University of Northern British Columbia Institutional Repository

Search results

Search Results

Displaying (1 - 17 of 17)

Title: Characterizing the CRD-BP-RNA interaction in-vitro and in cells.
Contributors: Mark Barnes (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: The highly conserved family of RNA-binding proteins known as the VICKZ RNA-binding proteins play an integral role in the formation of cytoplasmic RNPs which leads to the stabilization, localization and translational control of many mRNA transcripts in the cell. The key investigation of this thesis was to analyse the binding ability of the VICKZ protein family member, the coding region determinant-binding protein (CRD-BP), both in-vitro and in cells. CRD-BP has four K-homology (KH) domains and two RNA-recognition motif (RRM) domains. Deletion studies in CRD-BP orthologs have shown that the KH domains, and not the RRM domains, are predominantly responsible for binding to RNA substrates. However, it is still unclear to what extent each of the KH domains play in their physical interaction with RNA molecules, nor is it known if each of the KH domains an play equal role in interacting with different RNA substrates. In an effort to address the above questions, we used site-directed mutagenesis to mutate the first glycine of the G-X-X-G motif in each KH domain separately, and in combinations. We mutated the glycine to an aspartate to introduce both physical and electrostatic hindrance for binding at the G-X-X-G motif. The goal was to determine if such a mutation can disrupt CRD-BP's ability to bind its RNA substrates both in-vitro and in cells. Our results showed that KG single mutants KH2, KH3 and KH4 did not disrupt the CRD-BP-c-myc CRD RNA interaction in-vitro. CRD-BP KH1 single mutant exhibited a modest reduction in binding to the c-myc CRD RNA substrate in-vitro. However, double KH domain mutations (KH1-2, KH1-3, and KH2-4) resulted in a complete abrogation of CRD-BP's ability to bind the c-myc CRD RNA substrate, suggesting these KH domains work in tandem to bind to the c-myc CRD RNA substrate in-vitro. Interestingly, the CRD-BP KH domain double mutant, KH3-4, showed only a modest reduction in the c-myc CRD RNA substrate binding, suggesting that the first glycine in the G-X-X-G motif of KH3 and KH4 doe
Discipline: Chemistry
Date added: 2017-03-30T17:13:16.202Z

Title: The coding region determinant binding protein shields c-myc coding region determinant and MDR-1 RNAs from endonucleolytic attack by a novel mammalian endoribonuclease in vitro.
Contributors: Dan S. Sparanese (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Date added: 2017-03-30T17:02:32.014Z

Title: Characterization of a novel endoribonuclease capable of degrading c-myc mRNA in vitro.
Contributors: Joel Christopher Urquhart (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Date added: 2017-03-30T17:01:10.237Z

Title: Study of mechanisms of methylmercury neurotoxicity and association with Parkinson's disease
Contributors: Yueting Shao (author), Laurie Chan (Thesis advisor), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Methylmercury (MeHg) is a well-known neurotoxicant but the diverse mechanistic events associated with MeHg neurotoxicity have yet been fully elucidated. In this study, we explored the mechanisms of MeHg neurotoxicity particularly its possible roles in neurodegenerative diseases like Parkinson’s disease (PD) using dopaminergic neuronal cells and a non-human primate model. In the cell culture model, we compared effects of MeHg to those induced by 1-methyl-4-phenylpyridinium (MPP⁺), a well-established drug that can induce Parkinsonism-like symptoms. A proteomic approach was used to identify and analyze MeHg affected proteins and their biological functions and associated pathways in both the cellular and marmoset models. Our results showed that MeHg induced changes of gene/protein profiles are similar to the effects as MPP⁺. Evidence from proteomic results suggested MeHg caused neurodegenerative effects not only associated with PD but also other neurodegenerative disorders such as Huntington’s disease (HD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS). We also found brain regional specific response to MeHg stimuli, based on the protein profiles affected in the following order: cerebellum > occipital lobe (OL) > frontal lobe (FL) of the cerebrum. In the cerebellum, carbohydrate derivative metabolic process, synaptic transmission, cell development and calcium signalling are dominant functions and pathways contributing to the motor deficit in MeHg-treated marmoset. MeHg was found to selectively target membrane proteins in the cerebellum particularly in synaptic membranes. MeHg affected proteins involved in energy metabolism in both OL and FL of the cerebrum through different proteins and biochemical pathways. In the OL, proteins were enriched in functions of carbohydrate metabolic process, lipid metabolic process, cellular amino acid metabolic process, homeostatic process, transportation, and regulation of body fluid level. In the FL, differentially expressed proteins were mainly involved in the cell cycle and cell division, glycerolipid metabolic process, sulfur compound metabolic process, cellular amino acid metabolic process, microtubule-based process, and proteolysis. The dyshomeostasis of water transport and associated pathways observed in OL and FL was found to be the underlying mechanism for brain edema observed in the MeHg exposed marmoset. Novel proteins such as DLG4 (PSP95) in the cerebellum and APOE in OL were exhibited to be core proteins in linking multifunction targeted by MeHg. This study provides a new perspective upon understanding mechanisms behind MeHg mediated neurotoxic deficits, and suggests potential links between MeHg exposure and neurodegenerative disorders in humans.
Discipline: Natural Resources & Environmental Studies
Date added: 2017-03-29T17:28:40.654Z

