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Mechanisms of Post-transcriptional Regulation of Cat-1 Gene Expression by Amino Acid StarvationYaman, Ibrahim 05 July 2005 (has links)
No description available.
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Growth-regulated expression and G0-specific turnover of the mRNA that encodes AH49, a mammalian protein highly related to the mRNA export protein UAP56Pryor, Anne M. January 2003 (has links)
No description available.
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Transcriptional and Post-transcriptional Control of Nhlh2 with Differing Energy StatusAl-Rayyan, Numan A. 19 August 2011 (has links)
Nescient Helix Loop Helix 2 (Nhlh2) is a member of the basic helix-loop-helix transcription factor family. Mice with a targeted deletion of Nhlh2, called N2KO mice, show adult onset obesity in both males and females. Nhlh2 regulates other genes by binding to the E-box in the promoter region of these genes. This transcription factor regulates many other transcription factors including MC4R and PC1/3 which are associated with human obesity. The Nhlh2 promoter has been analyzed for putative transcription factors binding sites. These putative binding sites have been tested to be the regulators of Nhlh2 by transactivation assays with mutant promoters, Electrophoretic Shift Assay (EMSA), and Chromatin Immunoprecipitation Assay (ChIP) as methods to investigate the DNA-protein binding.
The results of these experiments showed that the Nhlh2 promoter has five Signal Transducer and Activator of Transcription 3 (Stat3) binding site motifs at -47, -65, -80, -281, -294 and two Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) binding site motifs at -67 and -135. While NFκB acts as a negative regulator of Nhlh2, this research showed that Stat3 acts as a regulator for the Nhlh2 basal expression and leptin stimulation. The ChIP assay using chromatin from mouse hypothalamus and antibodies against Stat3 and the NFκB subunits P50, P65, and c-Rel demonstrated that all of these antibodies were able to pull down the part of the Nhlh2 promoter containing the binding sites of Stat3 and NFκB. The EMSA results not only demonstrated that NFκB and Stat3 binding site motifs are real binding sites, but also exists the possibility of a relationship between these transcription factors to regulate Nhlh2 expression with leptin stimulation.
An effort in analyzing the human NHLH2 3'UTR showed that one of the SNPs located at position 1568 in the NHLH2 mRNA (NHLH2A<sup>1568G</sup>) which converts adenosine to guanine might have the potential to decrease the mRNA stability. For more investigation about this SNP, the mouse Nhlh2 tail was cloned into 2 different vectors and these vectors were subjected to site directed mutagenesis to create the 3'UTR SNP that convert A to G. One of these vectors used luciferase as a reporter gene for expression while the other one was used to measure Nhlh2 mRNA stability. These vectors were transfected into hypothalamic cell line N29/2 to test the effect of this SNP on Nhlh2 expression. This study demonstrated that this SNP down regulated luciferase expression and also decreased Nhlh2 mRNA stability.
Taken together, this study demonstrated that Nhlh2 could be regulated transcriptionally by both NFκB and Stat3 transcription factors and post-transcripitionally by the 3'UTR SNP that converts adenosine to guanine. / Ph. D.
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Role of the 3'UTR in translation and stability of HCV and HPV mRNAsWiklund, Lisa January 2002 (has links)
<p>Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. </p><p>A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised <i>in vitro </i>and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an <i>in vitro </i>stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation.</p><p>In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.</p>
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Role of the 3'UTR in translation and stability of HCV and HPV mRNAsWiklund, Lisa January 2002 (has links)
Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised in vitro and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an in vitro stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation. In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.
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The Effect of RNA Secondary Structures on RNA-Ligand Binding and the Modifier RNA Mechanism: A Quantitative ModelHackermüller, Jörg, Meisner, Nicole-Claudia, Auer, Manfred, Jaritz, Markus, Stadler, Peter F. 31 January 2019 (has links)
RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small \modifier RNAs' to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein HuR with AU-rich elements [Meisner et al. (2004), Chem. Biochem. in press]. We discuss our model and recent experimental findings demonstrating the ffectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences.
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Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuRSingh, Mamata 31 March 2011 (has links)
No description available.
