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Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs

Estrada-Rivadeneyra, Diego

[Thesis]. Manchester, UK: The University of Manchester; 2019.

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Abstract

Analysis of eukaryotic transcriptomes has revealed the existence of thousands of previously unannotated noncoding RNAs (ncRNAs), most of them with unknown functions. Recent evidence suggests that these novel transcripts play important roles in gene regulation, particularly under stress conditions. The main objective of this study was to use the recently developed ncRNA deletion and overexpression collections in Saccharomyces cerevisiae to study the functions of ncRNAs in eukaryotes and obtain information on the drug mode of action and possible ncRNA targets of orphan drugs. A high-throughput assay on solid media was used to systematically test the haploid (MATa) and diploid heterozygous ncRNA deletion collections in the presence of two orphan drugs: lithium citrate and riluzole. A total of seventeen ncRNAs were identified from this initial screening and subjected to liquid growth assays to confirm the results observed on solid media, quantify the growth differences, and detect the growth stages being affected. Moreover, by cloning and transforming the previously deleted ncRNAs back into the corresponding deletion strains we discovered evidence that suggests that four of the ncRNAs identified in this study might act in trans. Furthermore, high-throughput screening of the ncRNA overexpression collection in yeast revealed the existence of ncRNAs that conferred increased or decreased resistance to lithium when overexpressed. Analysis of gene expression by qPCR revealed that overexpression of the ncRNA SUT378 leads to downregulation of the overlapping gene TUM1, which confers increased resistance to lithium. Additionally, we present in this study results and methods from experiments performed with the objective of providing further evidence and resources that will serve as a base for the future study of ncRNAs. In conclusion, the yeast ncRNA deletion and overexpression collections have proven to be effective and inexpensive resources to obtain information on the functions of ncRNAs and discover possible ncRNA targets. Given that most basic biological processes are conserved within eukaryotes, data obtained from these assays could help us better understand the regulatory functions of ncRNAs in other eukaryotes and reveal the drug mode of action and targets of orphan drugs in humans.