miRNAs regulate the circadian clock

We have recently demonstrated that miRNAs are key regulators of the circadian clock. When we knocked down the miRNA pathway (by inhibiting the miRNA processing enzyme dicer-1), flies display very weak circadian rhythms.

In order to determine which circadian mRNA are controlled by miRNAs we developed a biochemical method for identifying mRNAs associated with the miRNA-silencing machinery. It consisted in the purification of the miRNA-mRNAs complexes from fly brains using a specific antibody. Our procedure turned out to work very well and using it we could determine that three core Clock components are regulated by the RISC (miRNA-silencing complex) in vivo. Among them is the master clock regulator clk. We complemented this data by developing a method that allows us to identify miRNAs expressed in fly head's circadian tissue.

It consisted in the knock-down of the miRNA processing pathway ONLY in the circadian clock cells (by inhibiting the proteins drosha and pasha) followed by detection of miRNA precursors (by the use of tilling arrays).

This inhibition leads to the accumulation of the miRNA precursors expressed ONLY in these cells. This strategy identified 27 miRNAs highly expressed in the circadian cells

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Finally, we showed that bantam regulation is essential for the circadian clock. This is because that mutation of the 3 binding sites for this miRNA on clk 3'UTR inhibit circadian rhythmicity.

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