Embedding mRNA Stability in Correlation Analysis of Time-Series Gene Expression Data

Current methods for the identification of putatively co-regulated genes directly from gene expression time profiles are based on the similarity of the time profile. Such association metrics, despite their central role in gene network inference and machine learning, have largely ignored the impact of dynamics or variation in mRNA stability. Here we introduce a simple, but powerful, new similarity metric called lead-lag R2 that successfully accounts for the properties of gene dynamics, including varying mRNA degradation and delays. Using yeast cell-cycle time-series gene expression data, we demonstrate that the predictive power of lead-lag R2 for the identification of co-regulated genes is significantly higher than that of standard similarity measures, thus allowing the selection of a large number of entirely new putatively co-regulated genes. Furthermore, the lead-lag metric can also be used to uncover the relationship between gene expression time-series and the dynamics of formation of multiple protein complexes. Remarkably, we found a high lead-lag R2 value among genes coding for a transient complex

Biochemical and cellular characterization of the coding region determinant-binding protein (CRD-BP)-mRNA interaction.

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.The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b194718

Inference in systems biology: modelling approaches and applications

The main topic of this thesis is the study of biological regulatory systems using different computational modelling approaches in order to gain new insights into not yet completely understood biological processes. In "systems biology", mathematical models represent a powerful tool to study biological processes. Models are abstractions of reality always including some degree of simplification: an important ingredient of the modelling process, having a major role in suggesting the appropriate level of abstraction and simplification, is the purpose of the model, that is the question they have to answer. This thesis is focused on the analysis of how models of different complexity appropriately describe the available data to achieve a given purpose. Such analysis guides the choice of the most appropriate degree of simplification of the system under study that allows neglecting some aspects without compromising the results of the model. Three levels of detail for inference and modelling are analyzed in this thesis depending on the system under consideration. The first level is the network level, where molecules are nodes connected by edges and the interest is in the inference of the topology of connections at large scale. In the second level the network is interpreted as a mean to produce qualitative simulations and predictions which can be compared with experimental data. The third level of detail consist in a more mechanistic dynamic description of the system using ordinary differential equations but limiting the analysis to small subsystems. For each level of detail, appropriate approaches have been developed and applied to in silico and real data of different biological systems. Finally, different modelling appraches have been integrated to analyze insulin signalling pathway on different levels of simplification using a novel experimental dataset collected specifically for this purpos

Characterizing the CRD-BP-RNA interaction in-vitro and in cells.

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 doeThe original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b186284