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Identifizierung und Charakterisierung einer alternativ gespleißten mRNA der Interleukin-4 Rezeptor alpha-Kette und Untersuchung der biologischen Funktion der verkürzten RezeptorvarianteMöricke, Anja 15 April 2002 (has links)
Alternatives mRNA-Splicing ist ein häufig beobachtetes Phänomen, das es der Zelle ermöglicht, unterschiedliche Proteine aus einem Gen zu generieren. In den letzten Jahren wurden immer mehr alternativ gespleißte Transkripte entdeckt, und einigen der daraus resultierenden Protein-Isoformen konnten geänderte biologische Funktionen zugeordnet werden. In dieser Arbeit ist erstmals ein alternativ gespleißtes Transkript der Interleukin-4 Rezeptor alpha (IL-4R-alpha) Kette beschrieben. Dieser mRNA Splice-Variante, genannt IL-4R-alpha-IT, fehlt im membranproximalen Bereich der zytoplasmatischen Domäne ein komplettes Exon. Dies führt zur Verschiebung des Leserasters und so zur Entstehung eines vorzeiten Stop-Codons. Der resultierenden Protein-Isoform fehlt der größte Teil der intrazellulären Kette mit den dort enthaltenen, für die Signaltransduktion essentiellen Domänen. Die Untersuchung der biologischen Funktion der Rezeptor-Varianten in einem geeigneten Zellsystem der Maus zeigte, daß die Splice-Variante IL-4R-alpha-IT keine Proliferation der Zellen vermitteln und auch den Übergang der Zellen in die Apoptose nicht verhindern kann. Bei der Quantifizierung der Expression von IL-4R-alpha-IT-mRNA in Relation zum IL-4R-alpha voller Länge mit einer kompetitiven RT-PCR an Knochenmark und peripheren Blutlymphozyten von Kindern mit ALL zeigte sich zunächst ein irreführender Unterschied zwischen Proben von Kindern mit ALL-Ersterkrankung und Rezidiv. Weitere Untersuchungen ergaben jedoch, daß der Zeitraum zwischen Abnahme und Aufarbeitung des Untersuchungsmaterials für diesen scheinbaren Zusammenhang verantwortlich war. Während direkt nach Abnahme aufgearbeitetes Untersuchungsmaterial eine nur niedrige relative Expression der Splice-Variante zeigte, nahm diese bei verzögerter Aufarbeitung drastisch zu. Diese Beobachtung wurde experimentell an Proben gesunder Probanden wiederholt bestätigt. Interessanterweise konnte derselbe Effekt in unterschiedlicher Ausprägung auch bei Splice-Variante anderer Zytokine und -Rezeptoren wie IL-7, IL-7R und beta-C beobachtet werden. mRNA-Stabilitäts-Assays und die Bestimmung der einzelnen Transkripte mit einer semiquantitativen RT-PCR zeigten, daß es tatsächlich zu einer absoluten Hochregulation der IL-4R-alpha-IT-mRNA in den verzögerte aufgearbeiteten Proben kommt. Wurden die Zellen wieder in Kultur genommen, war dies innerhalb weniger Stunden reversibel. Desweiteren scheinen auch unterschiedlichen mRNA-Stabilitäten eine Rolle zu spielen. / Alternative pre-mRNA splicing is a widespread mechanism contributing to the diversity of gene expression. The number of newly detected alternatively spliced transcripts has continuously risen, and distinct biological functions have been attributed to some protein isoforms resulting from these mRNA variants. We report on the detection of a novel alternatively spliced transcript of the human interleukin-4 receptor alpha (IL-4R-alpha) chain, which has been called IL-4R-alpha-IT mRNA. A premature stop codon due to omission of one exon in the membrane-proximal region of the cytoplasmic domain leads to an mRNA variant, which encodes an intracellular truncated receptor protein lacking domains which are essential for signal transduction. The investigation of the biological function of the IL-4Ra splice variants in a suitable mouse cell system showed, that the truncated receptor variant is not able to mediate cell proliferation or prevention of apoptosis. Bone marrow and peripheral blood samples from children with acute lymphoblastic leukemia were analyzed for the expression of IL-4R-alpha-IT mRNA relative to the full-length receptor transcript by competitive RT-PCR. Initially, there was found a difference of IL-4R-alpha-IT mRNA expression in patients with initial ALL versus relapsed ALL. However, this difference turned out to be due to the time interval between collection and preparation of samples. While freshly isolated material was associated with low levels of IL-4R-alpha-IT mRNA, samples with a longer period until cell preparation exhibited a drastic increase of IL-4R-alpha-IT mRNA levels. The same results were obtained for peripheral blood samples from healthy donors by imitating a prolonged time of transport until cell preparation. Interestingly, a similar effect could be demonstrated for splice variants of other cytokine receptors and cytokines (beta-C, IL-7R, and IL-7), although to different extents. mRNA stability assays and semiquantitative RT-PCR specific for IL-4Ra or IL-4R-alpha-IT, respectively, indicated that the expression of IL-4R-alpha-IT mRNA increases absolutely in these samples, although mRNA degradation may be of importance as well.
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The Role of Polyadenylation in Human Papillomavirus Type 16 Late Gene ExpressionÖberg, Daniel January 2005 (has links)
<p>High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation. </p><p>Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus.</p><p>In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells. </p><p>Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.</p>
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The Role of Polyadenylation in Human Papillomavirus Type 16 Late Gene ExpressionÖberg, Daniel January 2005 (has links)
High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation. Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus. In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells. Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.
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