Title: British Columbian wild mushrooms as potent source of novel natural anti-cancer compounds
Contributors: Ankush A Barad (author), Chow Lee (Thesis advisor), Kerry Reimer (Thesis advisor), University of Northern British Columbia College of Science and Management (Degree granting institution), Hugues Massicotte (Committee member), Keith Egger (Committee member)
Abstract: Worldwide, many fungal species have been used for their medicinal properties by various cultures for centuries. Major challenges with cancer chemotherapy, which includes toxicity, non-specificity and several adverse effects have motivated researchers to search for novel anti-cancer compounds from natural sources. Several studies have identified various anti-cancer compounds from mushrooms in many parts of the world, but mushrooms occurring in British Columbia remain unstudied for their anti-cancer activities. In this thesis, five mushroom species were collected, extracted, and assessed for anti-proliferative and immuno-stimulatory activities. Eleven extracts from these species showed anti-proliferative or immuno-stimulatory activity or both. Sodium hydroxide extract from Paxillus obscurisporus exhibited potent anti-proliferative activity and was further purified using size-exclusion and anion-exchange chromatography. Analysis of the relatively pure anti-proliferative fractions suggests the presence of protein and glycoprotein moieties. Further purification and characterisation is needed before these compounds can be tested for biomedical purposes in the future.
Discipline: Biochemistry
Date added: 2017-05-15T21:02:31.595Z

Title: Characterizing the endoribonuclease activity of APE1.
Contributors: Wan Cheol Kim (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Recent evidence shows that mRNA stability and turn-over is an integral control point in the regulation of gene expression. The stability of various mRNAs within a eukaryotic cell can differ and this results in a magnitude of difference in mRNA abundance. An enzyme known as APE1, apurinic/apyrimidinic DNA endonuclease 1, has recently been discovered to possess an endoribonuclease activity against c-myc messenger RNA (mRNA) in vitro. The identification of APE1 as an endoribonuclease warranted this research to further characterize this novel activity both in vitro and in vivo. Previous studies have discovered the residues constituting an active site for apurinic/aprymidinic [sic] DNA (AP-DNA) incision activity of APE1. Whether these residues are shared in the RNA-cleaving activity of APE1 was unknown. The first objective of this thesis was to assess the role of these amino acid residues in contributing to the endoribonuclease activity of APE1. Our results revealed that APE1 indeed shared these residues to cleave both RNA and AP-DNA. However, we also discovered certain differences in the activities of one mutant (D283N) in carrying out AP-DNA and RNA incisions. This suggested that the roles of active site residues in each reaction are not entirely identical. In addition, we have assessed the RNA-cleaving activities of APE1 variants identified in the human population. For a few variants, RNA-cleaving activities were severely reduced while its AP-DNA incision activities were functional. These results suggested a possible unrecognized link between the reductions in the RNA-cleaving activity of the variants and their reported association in certain diseases. The second objective of this thesis was to establish the RNA secondary structures and sequences that are preferentially cleaved by APE1. Our results revealed that APE1 has preference for cleaving the single stranded regions or weakly base paired regions of the RNA. Also, preferred sequences of cleavage were determined to be UA, UG, and CA dinucleotides. Prevalent A
Discipline: Chemistry
Date added: 2017-03-30T17:06:22.042Z

