 showed specific cell cycle defects. RPS19 mutant fibroblasts accumulate in the G1 phase whereas the RPS24 mutant cells show a defect in G2/M phase. The G1 phase arrest is associated with a reduced level of phosphorylated retinoblastoma (Rb) protein, cyclin E and cdk2 whereas the G2/M phase defect is associated with increased levels of p21, cyclin E, cdk4 and cdk6. RPS19 interacts with PIM-1 kinase. We investigated the effects of targeted disruptions of both Rps19 and Pim-1 in mice. Double mutant (Rps19+/-, Pim-1-/-) mice have increased peripheral white- and red blood cell counts when compared to the wild-type mice (Rps19+/+, Pim-1+/+). Bone marrow cells in Rps19+/-, Pim-1-/- mice showed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl2, Bcl-xl and Mcl-1 and reduced levels of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. In summary, this thesis clarifies several mechanisms in the pathogenesis of DBA. Mutations in RPS19 results in coordinated down-regulation of several small subunit r-proteins causing haploinsufficiency for the small ribosomal subunit. RPS19 have multiple transcriptional start sites and mutations in the RPS19 5’UTR found in DBA patients result in reduced translational activity. At the cellular level, mutations in RPS19 and RPS24 cause distinct cell cycle defects and reduced cell proliferation. Finally, PIM-1 kinase and RPS19 cooperates in the proliferation of myeloid cells.
24

Structure determination of ribosomal proteins and development of new methods in biomolecular NMR

Helgstrand, Magnus January 2001 (has links)
<p>This thesis concerns different areas of biomolecular nuclearmagnetic resonance spectroscopy (NMR). In the first part of thethesis a new formalism for simulations of NMR pulse sequencesis introduced. The formalism is derived both from classicalmechanics and quantum mechanics and is presented forhomonuclear and heteronuclear spin systems. The formalism hasalso been adapted to systems in chemical exchange. Simulationsof pulse sequences should be more straightforward using the newformalism.</p><p>In the second part of the thesis the NMR solution structuresof two ribosomal proteins are described. The ribosome isresponsible for protein production in all living cells and tounderstand the mechanism of the ribosome it is important toknow the three dimensional structure. In this thesis thestructures of S16 and S19, two of the proteins in the smallribosomal subunit, are presented. S16 is a mixed α /βprotein with a five-stranded parallel-antiparallel β-sheetand two α -helices. S19 is s mixed α/β proteinwith a three-stranded parallel-antiparallel β -sheet, oneα -helix and a short 3<sub>10</sub>-helix.</p><p>In the third part of the thesis a program for semiautomaticassignment of NMR-spectra is presented. Assigning resonances inthe NMR spectrum is a labor-intensive process, which can takelong time. In semiautomatic assignment a computer program aidsthe user in finding assignments but leaves all decisions to theuser, thus speeding up the process. The program described inthis thesis is a new version of ANSIG, called Ansig forWindows. The program runs on PCs under Windows and has severaltools for semiautomatic assignment.</p><p><b>Keywords:</b>nuclear magnetic resonance, structuredetermination, ribosomal proteins, NMR simulations, NMR theory,NMR assignment software, semiautomatic assignment</p>
25

The Epstein-Barr virus nuclear antigens 1 & 5 : study of virus-host cellular protein interactions /

Forsman, Alma, January 2009 (has links)
Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.
26

