MicroRNA biogenesis proteins follow tissue-dependent expression during freezing in Dryophytes versicolor

Grey tree frogs (Dryophytes versicolor) have the remarkable ability to endure full-body freezing over the winter, with up to 42% of total body water converted into extracellular ice. Survival is aided by metabolic rate depression that greatly reduces tissue energy costs over the winter. Post-transcriptional controls on gene expression which include miRNA regulation of gene transcripts can aid implementation of the reversible changes required for freeze tolerance, since miRNAs are ideal for facilitating the rapid metabolic reorganization needed for this process. The energy cost for synthesizing new miRNAs is low, and miRNAs' ability to target more than one mRNA transcript (and vice versa) allows a wide versatility in their capability for metabolic restructuring. Western immunoblotting was used to examine protein expression levels of members of the miRNA biogenesis pathway in D. versicolor liver, skeletal muscle, and kidney. Four of these proteins (Dicer, Drosha, Trbp, Xpo5) were upregulated in liver of frozen frogs, suggesting enhanced capacity for miRNA biogenesis, whereas expression of four proteins in frozen muscle (Ago1, Ago2, Dgcr8, Xpo5) and six proteins in kidney (Ago1, Ago2, Ago3, Ago4, Dgcr8, Ran-GTP) were downregulated, indicating an opposite trend. Overall, the data show that miRNA biosynthesis is altered during freezing and differentially regulated across tissues. We suggest that miRNAs are central for the freeze tolerance strategy developed by D. versicolor, and future research will expound upon specific miRNAs and their roles in mediating responses to freezing stress.