Title: Assessing the role of Syntaxin18 in controlling c-myc mRNA expression and growth in human breast cancer cells.
Contributors: Tyler A. Bassett (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Messenger RNA stability is critical in the control of gene expression. Enzymes capable of cleaving RNA, termed ribonucleases, are gaining appreciation for their role in regulating gene expression. Our lab has discovered a novel protein which has been shown to cleave c-myc mRNA within the coding region determinant (CRD). This protein was tentatively identified as Syntaxin18, a known t-SNARE protein. Recombinant Syntaxin18 has been shown to possess endoribonucleolytic activity against c-myc mRNA in vitro. In order to determine whether Syntaxin18 functions as an endoribonucleae in cells, the effect of altering its expression on the proliferation, levels and stability of c-myc mRNA was examined. The expression of Syntaxin18 in various cell lines and tissues were also compared. Through Western analysis, it was discovered that the expression of Syntaxin18 was similar in all immortalized cancer cell lines examined. However, higher Syntaxin18 expression was observed in cancerous breast tissue when compared to the normal breast tissue. MTT assays revealed that modulation of Syntaxin18 was able to affect the proliferation of MCF-7 cells, and this effect was observed only upon incubation with 17-β-estradiol and insulin. Finally, ribonuclease protection assay analysis showed that modulation of Syntaxin18 may have an effect on the steady-state levels of c-myc and β-actin mRNAs; however, down-regulation of Syntaxin18 did not impact the stability of the c-myc transcript. Based on results obtained from this study, it remains unclear whether Syntaxin18 can function as an endoribonuclease in MCF-7 cells.
Discipline: Chemistry
Date added: 2017-03-30T17:06:07.442Z

Title: Investigating the Cellular Localization of APE1.
Contributors: Sang Eun Kim (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Apurinic/apyrimidinic endonuclease 1 (APE1) is a well-known DNA repair enzyme with multiple functions which include redox-activation, 3'-DNA phosphodiesterase, 3'-5' DNA exonuclease, and RNase H activities. Recently the novel function of APE1 in RNA metabolism has been demonstrated. APE1 was shown to possess the ability to cleaving single-stranded RNA as well as abasic RNA. It was also shown to physically interact with proteins involved in RNA metabolism. Previous studies have discovered the altered distribution and expression of APE1 in different cell types including cancer cells. The cytoplasmic distribution of APE1 has been shown to have correlation with aggressive carcinomas and poor prognoses for patients. This has led to an increasing number of studies on the undefined extra-nuclear roles of APE1 and APE1's potential implications in cancer development. In this thesis, I aimed (i) to confirm the importance of nuclear localization signal (NLS) of APE1 at the N-terminus, (ii) to discover the role of single nucleotide change of APE1 human population variants in its sub-cellular distribution, (iii) to assess the cellular localization of APE1 with processing bodies and stress granules under cellular stresses, and (iv) to initiate immune-fluorescence analysis of APE1 in breast cancer tissue. Our results revealed that APE1 indeed has an important NLS of 1-20 amino acids at the N-terminus and APE1 human population variants showed nuclear localizations identical to the wild-type APE1. There was no co-localization of APE1 with PBs and/or SBs implying that APE1 is unlikely to be involved in mRNA processing that is carried out in PBs and SGs. We observed redistribution of the ND20-APE1-GFP upon cellular stresses in HepG2 cells and this phenomenon has highlighted possible degradation of the cytoplasmic APE1 upon cellular stress. This finding is also consistent with previous discovery of the five critical lysine residues that are responsible for ubiquitination and APE1 degradation upon cellular stress. In summary, this
Discipline: Chemistry
Date added: 2017-03-30T17:10:13.307Z

Title: Investigating the effects of expressing APE1 human population variants in cellular systems.
Contributors: Conan Ma (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Apurinic/apyrimidinic endonuclease 1 (APE1) is a multi-functional mammalian protein which has recently been shown to possess the ability to endonucleolytically cleave single-stranded RNA and abasic RNA. Several population variants of APE1 (L104R, E126D and D148E) are known to exist in the human population. L104R and E126D have been linked to Amyotrophic Lateral Sclerosis while D148E has been linked to various cancers. The exact molecular mechanisms which correlate these variants with human disease are currently unknown. Recent evidence has shown that the in vitro endoribonuclease activities of these variants are different from the wild-type APE1 protein. Here, we hypothesize that the altered endoribonuclease activity of APE1 population variants may be associated with phenotype changes leading to disease pathogenesis. The goal of this thesis was to determine whether APE1 population variants can cause an altered phenotype when expressed in prokaryotic (Origami™ (DE3) cells) and eukaryotic systems (HeLa cervical cancer and HepG2 hepatoma cancer cell lines). Subsequently, these changes were to be linked to altered endoribonuclease activity of these variants. Using two separate assays, it was shown that the L104R and E126D variants possess enhanced cytotoxicity to Origami™ (DE3) cells. This correlates with their distinct endoribonuclease activity demonstrated in vitro. The D148E variant, which had lost endoribonuclease activity, had no effect on colony formation and growth of Origami™(DE3) cells. Interestingly, this study also showed that, when over-expressed, the L104R and E126D variants are capable of causing enhanced growth in the mammalian HepG2 cells. Preliminary microarray and quantitative real time polymerase chain reaction experiments were conducted in an attempt to understand the mechanism for the L104R-induced cell growth in HepG2 cells. Unfortunately, the results were inconclusive. In summary, this thesis has demonstrated a solid correlation between having distinct endoribonuclease act
Discipline: Chemistry
Date added: 2017-03-30T17:10:24.53Z

