Micromanaging freeze tolerance: The biogenesis and regulation of microRNAs in frozen frogs
When temperatures plummet below 0°C, the wood frog (Rana sylvatica) freezes up to 65% of its body water in extracellular ice masses, displaying no measurable brain activity, no breathing, and a flat-lined heart. Various molecular mechanisms including microRNAs, a multifunctional group of short non-coding RNAs, are in place to facilitate freeze tolerance. This thesis provides the first large-scale investigation of microRNA in a freeze tolerant vertebrate. Immunoblotting was used to investigate protein abundance of key microRNA biogenesis factors in brain and liver of control, 24 h frozen, and 8 h thawed R. sylvatica. Biogenesis capacity was reduced in brains and elevated in livers during freezing and thawing. This correlated with RT-qPCR levels of ~110 microRNAs, where the majority of differentially expressed miRNAs were downregulated in brains and upregulated in livers. Bioinformatic miRNA targeting predicted brain miRNAs to play a neuroprotective role, while hepatic miRNAs suppressed energy-expensive pro-growth processes.