microRNAs and metabolites in naïve to primed human embryonic stem cell transition

Abstract

This dissertation research is focused on the small molecules of the cell: metabolites and miRNAs. The purpose is to gain a deeper understanding of how they control the cell by investigating the role of secondary structure during miRNA biogenesis and of the changes that occur in the metabolome during naïve to primed human embryonic stem cell (hESC) transition. The first chapter of the thesis focuses on miRNA regulation: Through analysis of miRNA secondary structure and miRNA expression levels in cells with different levels of the enzyme Drosha I have found that miRNAs with mismatches in the precursor region of the miRNA, 9-12 nucleotides from the Drosha cutting site show a higher sensitivity to changes in Drosha levels than miRNAs that lack mismatches in said regions. Through mutagenesis experiments I have shown that by altering the miRNA secondary structure its sensitivity to changes in Drosha levels can be changed. This shows how the cell can selectively regulate a group of miRNAs relative to another by changing Drosha expression and may explain the impact of point mutations outside of the seed region of certain miRNAs. miRNA therapies are also being developed, where the small size of miRNAs makes them excellent candidates for gene therapy. The results of this research can be applied to miRNA therapies by increasing the potency of select miRNAs in their target tissues through structural modifications. In cases where the target tissue has different levels of Drosha compared to surrounding tissues (such as in many types of cancer) the miRNA can also be designed to minimize side effects in surrounding healthy tissues. The second chapter of the thesis focuses on the metabolome in human embryonic stem cells: With the use of mass spectrometry combined with gene expression data I have analyzed the metabolome of naïve and primed hESCs. I show that the two developmental stages have distinct profiles in metabolite set up and I have identified pathways that show a change in activity during naïve to primed hESC transition. I show that tryptophan degradation is increased in primed cells leading to an accumulation of the tryptophan degradation product kynurenine, which is known to inhibit the antitumor response of the immune system by activting the transcription factor AhR and could be a mechanism which prevents rejection of the recently implanted embryo. I also show that primed hESCs display a decrease in activity of the enzyme NNMT, which uses the metabolite S-adenosyl methionine (SAM) to convert nicotinamide into 1-methyl nicotinamide. The decrease of NNMT activity makes SAM available as a substrate for other methylation mechanisms and increases H3K27me3 methylation. When knocking down NNMT the H3K27me3 marks are increased, supporting the hypothesis that the metabolome regulates the epigenetic landscape during naïve to primed hESC transition.