Title: Molecular characterization of the coding region determinant-binding protein interaction with KRAS oncogene mRNA
Contributors: Sebastian J. Mackedenski (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: The coding region determinant binding protein (CRD-BP) plays a role in some cancers, regulating gene expression through mRNA trafficking, turnover, and translational repression. Recent evidence reveals CRD-BP binding to KRAS mRNA '" a proto-oncogene frequently mutated and/or overexpressed in cancer, leading to up-regulation. A CRD-BP-KRAS mRNA interaction therefore presents a novel therapeutic opportunity to target KRAS-driven cancers and is the aim of this thesis. Electrophoretic mobility and immunoprecipitation experiments presented here using GXXG-mutant CRD-BP variants show four K-homology domains are required to bind a 57-nucleotide minimum binding sequence in the coding region of KRAS RNA these mutations do not compromise protein folding as evidenced by circular dichroism spectroscopy. DNA-based anti-sense oligonucleotide inhibitors were developed based on the minimum binding sequence that successfully abrogate the CRD-BP-KRAS mRNA interaction in vitro. Finally, a CRD-BP KH1toKH4 construct purified using a novel '~on-column'-refolding scheme suggests a possible role for RRM-domains as negative regulators of RNA-binding. --Leaf i.
Discipline: Biochemistry
Date added: 2017-03-29T17:28:16.116Z

Title: Investigation into class II P-glycoprotein messenger RNA decay in normal liver and liver tumours.
Contributors: Janis Alexandra Shandro (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Date added: 2017-03-30T16:58:31.601Z

Title: RNA sequences and structures cleaved by a novel mammalian endoribonuclease in vitro.
Contributors: Alaeddin Walid Tafech (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Date added: 2017-03-30T17:02:19.371Z

Title: Expression, purification and evaluation of recombinant human Syntaxin 18 as an endoribonuclease.
Contributors: William R. N. Bennett (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Regulation of mRNA decay is a major control point in gene expression. The processes and key players of mRNA decay in bacteria and yeast are relatively well established and characterized. In contrast, there are major gaps in our understanding of mRNA decay machineries in mammalian cells. Endonucleases appear to be key players in mammalian mRNA degradation, but the enzymes responsible are largely unknown. This is partly due to the fact that mRNA cleavage products are highly unstable and therefore difficult to detect. A novel mammalian endoribonuclease from rat liver that could cleave c-myc coding region determinant (CRD) RNA in vitro has recently been purified, and initial MALDI-MS data indicated that a candidate protein was Syntaxin 18 (Stx18), a soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE)-family member. Further work with recombinant human Stx18 also indicated that Stx18 possessed the ability to cleave c-myc CRD RNA in vitro. The main objective of this thesis was to determine the endonucleolytic domain of Stx18 utilizing a deletion mapping approach, whereby truncated mutant forms of Stx18 would be generated, purified, refolded, and tested for endonucleolytic activity. Following this, the key catalytic residues of Stx18 were to be determined using the alanine-scanning approach. Unfortunately, throughout the course of this work, new evidence came to light that questions if Stx18 does in fact possess endoribonucleolytic activity, and thus the objective of this thesis shifted somewhat towards definitive determination of whether or not Stx18 is an endoribonuclease. Irrefutably conclusive evidence that Stx18 does not possess endonuclease activity was not forthcoming, but other evidence presented herein strongly suggests that it is a small, as-yet unidentified co-purified protein that is responsible for the endonucleolytic activity seen.--Pii-iii.
Discipline: Chemistry
Date added: 2017-03-30T17:05:45.934Z

