Abstract
Many biological and behavioural processes of animals are governed by an endogenous circadian clock, which is dependent on transcriptional regulation. Here we address post-transcriptional regulation and the role of miRNAs in Drosophila circadian rhythms. At least six miRNAs show cycling expression levels within the pigment dispersing factor (PDF) cell-pacemaker neurons; only mir-92a peaks during the night. In vivo calcium monitoring, dynamics of PDF projections, ArcLight, GCaMP6 imaging and sleep assays indicate that mir-92a suppresses neuronal excitability. In addition, mir-92a levels within PDF cells respond to light pulses and also affect the phase shift response. Translating ribosome affinity purification (TRAP) and in vitro luciferase reporter assay indicate that mir-92a suppresses expression of sirt2, which is homologous to human sir2 and sirt3. sirt2 RNAi also phenocopies mir-92a overexpression. These experiments indicate that sirt2 is a functional mir-92a target and that mir-92a modulates PDF neuronal excitability via suppressing SIRT2 levels in a rhythmic manner.
Highlights
Many biological and behavioural processes of animals are governed by an endogenous circadian clock, which is dependent on transcriptional regulation
A few miRNAs show cycling expression levels in fly heads[22,23,24,25,26,27]; it was of interest to see whether there are more cycling miRNAs in discrete clock neurons like pigment dispersing factor (PDF) cells, where circadian regulation may be more important for behavioural rhythms and where RNA assays may be less compromised by cell and tissue heterogeneity
To identify PDF cell cycling miRNAs, green fluorescent protein (GFP)-labelled PDF cells were manually sorted for RNA extraction and miRNA libraries constructed with an optimized protocol[28]
Summary
Clock/light regulates mir-92a oscillations in PDF cells. To identify PDF cell cycling miRNAs, green fluorescent protein (GFP)-labelled PDF cells were manually sorted for RNA extraction and miRNA libraries constructed with an optimized protocol[28]. To address the functions of mir-92a, we first tested whether manipulating mir-92a levels affects the well-characterized circadian morphological changes in the PDF cell termini They undergo daily fasciculation–defasciculation cycles under circadian control[13]. Rat mir-92a is implicated in synaptic scaling[32], suggesting that fly mir-92a suppresses neuronal excitability To address this possibility more directly, we tested how mir-92a levels affect PDF cell depolarization with high concentration of KCl, by monitoring changes in fluorescence levels of the voltage sensor ArcLight (PDF-GAL4;UAS-ArcLight; UAS-mir-92aOE or UAS-mir-92aSP); they decrease when neurons are depolarized[11]. Changes among genotypes were not statistically significant, perhaps because of relatively large variations among brains as well as the weaker PDF-GSG driver compared to PDF-GAL4 (see below) This was despite a similar trend as the KCl stimulation, namely decreased responses with mir-92a overexpression and increases with mir-92a knockdown (Supplementary Fig. 5).
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