Evidence for cell cycle suppression and microRNA regulation of cyclin D1 during anoxia exposure in turtles.

The red-eared slider turtle (Trachemys scripta elegans) has a well-developed natural tolerance for oxygen deprivation that derives from biochemical adaptations, including anoxia-induced suppression of metabolic rate. We hypothesized that mechanisms that suppress ATP-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during anaerobiosis. Cyclin D1 is a critical regulator of the G 1 phase of the cell cycle and is regarded as key to initiating cell proliferation. The relative protein expression of cyclin D1 was analyzed in both whole-cell and nuclear fractions of liver, kidney and skeletal muscle from turtles exposed to 5 or 20 h of submergence anoxia. Expression of cyclin D1 in both total and nuclear fractions decreased significantly under anoxia in liver and kidney as compared with aerobic controls, but no significant change occurred in muscle. The relative phosphorylation state of cyclin D1 (threonine 286) was also unchanged during anoxia in all tissues. Since phosphorylation of threonine 286 is necessary for proteasomal degradation of cyclin D1, this implies that an alternative mechanism is responsible for cyclin D1 suppression in anoxia. Levels of cyclin D1 mRNA transcripts did not change under anoxia in any tissue, so a post-transcriptional method of regulation was implicated. Analysis of the 3'UTR of cyclin D1 showed the presence of both an AU-rich region and a conserved binding site for microRNA-16-1 and microRNA-15a. Levels of both microRNAs increased in liver and kidney (but not in muscle) under anoxic conditions, implicating microRNA inhibition of mRNA translation as the mechanism underlying the suppression of cyclin D1 protein levels in the anoxic turtle.