Molecular mechanisms of mRNA stability: the impact of codon optimality and temperature

In the grand scheme of gene regulation, RNA sits at the centre, being the metaphorical ‘middle man’ in the transfer of information from DNA to protein. This makes mRNA an important target for gene regulation processes. Gene expression can be controlled at the mRNA level by regulating transcription (affecting production), causing sequestration of mRNA (affecting local concentration), regulating mRNA decay rates, or impacting production of protein by translation. In this thesis, I study mRNA from three angles: codon dependence of mRNA stability, temperature dependence of mRNA stability and temperature dependence of hyphal transition in Candida albicans.
Firstly, I examine the molecular underpinnings of the linkage between codon optimality and mRNA half-lives. A link between mRNA decay and translation has been a common theme in several studies with optimal codons being reported to increase translation rates and lead to longer half-lives. In our investigation, we found that there is a threshold length, only above it do cells respond to codon optimality with differential mRNA stability. This threshold length corresponds to the preferential translation of the transcripts by polyribosomes and the untranslated regions of the mRNA were found to determine the position of this threshold length, or whether it will exist at all.
Secondly, I study the degradation and translation dynamics of mRNAs under heat shock as compared to standard temperatures. Accurate measurements of mRNA half-lives have been few and far between: more so at elevated temperatures. In addition, the datasets resulting from these studies have shown strong method-to-method variations and in several cases, even different studies using the same method have led to uncorrelated half-lives. I used approach to equilibrium technique of RNA metabolic labelling to generate high quality RNA half-life datasets in S. cerevisiae at standard and elevated temperatures. They show high inter-temperature correlation as well as high correlation with external measurements. In addition, I examine the translation status of transcripts at standard and heat shock temperatures in yeast and mammalian systems.
The third part of this thesis chronicles our search for a temperature dependent switch in RNA turnover in Candida, which would lead to hyphal induction. The hyphal transition is important for pathogeny and thus clinically relevant. I show that temperature alone is sufficient for the hyphal transition and that several RNA markers can predict this transition, before the morphology appears.