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- Description
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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 ...
Show moreThe 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
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1862840
Show less - Date
- 2013
- Contributors
- Mark Barnes (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- RNA-protein interactions., Protein binding., QP623.8.P75 B37 2013
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
No abstract available.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1304335
- Date
- 2007
- Contributors
- Dan S. Sparanese (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- RNA-protein interactions., Protein binding., QP623.8.P75 S63 2006
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
Arctic systems are expected to be impacted earlier and more severely by global warming than temperate ecosystems. However, much of the research on the impact of warming on arctic ecosystems has centered on plant communities. One objective of this thesi...
Show moreArctic systems are expected to be impacted earlier and more severely by global warming than temperate ecosystems. However, much of the research on the impact of warming on arctic ecosystems has centered on plant communities. One objective of this thesis was to examine how passive warming would impact the root-associated fungal community at Alexandra Fiord, Nunavut. The root-associated fungal community consists mostly of mycorrhizal, dark-septate and hyaline-septate fungi, which are considered important mutualists in arctic ecosystems. The objective was to compare the fungal community from plots warmed by open-top chambers to ambient plots, using two methodologies: 1) fungal DNA extracted directly from root tips with terminal restriction fragment length polymorphisms (T-RFLPs) used to estimate variation, and 2) fungal cultures isolated from root tips to which PCR-RFLP techniques were applied to assess variation. T-RFLPs were used to examine the root-associated fungal community on Salix arctica. Differences between the communities were analyzed using canonical correspondence analysis (CCA). Genotype diversity was tested using a 2-way, 2-stage, nested ANOVA. Warming did not significantly change genotype cumulative frequency or diversity of the root-associated fungal community, but cumulative frequency tended to increase on the warmed plots. Genotype richness was significantly different according to site, which was correlated with differences in soil chemistry. Again site, not warming, was the main factor that distinguished the root-associated fungal community of Salix arctica, Saxifraga oppositifolia, Cassiope tetragona, and Dryas integrifolia based on fungal cultures. Warming did not have a detectable impact on cumulative frequency and diversity, based on CCA and a nested, 3-way ANOVA. Fungal cultures were identified based on sequence analysis and morphology. Phialocephala fortinii was the most frequently identified taxon, but almost half of the fungal isolates remained unknown. The root-associated fungal community was examined along a glacier forefront characterized by a directional, non-replacement primary plant succession pattern. CCA was used to examine genotype frequency; linear regressions were used to test for changes of cumulative frequency and diversity as succession advanced. The fungal community on only one of the host plants increased in frequency and richness as succession advanced. The dark- and hyaline-septate endophyte communities were distinct on different host plants, providing evidence for host specificity and higher diversity than previously reported.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1301891
Show less - Date
- 1997
- Contributors
- Karla Staff (author), Balbinder Deo (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- Geriatric psychiatry -- British Columbia, Northern., RNA-protein interactions., Protein binding., Genetic regulation., Gene expression., Messenger RNA., RC451.4.A5 S73 1997
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
The Coding Region-Determinant Binding-Protein (CRDBP) belongs to a family of RNA-binding proteins involved in embryogenesis and oncogenesis. Accumulative evidence suggests that the physical interaction with target mRNAs is a critical determinant for CR...
Show moreThe Coding Region-Determinant Binding-Protein (CRDBP) belongs to a family of RNA-binding proteins involved in embryogenesis and oncogenesis. Accumulative evidence suggests that the physical interaction with target mRNAs is a critical determinant for CRDBP function. The goal of this study is to better understand the CRDBP-RNA interaction. The role of the glycine-X-X-glycine motif in each of the KH domains of CRDBP for RNA binding in vitro, in cells and in vivo was investigated. Smaller RNA fragments of c-myc, MITF, MAPK4 and β-TrCP1 bound by CRDBP were determined and used for in silico analysis. Overall, the results showed that the G-X-X-G motif in the KH domains of CRDBP are critical for RNA binding, and that the KH domains play differential roles in binding different RNA molecules. The mapped smaller RNA regions aid in discovering inhibitory molecules and in silico analysis leading to improved approach for understanding the CRDBP RNA interaction. --Leaf ii.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1953372
Show less - Date
- 2015
- Contributors
- Gerrit van Rensburg (author), Chow H. Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- RNA-protein interactions., Protein binding., QP623.8.P75 V36 2014
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
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...
Show moreApurinic/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
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1805625
Show less - Date
- 2012
- Contributors
- Conan Ma (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- RNA-protein interactions., Cells -- Growth., Ribonucleases -- Research., QP623.8.P75 M3 2012
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
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 de...
Show moreRegulation 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.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1435045
Show less - Date
- 2008
- Contributors
- William R. N. Bennett (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- RNA-protein interactions., Ribonucleases., QP623.8.P75 B46 2008
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
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 m...
Show moreEndoribonucleases 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.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1754497
Show less - Date
- 2012
- Contributors
- Suhua Ye (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- Ribonucleases., RNA-protein interactions., Recombinant proteins., Endonucleases., QP609.R53 Y4 2011
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
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 recombin...
Show moreRecently 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.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1517824
Show less - Date
- 2009
- Contributors
- Dani Michael-Didier (author), Chow Lee (Thesis advisor), Andrea Gorrell (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- Endonucleases., Rattus norvegicus., RNA-protein interactions., QP609.E44 M53 2008
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC
- Description
-
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 rep...
Show moreRNA-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.
The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b1947185
Show less - Date
- 2014
- Contributors
- Kashif Mehmood (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
- Subject
- RNA-protein interactions., Protein binding., Messenger RNA., QP623.8.P75 M44 2014
- Type
- thesis
- Collection
- info:fedora/unbc:dtc
- Source
- University of Northern BC