Title: Towards identifying New Human Ribonucleases that Cleave microRNA Using a High-Throughput Method.
Contributors: Suhua Ye (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Endoribonucleases were once thought of as only being key enzymes responsible for the degradation of prokaryotic mRNAs. They are now believed to play critical role in initiating eukaryotic/mammalian RNA decay and hence RNA abundance. To date, only few mammalian endoribonucleases that cleaved mRNA have been identified and studied. It is unknown if mammalian endoribonucleases can control microRNA (miRNA) abundance. The major goal of this MSc thesis was to develop a high-throughput method to identify new human endoribonucleases that cleave miR155. The first objective of this thesis was to develop a high-throughput method to express and purify human recombinant proteins from the hEx1 human fetal brain library. This was followed by development of a high-throughput fluorescence-based assay to screen purified recombinant proteins for activity against fluorogenic miR155 substrate. Through a series of optimization experiments, we have successfully established a high-throughput procedure and the criteria in selecting a preliminary list of positive candidates. --P.i.
Discipline: Chemistry
Date added: 2017-03-30T17:11:23.242Z

Title: Purification, identification and characterization of mammalian endoribonucleases that degrade c-myc mRNA in vitro.
Contributors: Tavish Richard MacKay Barnes (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: There is increasing evidence that mammalian endoribonucleases play a significant role in the degradation of messenger RNA (mRNA) and are key players in the regulation of gene expression particularly under conditions of cellular stress. One of the major challenges, however, is identifying these enzymes and assessing their significance within the context of normal/basal levels of gene expression. To this end, our understanding of this diverse set of enzymes, the mechanisms by which they operate, and their target substrates, remains somewhat mysterious. Previous studies in this laboratory (Bergstrom et al. 2006) have uncovered a mammalian hepatic-derived endoribonuclease with the ability to degrade c-myc CRD RNA in vitro. However, the identity of the enzyme(s) and the remaining co-purified proteins was not determined. The c-myc transcript is a regulator of cell proliferation, differentiation and apoptosis. Deregulated expression of c-myc prevents differentiation of many cell types, induces apoptosis, induces genomic instability, and is associated with several tumor phenotypes. In addition, there is widespread evidence that mRNA stability plays a critical role in the regulation of c-myc gene expression. The main goal of this thesis was to re-purify and conclusively identify the mammalian hepatic-derived endoribonuclease(s) and the proteins that co-purified with endonucleolytic activity against c-myc CRD RNA in vitro. The first aim of this investigation was to purify and identify enzyme(s) responsible for endoribonucleolytic activity. This portion of the study demonstrates that distinct mammalian proteins with molecular weights corresponding to 17 kDa and 35 kDa, respectively, exhibit endoribonuclease activity against c-myc CRD RNA. The second aim of this study was to further characterize the endoribonuclease(s) and to confirm the identity of the enzyme(s) by immunodepleting native endoribonuclease activity. This study revealed that the 17 kDa endoribonuclease activity was contributed by rat pancreatic ribonuclease
Discipline: Chemistry
Date added: 2017-03-30T17:05:58.89Z

Title: Assessing Rattus norvegicus recombinant syntaxin 18 for endonucleolytic function.
Contributors: Dani Michael-Didier (author), Chow Lee (Thesis advisor), Andrea Gorrell (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Recently a native endonuclease, purified from Rattus norvegicus liver, having the ability to cleave c-myc mRNA within the coding region, was tentatively identified as syntaxin 18. The objective of this research was to generate, purify and test recombinant R. norvegicus syntaxin 18 for endonucleolytic activity. To further investigate the role of syntaxin 18, homologs to R. norvegicus syntaxin 18 in a range of eukaryotes were selected for endonucleolytic testing: Mus musculus, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae. M. musculus, X. laevis, D. melanogaster and A. thaliana were expressed but not tested for endonucleolytic activity. C. elegans and S. cerevisiae were not expressed. R. norvegicus syntaxin 18 cDNA was amplified and subcloned into protein expression vector pHTT7K. E. coli BL21 were transformed with the modified vector and induced. R. norvegicus recombinant protein did express. However purification of recombinant protein was unobtainable in the pHTT7K vector. A Western blot analysis revealed that the recombinant protein was missing the 6X His-tag at the amino terminus. R. norvegicus was subcloned into new protein expression vector pET28a where the recombinant protein expressed with an attached 6X His-tag. Upon removal of the trans membrane domain, the recombinant protein was expressed, purified and tested for endonucleolytic activity against a portion of c-myc mRNA using an endonuclease assay. In contrast to the original report, a R. norvegicus recombinant syntaxin 18 preparation demonstrated only weak endonucleolytic activity. Analysis of the preparation showed although the vast majority of the yield was recombinant syntaxin 18, a number of undetermined proteins co-purified. The results shed doubt on R. norvegicus recombinant syntaxin 18 protein's function as an endoribonuclease. --P.ii.
Discipline: Chemistry
Date added: 2017-03-30T17:04:33.401Z

