Spelling suggestions: "subject:"abinding 2proteins"" "subject:"abinding 1proteins""
101 |
Understanding Assembly of AGO2 RISC: the RNAi enzyme: a DissertationMatranga, Christian B. 17 September 2007 (has links)
In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double stranded RNA (dsRNA) can trigger silencing of cognate sequences when injected into the nematode, Caenorhabditis elegans (Fire et al. 1998). This data explained observations seen years earlier by other worm researchers, and suggested that repression of pigmentation in plants was caused by a dsRNA-intermediate (Guo and Kemphues 1995; Napoli et al. 1990). The phenomenon––which soon after was coined RNA interference (RNAi)––was soon discovered to be a post-transcriptional surveillance system in plants and animals to remove foreign nucleic acids.
|
102 |
Purification of UV cross-linked RNA-protein complexes by phenol-toluol extractionUrdaneta Zurbarán, Erika Cristina 24 April 2020 (has links)
RNA-Bindungsproteine spielen Schlüsselfunktionen bei der post-transkriptionellen Regulation der Genexpression. Durch Bindung an RNA steuern sie die RNA-Aufbereitung, den Transport, die Stabilität und die Translation. In den letzten zehn Jahren wurden bedeutende Fortschritte bei der Aufklärung bakterieller post-transkriptioneller Mechanismen erzielt. Es wird immer deutlicher, dass diese Regulierungsebene auch bei der Pathogenese und Antibiotikaresistenz eine wichtige Rolle spielt. Die Analyse von RNA-Protein-Komplexen (RNPs) auf Proteomebene wurde durch die (m)RNA-interactome-capture Technologie vorangetrieben, die den Teil des Proteoms isoliert, welcher mit polyadenylierter (m)RNA vernetzt ist. Dies hat zur Identifizierung von Hunderten von neuen RBPs in einer Vielzahl von eukaryontischen Arten, vom Menschen bis zur Hefe, geführt. Allerdings fehlt die Poly-Adenylierung in der funktionellen RNA von Bakterien und anderen Klassen von -eukaryontischen- regulatorischen RNAs. Ziel dieser Arbeit war es, diese Einschränkung durch die Entwicklung einer neuartigen und unvoreingenommenen Methode zur Aufreinigung von UV-vernetzten RNPs in lebenden Zellen zu überwinden: PTex (Phenol-Toluol-Extraktion). Das Reinigungsprinzip basiert ausschließlich auf den physikalisch-chemischen Eigenschaften von vernetzten RNPs gegenüber ungebundenen Proteinen oder RNA; es ist dabei unparteiisch gegenüber spezifischen RNAs oder Proteinen und ermöglicht somit erstmals eine systemweite Analyse von nicht-poly-(A)-RNA-interagierenden Proteinen sowohl in eukaryontischen (HEK293) als auch in prokaryontischen (Salmonella Typhimurium) Zellen. / RNA binding proteins play key functions in post-transcriptional regulation of gene expression. By binding to RNA, they control RNA editing, transport, stability and translation. In the last decade, significant advances have been made in the elucidation of bacterial post-transcriptional mechanisms. It is becoming increasingly clear that this layer of regulation also plays an important role in pathogenesis and antibiotic resistance. The analysis of RNA-protein complexes (RNPs) at the proteome level has been driven by the (m)RNA interactome capture technology which isolates the proteome cross-linked to poly-adenylated (m)RNA. This has resulted in the identification of hundreds of novel RBPs in a diversity of eukaryotic species ranging from humans to yeast. However, poly-adenylation is absent in functional RNA from bacteria and other classes of -eukaryotic- regulatory RNAs. This work was aimed to overcome that limitation by developing a novel and unbiased method for the purification of UV-cross-linked RNPs in living cells: PTex (Phenol Toluol extraction). The purification principle is solely based on physicochemical properties of cross-linked RNPs versus unbound proteins or RNA, and it is impartial towards specific RNA or proteins; enabling for the first time a system-wide analysis of non-poly(A) RNA interacting proteins in both eukaryotic (HEK293) and prokaryotic (Salmonella Typhimurium) cells.
|
103 |
Role of CPEB in Senescence and Inflammation: A DissertationIvshina, Maria 28 July 2010 (has links)
Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16INK4A; however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.
CPEB is a sequence-specific RNA binding protein that regulates cytoplasmic polyadenylation-induced translation. We report here that CPEB KO mice are hypersensitive to LPS-induced endotoxic shock, which correlates with elevated serum levels of the proinflammatory cytokines IL-6, IL-8 and IL-12. Peritoneal macrophages from the KO mice, as well as a CPEB-depleted macrophage cell line, not only secrete more IL-6 than control cells in response to LPS, but also have prolonged retention of NFϰB in the nucleus, which is responsible for elevated IL-6 transcription. The amount of nuclear NFϰB correlates with reduced levels of IϰBα, which is hyperphosphorylated and rapidly degraded. Collectively, these data suggest that CPEB deficiency enhances the inflammatory response via delayed resolution of NFϰB signaling.
|
104 |
The Dynamic Fate of the Exon Junction ComplexPatton, Robert Dennison 13 November 2020 (has links)
No description available.
