Spelling suggestions: "subject:"trans a_ecting 5factors"" "subject:"trans a_ecting allactors""
1 |
Regulation Of Interferon Regulatory Factor-2 mRNA Translation By 'IRES' Element : Possible Role Of trans Acting FactorsDhar, Debojyoti January 2007 (has links)
Cellular response to various stress conditions involves regulation of gene expression by different mechanisms. Translation is the final step in the flow of genetic information and regulation at this level allows an early response to changes in physiological conditions. Initiation of translation is the rate-limiting step of protein synthesis and hence is tightly regulated. Translation initiation in mammalian cells is mainly by “cap dependent pathway” wherein the 5’methyl guanosine “cap” structure is recognized by certain canonical initiation factors along with 40S ribosomal subunit and the complex scans the 5’UTR till it recognizes initiator AUG. This leads to the joining of the 60S ribosomal subunit and the initiation of translation. In an alternate mode of translation initiation called as the Internal ribosome entry site mediated translation (IRES), the ribosomes are recruited closer to the initiator AUG in a 5’ cap independent manner. Efficient translation by IRES mode requires some canonical initiation factors like eIF2 and eIF3 and other non-canonical IRES-trans-acting factors (ITAFs), which include human La antigen, polypyrimidine-tract binding protein (PTB),Upstream of N-Ras (Unr), Poly (rC) binding protein (PCBP2) etc. Various types of stress conditions, such as starvation of growth factors, heat shock, hypoxia, viral infection lead to down regulation of protein synthesis. However, translation of a subset of mRNAs continues or is up-regulated. Many of these mRNA may be translated by an IRES mode. It is believed that cellular IRESs become active during such conditions that abrogate the cap-dependent mode of translation so that the pool of vital proteins is maintained in the cell. In this thesis, presence of ‘IRES’ element has been investigated in the 5’UTR of Interferon regulatory factor -2 (IRF2) mRNA and the possible physiological significance has been studied. Further, it has been shown that polypyrimidine tract binding protein or PTB is important for the IRES activity. The probable mechanism of action of PTB has also been investigated which suggests that PTB interaction alters the IRF2 IRES conformation thus facilitating translation initiation.
In the first part of the thesis, mRNAs that continue to be translated under heat-shocked condition, which is known to abrogate cap-dependent translation initiation, has been investigated by cDNA micro-array hybridization analysis of the ribosome bound RNA. The global protein synthesis was severely impaired under heat shock; however a number of mRNAs continued translation under this condition. Some of these mRNAs encode proteins that are likely to be involved in the heat shock response. Few of these genes are also reported to contain IRES element. Since the micro-array was performed from the RNA extracted from ribosome bound mRNA fraction in a condition when cap-dependent translation is impaired, it was hypothesized that some of the genes, which are up regulated under such condition, might operate via cap-independent mode of translation initiation. Based on this study, one candidate gene, the ‘interferon regulatory factor 2 (IRF2)’ was selected from the pool of up regulated genes and presence of an IRES element was investigated. Interferon regulatory factors are DNA-binding proteins that control interferon (IFN) gene expression. IRF2 has been shown to function as repressor of IFN and IFN-inducible genes. Real–Time and semi-quantitative RT-PCR assays were performed which validated the micro-array data.
In the second part of the thesis, the presence of IRES element in the 5’UTR of IRF2 was investigated. Bicistronic assay showed comparable IRES activity with a known representative IRES, BiP, thus suggesting the presence of an IRES element in the IRF2 5’UTR. Stringent assays were then performed to rule out cryptic promoter activity, re-initiation/scanning or alternative splicing in the 5’UTR of the IRF2. RNA transfections using in vitro synthesized bicistronic RNAs further validated the presence of the IRES element. To understand the physiological significance of an IRES element in IRF2 mRNA, the cells were subjected to various stress conditions and IRES activity was studied. It seems IRF2 IRES function might not be sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease, which is known to cleave eIF 4G and thus inhibit the cap-dependent translation. Incidentally, Hepatitis A virus IRES was affected under such condition. Additionally, it was observed that compared to HCV or Bip IRES, the effect of Interferon α treatment was not so pronounced on the IRF2 IRES. This was further evidenced by its unchanged protein level post-treatment with interferon α. Furthermore, in cells treated with tunicamycin (a known agent causing ER stress), the IRF2 IRES activity and the protein levels were unaffected, although the cap dependent translation was severely impaired. The observations so far suggested that the IRF2 protein level is practically unchanged under conditions of ER stress and interferon treatment. Metabolic labeling followed by immunoprecipitation of IRF2 in cells treated with either tunicamycin or interferon suggested that de novo synthesis of the protein is continued under the above conditions thus validating our earlier data.
In the third part of the thesis, the role of an IRES trans acting factor, PTB, in modulating the IRF2 IRES activity has been investigated. Analysis of the cellular protein binding with the IRF2 IRES suggested that certain cellular factors might influence its function under stress conditions. The IRF2 IRES was found to interact with a known trans-acting factor or PTB. To study the possible role of this trans acting factor, the PTB gene was partially silenced by PTB specific siRNA. This led to a decrease in the IRF2 IRES activity, suggesting that PTB is probably essential for the IRES activity. Interestingly, when Hela cells (with partially silenced PTB) were treated with tunicamycin (inducer of ER stress) the level of IRF2 protein was also found to be less thus pointing to an important role of PTB in IRF2 protein synthesis under such conditions. Western blot analysis and immunofluoroscence assay suggested that there was no significant nuclear-cytoplasmic relocalization of PTB under the condition studied. Primer extension inhibition assay or Toe-printing analysis was performed to detect the contact points of PTB on the IRF2 5’UTR. Many toe-prints were found on the 3’ end of the 5’UTR RNA. A 3’ deletion mutant was generated that showed reduced PTB binding. Incidentally the IRES activity of the mutant was also found to be less than the wt IRF2 RNA. Subsequently, structural analysis of the RNA was performed using enzymatic (CV1, RNase T1) and chemical modification (DMS) agents. Footprinting assay in presence of PTB suggested that there is change in the structure when PTB interacts with the RNA. To investigate this further, CD spectrum analysis of the IRF2 RNA in the presence of PTB was performed which indicated that there was a conformational change under such condition thus validating our earlier observation.
The thesis reveals a novel cellular IRES element in the 5’UTR of IRF2 mRNA. The characterization of the IRES and possible role played by PTB protein in modulating its activity suggests that the regulated expression of IRF2 protein by its IRES element under various stress conditions would have major implications on the cellular response. Incidentally, this study constitutes the first report on translational control of interferon regulatory factors by internal initiation. The results might have far reaching implications on the possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general.
|
2 |
Hledání lidských bílkovin ovlivňujících funkci IRES viru hepatitidy typu C / Screening for the HCV IRES interacting proteinsRoučová, Kristina January 2012 (has links)
Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...
|
Page generated in 0.0646 seconds