Gene expression analysis of miR-210 over-expressing flies at ZT12.. Drosophila melanogaster

MicroRNAs (miRNAs) are a novel class of small RNAs which act as modulators of gene expression either by inhibiting the translation or by inducing the degradation of their target mRNAs. Several studies suggest a role for miRNAs as regulators of the circadian clock in mammals and Drosophila. Based on computational predictions of target mRNAs of clock (or clock related) genes, we have selected the miR-210 as a putative regulator of the period clock gene. We demonstrated that flies over-expressing this miRNA in the canonical clock neurons show an impaired locomotor activity pattern in both light-dark (LD) and constant darkness conditions (DD). Moreover, the projections of the Pigment Dispersing Factor (PDF)-expressing clock neurons in the optic lobe are abnormal showing peculiar “star” shaped body cells. The microarray analysis performed in the adult fly brain, revealed that this miRNA is affecting indirectly the expression of some circadian genes (ie: pdf, npf, cry, tim, pdp1) but not the period gene, and directly genes like echinus and RhoGAp92B. The in-vivo down regulation of echinus indeed can be associated with a severe impairment of the locomotor activity of flies, while the GTPase RhoGAP92B, important for the regulation of the cellular shape of neurons, could be involved in the morphological development of the PDF-expressing neurons. Overall design: Gene expression profiling was carried out on controls (w;tim-GAL4 (UAS)/+) and miR-210 overexpressing flies (w;tim-GAL4(UAS)/UAS-miR-210) by using the Drosophila 2.0 custom platform (Agilent Technologies). Total RNA was obtained from approximately 25 flies brains for each genotype. Four biological replicates were analyzed for controls and miR-210 overexpressing samples respectively for a total of 8 microarray experiments.