|
105 |
Analýza regulace komplexů cytoplazmatických poly(A) polymeráz / Analýza regulace komplexů cytoplazmatických poly(A) polymerázNovák, Jakub January 2011 (has links)
The regulation of gene expression is achieved at many levels. Chromatin-based gene regulation has been the central focus of many decades of research; however, posttranscriptional control mechanisms are emerging as a fundamental complement to direct protein synthesis. This thesis is focused on a specific mechanism of posttranscriptional control - the translational regulation of mRNAs in the cell cytoplasm. This control is a consequence of the balance between translational repression and activation and hinges on the selective recognition of regulated mRNAs by RNA-binding proteins and their ability to recruit RNA modifying proteins. In this thesis, Caenorhabditis elegans germline was used to study translational control of the germ cell-enriched gene, gld-2. Mutants of known RNA-binding proteins of the PUF and CPB protein families were analyzed by performing Western blots, using anti-GLD-2 antibodies. Yeast 3-Hybrid system was used to identify the cis-regulatory sites in the gld-2 mRNA conferring translational regulation by members of PUF and CPB protein families. Potential autoregulatory loop of gld-2 gene expression was also investigated. This thesis shows that FBF proteins positively regulate expression of gld-2 and bind to a conserved sequence in the 3'UTR of its mRNA. Mutations of gld-2 negatively affect...
|
106 |
Analysis of CPEB Family Protein Member CPEB4 Function in Mammalian Neurons: A DissertationKan, Ming-Chung 01 June 2008 (has links)
Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating local protein synthesis, the RNA binding specificity of these homologs are studied by SELEX. The result shows CPEB2-4 bind to RNAs with consensus sequence that is distinct from CPE, the binding site of CPEB1. This distinction RNA binding specificity between CPEB1 and CPEB2-4 suggests CPEB2-4 cannot replace CPEB1 in mediating local protein synthesis. For CPEB2-4 have distinct RNA binding specificity compared to CPEB1, they are referred as CPEB-like proteins. One of CPEB-like protein, CPEB3, binds GluR2 mRNA and represses its translation. The subcellular localization of CPEB family proteins during glutamate over stimulation is also studied. The CPEB family proteins are identified as nucleus/cytoplasm shuttling proteins that depend on CRM1 for nuclear export. CPEB-like proteins share similar nuclear export ciselement that is not present in CPEB1. Over-stimulation of neuron by glutamate induces the nuclear accumulation of CPEB family proteins possibly through disrupted nuclear export. This nuclear accumulation of CPEB family protein is induced by imbalance of calcium metabolism in the neurons. Biochemical and cytological results suggest CPEB4 protein is associated with ER membrane peripherally in RNA independent manner. This research provides general description of biochemical, cytological properties of CPEB family proteins.
|
107 |
Dissecting Small RNA Loading Pathway in <em>Drosophila melanogaster</em>: A DissertationDu, Tingting 28 January 2008 (has links)
In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type.
Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways.
Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC.
|
108 |
Role of post-transcriptional regulation in human liverChaturvedi, Praneet 11 February 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / My thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD.
My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes.
|
109 |
Post-transcriptional control of Drosophila pole plasm component, germ cell-lessMoore, Jocelyn. January 2008 (has links)
No description available.
|
110 |
Defining the functions and mechanisms of mRNA targeting to the mitotic apparatusPatel, Dhara 07 1900 (has links)
La localisation des ARNm dans différents compartiments subcellulaires est conservée dans un large éventail d'espèces et de divers types cellulaires. Le trafic est médié par l'interaction entre les protéines de liaison à l'ARN (RBP) et l'ARNm. Les RBP reconnaissent les éléments cis-régulateurs de l'ARNm, également appelés éléments de localisation. Ceux-ci sont définis par leur séquence et/ou leurs caractéristiques structurelles résidant dans la molécule d'ARNm. La localisation des ARNm est essentielle pour la résolution subcellulaire et temporelle. De plus, les ARNm se sont avérés enrichis dans de nombreux compartiments cellulaires, notamment les mitochondries, l'appareil mitotique, et le réticulum endoplasmique. En outre, des études ont démontré que les RBP et les ARNm sont associés aux structures de l'appareil mitotique. Cependant, le rôle que joue la localisation de l'ARNm au cours de la mitose reste largement inexploré. Ma thèse de doctorat vise à comprendre comment le trafic d'ARNm est impliqué lors de la mitose.
La première partie de cette thèse porte sur l'interaction post-transcriptionnelle qui se produit entre les deux ARNm, cen et ik2. Les gènes qui se chevauchent sont une caractéristique frappante de la plupart des génomes. En fait, il a été constaté que le chevauchement des séquences génomiques module différents aspects de la régulation des gènes tels que l'empreinte génomique, la transcription, l'édition et la traduction de l'ARN. Cependant, la mesure dans laquelle cette organisation influence les événements réglementaires opérant au niveau post-transcriptionnel reste incertaine. En étudiant les gènes cen et ik2 de Drosophila melanogaster, qui sont transcrits de manière convergente avec des régions 3' non traduites qui se chevauchent, nous avons constaté que la liaison physique de ces gènes est un déterminant clé dans la co-localisation de leurs ARNm aux centrosomes cytoplasmiques. Le ciblage du transcrit ik2 dépend de la présence et de l'association physique avec l'ARNm de cen, qui est le principal moteur de la co-localisation centrosomale. En interrogeant les ensembles de données de séquençage de fractionnement, nous constatons que les ARNm codés par des gènes qui se chevauchent en 3' sont plus souvent co-localisés par rapport aux paires de transcrits aléatoires. Ce travail suggère que les interactions post-transcriptionnelles des ARNm avec des séquences complémentaires peuvent dicter leur destin de localisation dans le cytoplasme.