Title: Biochemical and cellular characterization of the coding region determinant-binding protein (CRD-BP)-mRNA interaction.
Contributors: Kashif Mehmood (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: RNA-binding proteins play critical role in the post-transcriptional processing of mRNAs. One such RNA binding protein is termed as the-Coding Region Determinant Binding Protein (CRD-BP). CRD-BP is an onco-fetal protein whose overexpression has been reported in various types of human cancers including, breast, colon, liver, skin, ovary, lung, brain, chorion, and testicular cancers. CRD-BP is a member of VICKZ family of RNA-binding proteins. In many instances, RNA binding leads to stabilization of the transcripts and an increase in their corresponding protein levels; the result is manifest in downstream effects and the cancer phenotype. The primary goal of this study was to obtain a better understanding of the interaction between CRD-BP and its three target mRNAs: GLI1, MDR1 and CD44. Radiolabelled electrophoretic mobility shift assay (EMSA) was performed with [³²P]-labeled truncated GLI1 and MDR1 RNAs to find smaller region of the transcripts which can still bind CRD-BP. It was found that GLI1 320-380 RNA is the minimum region required for binding CRD-BP, while MDR1 779-881 RNA is the minimum region which still has high affinity for CRD-BP. Previous deletion studies of CRD-BP orthologs revealed that the KH domains, and not the RRM domains, are critical for binding RNA substrates. However, it was unclear as to what extent each KH domain plays nor is it known if different KH domains are important in binding different RNAs. In this study, I used site-directed mutagenesis to mutate the GXXG to DXXG in each of the KH domains as an approach to investigate the role of each KH domains, in the context of the entire protein, in binding GLI1 and MDR1 RNAs. The K[subscript]d values of all the single and double KH variants that are capable of binding to GLI1 and CD44 RNAs was determined. In general, it was found that single mutation in KH domains may or may not affect the binding affinity of transcript, while mutations at any two KH domains totally abrogated the binding of RNA to CRD-BP, with the exception of KH3-4 which binds CD44 RNA but not GLI1 RNA. This finding also supports the hypothesis that KH domains generally work in tandem. The result also clearly showed that different RNAs bind CRD-BP differently in vitro. The secondary goal of my thesis was to design RNA oligonucleotides capable of breaking the specific CRD-BP-GLI1 RNA interaction in vitro and in cells. For in vitro studies, competition studies using [³²P] labelled GLI1 230-420 RNA and MFOLD designed RNA/DNA oligonucleotides were utilized. Amongst eight RNA oligonucleotides and one DNA oligonucleotide, S1 RNA was the best competitor against [³²P] labelled GLI 230-420 RNA in vitro. The T47D human breast cancer cell and HCT116 human colorectal cancer cell which expressed detectable level of GLI1 mRNA were chosen for further studies with the S1 RNA oligonucleotide. In both T47D and HCT116 cells where CRD-BP-GLI1 mRNA interaction was demonstrated to exists, S1 RNA oligonucleotide significantly and specifically down-regulate GLI1 mRNA expression. The results obtained support the model that CRD-BP protects GLI1 mRNA from degradation in T47D and HCT116 cells, and suggest that breaking CRD-BP-GLI1 mRNA interaction is a feasible approach to inhibit GLI1 expression. In summary, this work shows that different mRNAs indeed bind to CRD-BP differently and it is feasible to design/discover molecules capable of breaking specific CRD-BP-RNA interaction in vitro. Most importantly, molecule that breaks CRD-BP-RNA interaction in vitro was also capable of down-regulating specific the mRNA in cells. This work has provided further evidence to support the development of a new class of anti-cancer drugs that act by breaking specific protein-RNA interaction.
Discipline: Chemistry
Date added: 2017-04-11T21:12:50.903Z