Nuclear export and cytoplasmic maturation of the large ribosomal subunit

Lo, Kai-Yin, 1978- 24 March 2011 (has links)
The work in this thesis addresses the general problem of how ribosomal subunits are exported from the nucleus to mature in the cytoplasm. There are three parts in this dissertation. In the first part, I asked questions about the specificity for export receptors in the nuclear export of the large (60S) ribosomal subunit in yeast. In principle, I tethered different export receptors that are known to work in various unrelated export pathways to the ribosome by fusing them to the trans-acting factor Nmd3. Interestingly, all the chimeric receptors were able to support export, although to different degrees. Moreover, 60S export driven by these chimeric receptors was independent of Crm1, an export receptor that is essential for 60S export in wild-type cells. The second question I addressed in this project was whether or not a nuclear export signal could be provided in cis on ribosomal proteins (Rpls) rather than in trans by a transacting factor. The nuclear export signal (NES) of Nmd3 was fused to different ribosomal proteins and tested for support of 60S export. Several Rpl-NES fusion constructs worked to promote 60S export. Rpl3 gave the best efficiency. In conclusion, these results imply unexpected flexibility in the 60S export pathway. This may help explain how different export receptors could have evolved in different eukaryotic lineages. In the second part of my thesis, I identified the assembly pathway for the base of the ribosome stalk. The stalk is an important functional domain of the large ribosomal subunit because of its requirement for interaction with translation factors. Mrt4 is a nuclear paralog of P0, which is an essential part of the stalk. Here, I identified Yvh1 a novel ribosome biogenesis factor that is required for the release of Mrt4. Yvh1 is a conserved dual phosphatase, but the C-terminal zinc-binding domain rather than the phosphatase function was required for its activity to release Mrt4. Mrt4 localizes in the nucleus and nucleolus in the wild-type cells, but was persistent on cytoplasmic 60S subunits in yvh1[Delta] cells. The persistence of Mrt4 on the 60S subunits blocked the loading of P0 and assembly of the stalk. I also found the binding of Yvh1 depended on Rpl12, a protein that binds together with P0 to form the base of the stalk. Deletion of Rpl12 phenocopied yvh1[Delta]. These data identified the function of Yvh1 as a release factor of Mrt4. I also showed that the function of Yvh1 is conserved in human cells. In my final project, I analyzed the interdependence and order of the known cytoplasmic maturation events of the 60S subunit. 60S subunits require several maturation steps in the cytoplasm before they become competent in translation. There are four major steps involving two ATPases, Drg1 and Ssa1, and two GTPases, Efl1 and Lsg1. In my study, I ordered these steps into one serial pathway. Drg1 releases Rlp24 in the earliest step of 60S maturation in the cytoplasm. Truncation of the C-terminus of Rlp24 blocked cytoplasmic maturation of the large subunit by preventing the recruitment of Drg1 and led to a secondary defect in the release of Arx1 because of a failure to recruit Rei1. Deletion of REI1 mislocalized Tif6 from the nucleus and nucleolus to the cytoplasm and deletion of ARX1 suppressed the Tif6 mislocalization, indicating that the release of Arx1 was required for Tif6 release downstream. I found that mutation of efl1 or sdo1, the known release factors for Tif6, also blocked Nmd3 release. Tif6-V192F, which could bypass the growth defects of efl1 or sdo1 mutants, suppressed the defect of Nmd3 recycling. These results showed that the release of Tif6 was a prerequisite for Nmd3 release. Thus, the release of Nmd3 is downstream of the Tif6 release step. In conclusion, I have ordered the events of cytoplasmic maturation with Drg1 as the first step after ribosome export, followed by Rei1/Jji1 and then Sdo1/Efl1. The release of Nmd3 by Lsg1 appears to be the last step of ribosome maturation in the cytoplasm. Thus, the two ATPases Drg1 and Ssa work first and then the two GTPases Efl1 and Lsg1 work in a linear pathway of 60S maturation in the cytoplasm. / text
27

Étude de l'impact de l'activité traductionnelle sur le phénotype tumoral dans le cancer du côlon / Impact of translational activity on colon cancer cell phenotype

