The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

Polypurine·polypyridine (pur·pyr) tracts are a run of all purines on one strand and all pyrimidines on the complementary DNA strand. Statistical overrepresentation of the tracts in eukarocytes suggests a cellular role or roles. The tracts from triplex DNA <i>in vitro</i> and there is evidence for triplex DNA <i>in vivo</i>. Several cellular roles are possible for triplex DNA. The presence of the tracts in gene 5' flanking regions suggets a regulatory role. This work investigates the role of triplex DNA in the cell, particularly in the regulation of transcription. Proteins mediate DNA looping in the regulation of transcription and in its condensation in chromosomes. Such looping may also be mediated by transmolecular triplexes, formed between separated pur·pyr tracts. Formation of pyr·pur·pyr transmolecular triplexes was investigated using linear and circular plasmid models containing separated pur·pyr tracts able to form a triplex with each other, but not within a tract. Transmolecular triplex loops (T-loops) formed in circular DNA, suggesting a possible regulatory or structural role <i>in vivo</i>. The following model shows a T-loop formed at pH 4. At pH 6, a duplex partially reforms and single-stranded region(s) trap the structure. and single-stranded region(s) trap the structure. T-loops were used as a model to test the Idea that a single-strand extruded by triplex formation in the 5' flanking region of a gene could promote transcription. Transcription was inhibited in T-loops, suggesting such structures could block transcriptional elongation if formed <i>in vivo</i>. The ability of polyamine analogues to promote triplex formation was also tested using T-loops. Pentamines promoted T-loop formation at lower concentrations than tetramines. Spatial distribution of charge was also important. A triplex role in transcriptional regulation was investigated using two examples of human genes with 5' flanking pur·pyr tracts. The effect of triplex-specific antibodies on expression of c-' myc' was investigated using agarose-encapsulated nuclei. Triplex formation between c-'src' promoter pur·pyr tracts was visualized as gel band shift die to dimerization between linear plasmid fragments containing individual tracts. A transmolecular triplex was proposed as one way in which the c-'src' tracts could form a triplex <i>in vivo</i> which might be involved in the regulation of transcription.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

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.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 64-74.

No description available.

Angiogenesis is the formation of new blood vessels from existing vasculature. Vascular Endothelial Growth Factor (VEGF), a known angiogenic protein, stimulates endothelial cell proliferation and migration via interactions with its receptors, KDR and Flt-1. A secreted form of Flt-1 (sFlt-1), derived from alternatively-spliced RNA, can inhibit actions of VEGF in vivo. It has been suggested that alterations in sFlt-1 expression could significantly change the angiogenic VEGF activity. This project focuses on characterizing intronic elements that regulate Flt-1 mRNA splicing. A "wild-type" construct (pFIN13), containing the first 13 exons, intron 13 and exons 14-30 of mouse Flt-1, was shown to produce both forms of Flt-1 mRNA after transfection into HEK293 cells. To gauge the strength of the native splicing signals in intron 13 of Flt-1, a series of point mutations were made in the polypyrimidine tract using pFIN13. After transient transfection, the levels of Flt-1 and sFlt-1 protein and mRNA were compared using quantitative PCR, RNA hybridization analysis, and protein immunoblotting. Results from quantitative PCR showed that purine substitutions were associated with 120 to 350 fold decreases in Flt-1 mRNA (normalized against neor), consistent with less efficient splicing. These large decreases in Flt-1 mRNA were accompanied by increases in sFlt-1 mRNA. Modest (20 to 100%) increases in Flt-1 mRNA, reflecting improved splicing, resulted from increasing the uridine complement in the polypyrimidine tract. These results suggest that the wild-type polypyrimidine tract is of intermediate strength and may be a regulatory locus for modulating Flt-1: sFlt-1 ratios. / Master of Science

Alternative splicing provides the cell the ability to generate, from a single gene, multiple protein isoforms, sometimes with different or even antagonistic functions. This process is tightly regulated and alterations in the accurate balance of alternatively spliced mRNAs are a common cause of disease. The main objective of this thesis has been to understand the molecular mechanisms underlying disease-causing defective splicing. Skipping of Fas death receptor exon 6 leads to decreased Fas-ligand induced apoptosis. We have studied how this event is promoted by a mutation at the 3’ splice site and by the proto-oncogene SF2, leading to Autoimmune Lymphoproliferative Syndrome and possibly contributing to tumor progression, respectively. Moreover, we have determined the mechanism by which an antitumor drug, Spliceostatin A, alters 3’ splice site recognition and affects alternative splicing. This thesis underscores the importance of pre-mRNA splicing in disease and how the study of disease-causing aberrant splicing can be used as a tool to understand splicing mechanisms and vice versa. / El processament alternatiu del pre-ARNm proporciona a la cèl•lula l’habilitat de generar, a partir d’un únic gen, proteïnes amb funcions diferents i, fins i tot, antagòniques. Aquest procés està altament regulat i desequilibris en l’abundància de les diferent isoformes són causes comunes de malaltia. L’objectiu principal d’aquesta tesi ha estat entendre el mecanisme molecular a través del qual problemes en el processament del pre-ARNm causen malalties. L’exclusió de l’exó 6 del receptor de mort cel•lular Fas condueix a una disminució de l’apoptosi en resposta al lligand de Fas. Hem estudiat com una mutació al lloc de processament 3’ d’aquest exó i el proto-oncogén SF2 promouen aquest patró, causant el síndrome autoimmune lifoproliferatiu i possiblement contribuint a la progressió tumorogènica, respectivament. A més, hem estudiat el mecanisme pel qual la droga antitumoral Spliceostatin A altera el reconeixement del lloc de processament 3’ i causa canvis en el processament alternatiu de diversos gens. Aquesta tesi posa en evidència la importancia del processament del pre-ARNm en malalties i com l’estudi de mutacions que alteren aquest procés i són causa de malaties pot ser utilitzat con una eina per entendre el mecanisme d’aquest processament i viceversa.