MicroRNA regulation below zero: differential expression of miRNA-21 and miRNA-16 during freezing in wood frogs.

Natural freeze tolerance depends on numerous biochemical adaptations that address the multiple stresses imposed on cells by freezing and preserves viability by suppressing energy-expensive cell functions in the frozen state. We hypothesized that microRNAs, small non-coding RNA transcripts that bind to mRNA, could act to establish rapid biological controls that aid the reorganization of metabolic priorities for freezing survival. Selected microRNA species (miR-16 and miR-21) were evaluated using RT-PCR in liver and skeletal muscle of wood frogs (Rana sylvatica) comparing controls (5 degrees C acclimated) with animals frozen for 24h at -3 degrees C. Levels of miR-21 increased significantly during freezing by 1.5-fold and 1.3-fold in liver and skeletal muscle, respectively. MiR-16 transcripts also rose significantly by 1.5-fold in liver of frozen frogs but fell by 50% in skeletal muscle. Protein levels of Dicer, a type III RNase that is responsible for mature microRNA processing in the cytoplasm, were unchanged in liver and decreased significantly by 50% in muscle. This data provides the first report of differential regulation of microRNA species in a freeze tolerant vertebrate and suggest a mechanism for rapid, yet reversible, gene silencing when animals transition into the frozen state.