Yazdani, Laura 22 September 2017 (has links)
Avec près d’un million de nouveaux cas par an à travers le monde, le cancer colorectal est un problème de santé publique majeur. Il est la 2ème cause de mortalité par cancer en France, ce fort taux de mortalité étant relié à un pourcentage important de récidives et de métastases. L’hétérogénéité tumorale, la dissémination, la résistance aux traitements et la récidive seraient notamment dues à une population particulière de cellules tumorales appelées cellules souches cancéreuses (CSC). Ces cellules sont dotées d’une capacité d’adaptation extraordinaire et la compréhension des mécanismes moléculaires sous-tendant cette plasticité cellulaire est un objectif majeur pour concevoir de nouvelles stratégies thérapeutiques les ciblant spécifiquement. La synthèse protéique joue un rôle clé dans la carcinogénèse : d’une part, une synthèse protéique élevée est nécessaire à la prolifération des cellules tumorales, d’autre part, la traduction sélective favorise l’expression de protéines pro-oncogéniques. Considéré pendant des années comme un acteur passif de la traduction, le ribosome semblerait jouer un rôle majeur dans la régulation de la synthèse protéique. En effet, des études récentes ont montré que la composition du ribosome était « flexible » et permettrait de favoriser la traduction de certains ARN messager (ARNm). De plus, l’expression de certaines protéines ribosomiques (PR) varie entre le tissu sain et le tissu cancéreux, parfois même entre la tumeur initiale et la métastase. Ce projet vise à déterminer de quelle manière le contrôle traductionnel intervient dans plusieurs étapes clé du cancer colorectal, en se focalisant tout particulièrement sur l’acquisition ou la perte de propriétés propres aux CSC. Cette étude permettra de mieux comprendre les mécanismes moléculaires exploités par les CSC afin de résister aux traitements et s’adapter à leur environnement. De plus, ce projet pourrait mettre en évidence un rôle exercé par certaines protéines ribosomiques dans le contrôle traductionnel, à travers la filtration des ARNm par le ribosome. A plus long terme, il pourrait déboucher sur le développement de nouveaux traitements permettant de cibler spécifiquement la machinerie traductionnelle des CSC. / Despite significant advances in diagnostics and treatment, colorectal cancer (CRC) remains a major cause of mortality worldwide and occurrence of metastasis represents the primary cause of death. Metastasis process, chemoresistance and tumor recurrence is powered by a minor subpopulation of tumor cells endowed with self‐renewal and multi‐lineage differentiation ability: the cancer stem cells (CSC).Translation of mRNA into protein is the final step in gene-expression process, which mediates the formation of the translatome from genomic information. Several mechanisms, such as signaling pathways, translation factors availability, alternative open reading frame and alternative initiation pathways account for real time translatome remodeling. Moreover, an emerging concept suggests that ribosome is heterogeneous and can be "reprogrammed". These "specialized ribosomes" would preferentially engage certain mRNA at the expense of others and therefore drive cell phenotype and favor cell adaptation. Many studies have correlated deregulation of both translation machinery composition and activity with cancer initiation and evolution. From that perspective, CSC might well represent a perfect model to test whether the translation apparatus takes an active part in tumor initiation, progression, and metastasis. Our goal is to demonstrate that protein synthesis is differentially regulated depending on cancer cell subpopulation and determine whether ribosomal heterogeneity could influence tumoral evolution and plasticity. In a long run, we envision the development of novel therapies based on specific targeting of translational control in CSC.
28

Functional diversity within a ribosomal-like protein family in Arabidopsis thaliana / Diversité fonctionnelle au sein d'une famille de protéines de type ribosomique chez Arabidopsis thaliana

