Project description:Using high throughput sequencing of Drosophila head RNA, a small set of miRNAs that undergo robust circadian oscillations in levels were discovered. We concentrated on a cluster of six miRNAs, mir-959-964, all of which peak at about ZT12 or lights-off. The data indicate that the cluster pri-miRNA is transcribed under bona fide circadian transcriptional control and that all 6 mature miRNAs have short half-lives, a requirement for oscillating. Manipulation of food intake dramatically affects the levels and timing of cluster miRNA transcription with no more than minor effects on the core circadian oscillator. This indicates that the central clock regulates feeding, which in turn regulates proper levels and cycling of the cluster miRNAs. Viable Gal4 knock-in as well as cluster knock-out and over-expression strains were used to localize cluster miRNA expression as well as explore their functions. The adult head fat body is a major site of expression, and feeding behavior, innate immunity, metabolism, and perhaps stress responses are under cluster miRNA regulation. The feeding behavior results indicate that there is a feedback circuit between feeding time and cluster miRNA function as well as a surprising role of post-transcriptional regulation in these behaviors and physiology. To address possible functions of the cluster miRNAs, the knock-out strain (mir959-952-KO) as well as a cluster over-expression strain (UAS-cluster, tim-gal4) was assayed for mRNA changes relative to their WT counterparts on Affymetrix expression arrays. The strategy was based on the observation that miRNAs often cause a decrease in the steady-state levels of their target mRNAs (Guo et al., 2010; Lim et al., 2005). Because of the circadian regulation and the possibility of non-fat body expression, the over-expression strain was generated with the broad circadian driver tim-gal4 rather than a fat body driver. To accommodate the possibility that important mRNA changes only appear at certain circadian times, RNA was assayed from heads collected at two different times, ZT4 and ZT16. Analyzed expression data was assayed from total RNA from Drosophila heads in two sample groups consisting of four samples (with eight samples in total): knock-out strain at ZT4 and ZT16, and its respective wildtype control at ZT4 and ZT16; cluster over-expression strain at ZT4 and ZT16 and its respective wildtype control at ZT4 and ZT16.

Project description:Using high throughput sequencing of Drosophila head RNA, a small set of miRNAs that undergo robust circadian oscillations in levels were discovered. We concentrated on a cluster of six miRNAs, mir-959-964, all of which peak at about ZT12 or lights-off. The data indicate that the cluster pri-miRNA is transcribed under bona fide circadian transcriptional control and that all 6 mature miRNAs have short half-lives, a requirement for oscillating. Manipulation of food intake dramatically affects the levels and timing of cluster miRNA transcription with no more than minor effects on the core circadian oscillator. This indicates that the central clock regulates feeding, which in turn regulates proper levels and cycling of the cluster miRNAs. Viable Gal4 knock-in as well as cluster knock-out and over-expression strains were used to localize cluster miRNA expression as well as explore their functions. The adult head fat body is a major site of expression, and feeding behavior, innate immunity, metabolism, and perhaps stress responses are under cluster miRNA regulation. The feeding behavior results indicate that there is a feedback circuit between feeding time and cluster miRNA function as well as a surprising role of post-transcriptional regulation in these behaviors and physiology. To address possible functions of the cluster miRNAs, the knock-out strain (mir959-952-KO) as well as a cluster over-expression strain (UAS-cluster, tim-gal4) was assayed for mRNA changes relative to their WT counterparts on Affymetrix expression arrays. The strategy was based on the observation that miRNAs often cause a decrease in the steady-state levels of their target mRNAs (Guo et al., 2010; Lim et al., 2005). Because of the circadian regulation and the possibility of non-fat body expression, the over-expression strain was generated with the broad circadian driver tim-gal4 rather than a fat body driver. To accommodate the possibility that important mRNA changes only appear at certain circadian times, RNA was assayed from heads collected at two different times, ZT4 and ZT16.

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