Photoperiod-Dependent Expression of MicroRNA in Drosophila

Abstract

Like many other insects in temperate regions, Drosophila melanogaster exploits the photoperiod shortening that occurs during the autumn as an important cue to trigger a seasonal response. Flies survive the winter by entering a state of reproductive arrest (diapause), which drives the relocation of resources from reproduction to survival. Here, we profiled the expression of microRNA (miRNA) in long and short photoperiods and identified seven differentially expressed miRNAs (dme-mir-2b, dme-mir-11, dme-mir-34, dme-mir-274, dme-mir-184, dme-mir-184*, and dme-mir-285). Misexpression of dme-mir-2b, dme-mir-184, and dme-mir-274 in pigment-dispersing, factor-expressing neurons largely disrupted the normal photoperiodic response, suggesting that these miRNAs play functional roles in photoperiodic timing. We also analyzed the targets of photoperiodic miRNA by both computational predication and by Argonaute-1-mediated immunoprecipitation of long- and short-day RNA samples. Together with global transcriptome profiling, our results expand existing data on other Drosophila species, identifying genes and pathways that are differentially regulated in different photoperiods and reproductive status. Our data suggest that post-transcriptional regulation by miRNA is an important facet of photoperiodic timing.

Keywords: Drosophila; RNA immunoprecipitation; diapause; microRNA; photoperiodism; seasonal timing.

Figure 1

Transcriptional response to diapause and photoperiod in Drosophila. (A) Volcano plots depicting the statistical significance (y axis) vs. the fold change (x axis) of differential expression in female heads (red: p < 0.01; blue: NS). In the diapause experiment (left), positive fold changes represent upregulation in diapause. In the photoperiodic experiment (right), positive fold changes represent upregulation in long-day readings. (B) Overlapping Venn diagrams of photoperiodic and diapause DEGs.

Figure 2

Differences in miRNA expression. (A) Volcano plot showing differentially expressed miRNA in female heads (p < 0.01, red; dme-mir184* p = 0.0104) exposed to long-day (LD) and short-day (SD) conditions. Positive fold changes indicate upregulation in short-day readings. (B) Pie chart showing enriched biological functions (DAVID, Benjamini p < 0.05) of the 20 most probable targets for each DEM. The size of the pie section is proportional to the number of genes for that biological function. SD: short day. LD: long day.

Figure 3

Intersection of yeast two-hybrid interaction networks. The intersection of the three yeast two-hybrid interaction networks (diapause, photoperiodic, and miRNA) resulted in two interaction pathways. The largest interaction pathway (top) has at its center at cdc2-related-kinase, a diapause DEG (Table S1). The second interaction pathway has at its center at the gene for the C-terminal-binding protein. Red nodes are photoperiodic DEGs, light blue nodes are diapause DEGs, purple nodes are DEMs targets, and yellow nodes are both DEMs targets and photoperiodic DEGs.

Figure 4

miRNA overexpression causes a loss of photoperiodic response. Average diapause [%] in flies overexpressing (A) dme-mir-2b, (B) dme-mir-184, (C) dme-mir-274, and dme-mir-100 (D), in PDF-expressing cells. Error bars represent SEM. Significant Phot:Gen interactions [p < 0.001 (***), p < 0.05 (*)] indicate a different photoperiodic behavior between overexpressing lines and controls. dme-mir-100 is not a photoperiodic miRNA, and the experiment serves as a negative control.