MicroRNAs are important coordinators of circadian regulation that mediate the finetuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W 1118 and a mutant of the key circadian transcriptional regulator Clock (Clk jrk). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clk jrk flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm. K E Y W O R D S CLEAR-CLIP, circadian rhythm, miRNAs, miR-375, sleep | 16537 XIA et Al.

41MicroRNA is critical coordinator to circadian regulation by silencing gene 42 expression. Although many circadian related miRNAs and some of its target are 43 known, the global functional miRNA-mRNA interaction networks remain poorly 44 understand which is hindered by imperfect base-pairing between miRNA and target 45 mRNA. In this study, we used CLEAR (Covalent Ligation of Endogenous 46Argonaute-bound RNAs) -CLIP (Cross-Linking and Immuno-Precipitation) to explore 47 the regulatory functions of miRNAs in the circadian system by comparing the 48 miRNA-mRNA interactions between the Drosophila wild-type strain w 1118 and the Clk 49 mutant Clk jrk . We unambiguously identified thousands of miRNA-mRNA interactions 50 from CLEAR-CLIP data set at unprecedented depth in vivo for the first time. Among 51 them, about 300 miRNA-mRNA interactions were involved in the regulation of 52 circadian, in which miRNAs targeting core clock genes pdp1, tim and vri presented 53 distinct changes in response to Clk jrk . Particularly, the mir-375-timeless interaction 54 from CLER-CLIP shows important effects on circadian, this functional event occurred 55 in the l-LNv neurons. Overexpression of mir-375 in tim neurons caused decreases in 56 TIM content resulting in arrhythmicity of daily locomotion and changes of sleep. This 57 present work provides a global view of miRNA targeting in the circadian rhythm. 58 59 Keywords: CLEAR-CLIP, miRNAs, circadian rhythm, sleep, miR-375 60 61 62 In animals, the intrinsic circadian clock regulates daily rhythms in physiology and 64 behavior, which are entrained by environmental stimuli such as light and 65 temperature 1, 2 . This robust timing system maintains rhythmic oscillation even under 66 constant darkness conditions in the fruit fly Drosophila melanogaster. 67 Sleep is one of the established circadian behavior, which is highly associated with 68 health status 3 . Sleep in Drosophila has been well characterized by certain parameters 69 such as total sleep, sleep bout duration and sleep bout number 4-6 . Throughout the daily 70 sleep-wake cycle, the flies exhibit two peaks of activity -one before lights on and one 71 before lights off. Genetic dissection in Drosophila indicates that sleep is regulated by 72 the circadian clock. The mutations of the core clock genes result in abnormal sleep 7, 8 , 73 while the neuropeptide pigment dispersing factor (PDF)-expressing peptidergic clock 74 neurons regulate arousal as well as sleep stability 9 . 75 The Drosophila rhythmic behavior is maintained by a central clock network in 76 the brain, which is consisted of ~150 circadian neurons 10 . These clock neurons 77 include ventral lateral neurons (LNvs) (I-LNvs, s-LNvs, and 5 th s-LNvs), dorsal 78 lateral neurons (LNds), lateral posterior neurons (LPN) and dorsal neurons (DN1, 79 DN2, and DN3), classified based on anatomical locations and varied expression of 80 core clock genes 10-13 . Among them, the PDF-positive neurons (I-LNvs and s-LNvs) 81 are essential for normal circadian activity and daily sleep. ...