Wang, ChuanDe 27 November 2018 (has links)
L'expression des ARN mitochondriaux et chloroplastiques des plantes implique un grand nombre de modifications post-transcriptionnelles, parmi lesquelles l'épissage des introns est un processus essentiel. Sur la base de leur structure et des mécanismes d'épissage associés, les introns peuvent être classés en deux familles et ceux présents dans les organites des plantes appartiennent au groupe II. Les introns mitochondriaux et chloroplastiques de groupe II sont fortement dégénérés et ont perdu la capacité de s'auto-épisser in vivo. Leur élimination nécessite l’action de nombreux facteurs protéiques codés dans le noyau et importés dans les organites. Les protéines de liaison à l'ARN jouent un rôle prédominant dans ce processus complexe. Les protéines ribosomales sont des protéines abondantes se liant à l'ARN et peuvent être recrutées pour remplir diverses fonctions annexes. Au cours de ma thèse, j’ai étudié la fonction des protéines de type uL18 chez Arabidopsis, qui comprend 8 membres. Ces protéines partagent un domaine uL18 plutôt dégénéré, mais dont la structure est conservée, et dont la fonction initiale est de permettre l’association avec l’ARNr 5S. Nos résultats ont montré que cinq protéines de type uL18 sont adressées aux mitochondries et trois aux chloroplastes. Deux d’entre elles correspondent à de véritables protéines ribosomales uL18 associées aux ribosomes des organites, tandis que deux autres (uL18-L1 et uL18-L8) se sont transformées en facteurs d'épissage et sont nécessaires à l'élimination d’introns mitochondriaux ou chloroplastiques spécifiques. L'analyse d'un troisième membre de la famille, uL18-L5, a révélé qu'il participait à l'épissage de nombreux introns mitochondriaux. Mes résultats ont permis de révéler que les facteurs dérivés des protéines ribosomales uL18 jouent un rôle essentiel dans l’épissage des introns du groupe II mitochondriaux ou chloroplastiques chez les végétaux et que ces fonctions ciblent sot un seul intron ou bien plusieurs d’entre eux. / RNA expression in plant organelles implies a large number of post-transcriptional modifications in which intron splicing is an essential process. Based on RNA structures and splicing mechanisms, introns can be classified into two families and organellar introns of seed plants are categorized as group II. Organellar group II introns are highly degenerate and have lost the ability to self-splice in vivo. Their removal from transcripts is thus facilitated by numerous nuclear-encoded proteins that are post-translationaly imported into organelles. Among them, RNA binding proteins play predominant roles in this complex process. Ribosomal proteins are abundant RNA-binding proteins and could be recruited to carry out multifarious auxiliary functions. During my thesis, I investigated the function of the uL18 ribosomal-like protein family in Arabidopsis that comprises 8 members. The members of this protein family share a rather degenerate but structurally conserved uL18 domain whose original function is to permit association with the 5S rRNA. Our results showed that five uL18-Like proteins are targeted to mitochondria and three to chloroplasts. Two of these proteins correspond to real ribosomal uL18 proteins that incorporate into organellar ribosomes, while two other members (uL18-L1 and uL18-L8) have turned into splicing factors and are required for the removal of specific mitochondrial or plastid group II introns. The analysis of a third member, uL18-L5, revealed that it participated in the splicing of numerous mitochondrial introns. Our results revealed that uL18-like factors play essential roles in group II intron splicing in both mitochondria and plastids of plants and that these functions could target a single or multiple introns.
29

Identification de déterminants impliqués dans la différenciation des cellules souches embryonnaires