La deuxième partie de cette thèse consiste à étudier le rôle que jouent les RBP au cours de la mitose. Auparavant, les RBP se sont avérés être associés au fuseau et aux centrosomes. Cependant, leur rôle fonctionnel au niveau de ces structures reste à étudier. Grâce à un criblage par imagerie avec plus de 300 anticorps, nous avons identifié 30 RBP localisés dans les structures mitotiques des cellules HeLa. Ensuite, pour évaluer les rôles fonctionnels de ces RBP, nous avons utilisé l'interférence ARN (ARNi) pour évaluer si la fidélité du cycle cellulaire était compromise dans les cellules HeLa et les embryons de Drosophila melanogaster. Fait intéressant, nous avons identifié plusieurs candidats RBP pour lesquels le knockdown perturbe la mitose et la localisation de l'ARNm dans les cellules HeLa. De plus, la perte des orthologues a entraîné des défauts de développement chez l'embryon de mouche. Grâce à ce travail, nous avons démontré que les RBP sont impliquées pour assurer une mitose sans erreur.
En résumé, les travaux que j'ai menés mettent en lumière l'implication de la régulation post-transcriptionnelle au cours de la mitose. En définissant les fonctions et le mécanisme de localisation des ARNm en mitose, ce travail permettra de définir de nouvelles voies moléculaires impliquées dans la régulation de la mitose. Puisque la division cellulaire non contrôlée peut mener à des maladies tel le cancer, étudier le contrôle du cycle cellulaire sous cet angle « centré sur l'ARN » peut aider à développer de nouvelles approches thérapeutiques pour trouver des solutions aux problèmes de santé. / The localization of mRNAs to different subcellular compartments is conserved in a wide range of species and diverse cell types. Trafficking is mediated by the interaction between RNA binding proteins (RBPs) and mRNA. RBPs recognize mRNA cis regulatory motifs, otherwise known as localization elements. These are defined by their sequence and/or structural features residing within the mRNA molecule. Localization of mRNAs is essential for subcellular and temporal resolution. Furthermore, mRNAs have been found to be enriched in many cellular compartments including the mitochondria, mitotic apparatus, and endoplasmic reticulum. Moreover, studies have demonstrated that RBPs and mRNAs are associated with mitotic apparatus structures. However, the role that mRNA localization plays during mitosis remains largely unexplored. My PhD thesis aims to understand how the trafficking of mRNAs is implicated during mitosis.
The first part of this thesis encompasses the post-transcriptional interaction that occurs between the two mRNAs, cen and ik2. Overlapping genes are a striking feature of most genomes. In fact, genomic sequence overlap has been found to modulate different aspects of gene regulation such as genomic imprinting, transcription, RNA editing and translation. However, the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. By studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed with overlapping 3’untranslated regions, we found that the physical linkage of these genes is a key determinant in co-localizing their mRNAs to cytoplasmic centrosomes. Targeting of the ik2 transcript is dependent on the presence and physical association with cen mRNA, which serves as the main driver of centrosomal colocalization. By interrogating global fractionation-sequencing datasets, we find that mRNAs encoded by 3’overlapping genes are more often co-localized as compared to random transcript pairs. This work suggests that post-transcriptional interactions of mRNAs with complementary sequences can dictate their localization fate in the cytoplasm.
The second part of this thesis involves investigating the role that RBPs play during mitosis. Previously, RBPs have been found to be associated with the spindle and centrosomes. However, their functional role at these structures was yet to be investigated. Through an imaging screen with >300 antibodies, we identified 30 RBPs localized to mitotic structures in HeLa cells. Then, to assess the functional roles of these RBPs, we used RNA interference (RNAi) to assess whether cell cycle fidelity was compromised in HeLa cells and Drosophila melanogaster embryos. Interestingly, we identified several RBP candidates for which the knockdown disrupted mitosis and mRNA localization in HeLa cells. Furthermore, loss of the orthologs led to developmental defects in the fly embryo. Through this work, we demonstrated that RBPs are involved in ensuring an error-free mitosis.
In summary, the work that I have conducted sheds light on the involvement of post-transcriptional regulation during mitosis. By defining the functions and mechanism of mRNA localization in mitosis, this work will help define new molecular pathways involved in mitosis regulation. As uncontrolled cell division can lead to diseases such as cancer, studying cell cycle control from this ‘RNA-centric’ angle may help to develop new therapeutic approaches to find solutions to health problems.
|
Page generated in 0.109 seconds