dc.contributor.authorTan, Jiazi
dc.identifier.citationTan, J. (2014). Gene expression regulation by well-known non-coding RNAs. Doctoral thesis, Nanyang Technological University, Singapore.
dc.description.abstractIn this thesis, I studied the functions of two well-known non-coding ribonucleic acids (ncRNAs), namely U1 small nuclear ribonucleic acid (snRNA) and Xist, which exert their influence over gene expression via different pathways. 5’ splice site (5’ss) recognition by U1 snRNA binding is one of the first steps in pre-mRNA splicing, which is critical for gene expression in eukaryotes. U1 classically base pairs to the 5’ss in a specific ‘canonical’ register, yet there is proof that other non-canonical registers exist. In this thesis, we verify the existence of non-canonical 1-nucleotide asymmetric loop registers, as well as present proof for the usage of non-canonical registers with 2 bulged nucleotides. We also show evidence which implies that bulge registers longer than 2 may not be tolerated. We also demonstrate that if the fifth intronic nucleotide of the 5’ss is a guanine, U1 always base pairs with it in the canonical register, despite thermodynamic predictions to the contrary. In addition, we report that a uridine residue at position +4 of the 5’ss can establish a non-canonical base pair with a pseudouridine in U1, thus contributing to 5’ss recognition. Our results extend our knowledge on the flexibility of the 5’ss/U1 RNA duplex structure that leads to productive splicing. The Xist long ncRNA is essential for random X chromosome inactivation (XCI). XCI acts upon one of the two X chromosomes in female mammalian cells during differentiation, silencing most of the genes on that chromosome. In one project, we attempted to insert a second Xist gene into the single X chromosome of male murine embryonic stem (ES) cells to cause ectopic XCI upon differentiation, leading to cell lethality as important X-linked genes were inactivated. From there, we could screen for genes important for XCI by rescuing the differentiated transgenic ES cells by gene silencing. Although ectopic XCI was achieved, the expected 100% lethality did not materialize, preventing us from establishing the screen. In another project, we screened for activators of XCI. By transfecting male ES cells with sequences derived from a region of the X-chromosome known to be important for XCI, and screening for induction of ectopic XCI, our lab had previously identified four novel sequences that may contribute to XCI activation. Using the same strategy, we may have located yet another genomic sequence that fuels XCI activation.en_US
dc.format.extent167 p.en_US
dc.subjectDRNTU::Science::Biological sciencesen_US
dc.titleGene expression regulation by well-known non-coding RNAsen_US
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.contributor.supervisorFrancesc Xavier Roca Castellaen_US
dc.description.degreeDOCTOR OF PHILOSOPHY (SBS)en_US

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