Fortier, Simon 12 1900 (has links)
Les cellules souches ont attiré l’attention du public ces dernières années, grâce non-seulement à leur utilisation comme thérapies visant à s’attaquer à certains types de cancers, mais aussi en relation avec leur potentiel dans le domaine de la médecine regénérative. Il est établi que le destin cellulaire des cellules souches embryonnaires (ESC) est régulé de façon intensive par un groupe de facteur clés agissant sur leur pluripotence. Il est néanmoins envisageable que certains déterminants influençant l’auto-renouvellement et la différenciation de ces cellules soient toujours inconnus. Afin de tester cette hypothèse, nous avons généré, en utilisant une méthode par infections virales, une collection de ESC contenant des délétions chromosomales chevauchantes que nous avons baptisée DelES (Deletion in ES cells). Cette librairie contient plus de 1000 clones indépendants dont les régions délétées couvrent environ 25% du génome murin. À l’aide de cette ressource, nous avons conduit un criblage de formation de corps embryoïdes (EB), démontrant que plusieurs clones délétés avaient un phénotype de différenciation anormal. Nos études de complémentation sur un groupe de clones ont par la suite permis l’identification de Rps14 - un gène codant pour une protéine ribosomale (RP) comme étant haploinsuffisant pour la formation de EB. Dans un deuxième temps, l’analyse approfondie des résultats de notre crible a permis d’identifier un groupe de gènes codants pour des RP qui semblent essentiels pour la différenciation des ESC, mais dispensables pour leur auto-renouvellement. De manière intéressante, les phénotypes anormaux de formation en EB les plus marqués sont associés à des délétions de RP qui se retrouvent au site de sortie des ARN messagers (ARNm) du ribosome, soit Rps5, Rps14 et Rps28. Étonnament, alors qu’un débalancement des RP conduit généralement à une réponse de type p53, l’haploinsuffisance de ces trois gènes ne peut être renversée par une simple réduction des niveaux d’expression de ce gène suppresseur de tumeurs. Finalement, nos études de profilage polysomal et de séquençage à haut-débit montrent une signature spécifique de gènes liés au mésoderme chez un clone hétérozygote pour Rps5, suggérant ainsi une explication au phénotype de différenciation p53-indépendant identifié chez ces ESC. Nos travaux rapportent donc la création d’une ressource intéressante de génomique fonctionnelle qui a permis de mettre à jour le rôle essentiel que jouent les RP dans le processus de formation de EB. Nos résultats permettent aussi de documenter une réponse p53-indépendante suite à un débalancement de RP dans un contexte opposant l’auto-renouvellement et la différenciation des ESC. / Stem cells have captured public’s attention in the last years, thanks to their involvement in cancer therapies and also their huge theoretical potential in the regenerative medicine field. In order to translate this new technology to the clinic, a better understanding of their regulatory mechanisms is still needed. It is well established that mouse embryonic stem cell (ESC) fate is highly regulated by core pluripotency factors. However, it is conceivable that novel self-renewal or differentiation regulators are not yet described. To investigate this possibility, we used a viral-based approach to generate a collection of ESC with nested chromosomal deletions called DelES (Deletion in ES cells). This library contains more than a thousand independent ESC clones highly enriched in chromosomal deletions which together cover ~25% of the mouse genome. Using this resource, we conducted an embryoid body (EB) differentiation screen and showed that several clones were having an abnormal EB formation phenotype. Complementation studies later identified Rps14-a ribosomal protein (RP) coding gene- as a novel haploinsufficient gene in EB formation from undifferentiated ESC. Further analyses of our screen results showed a strong bias for a subset of small subunit ribosomal protein genes which are critical for ESC differentiation but not for their self-renewal activity. Interestingly, the most severe differentiation phenotypes were found with ribosomal proteins associated to the ribosome’s mRNA exit site, namely Rps5, Rps14 and Rps28. While RP gene imbalance often leads to a p53 response that can be corrected by p53 suppression, ESC clones with decreased expression of mRNA exit site RP genes were surprisingly insensitive to p53 reduction, but were rescued by BAC or cDNA complementation, thus confirming the causative nature of these genes in the ESC phenotype. Finally, polysomal profiling and RNA-Seq studies showed that Rps5 deleted ESC exhibit an abnormal mesodermal gene signature. Together, our work presents a highly valuable resource for functional genomic studies in ESC and also highlights a novel p53-independent role linked to RP gene imbalance. Our results shed light on the relevance of these subunits for the developmental transition of ESC from a pluripotent to a differentiated state.
30

Removal and Replacement of Ribosomal Proteins : Effects on Bacterial Fitness and Ribosome Function

Tobin, Christina January 2011 (has links)
Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.

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