Deep sequencing the circadian and light-dependent transcriptome of Drosophila brain. Drosophila melanogaster

Eukaryotic circadian clocks include transcriptional/translational feedback loops that drive 24-hour rhythms of transcription.These transcriptional rhythms underlie oscillations of protein abundance, thereby mediating circadian rhythms of behavior, physiology, and metabolism. Numerous studies over the last decade have employed microarrays to profile circadian transcriptional rhythms in various organisms and tissues. Here we use RNA sequencing (RNA-Seq) to profile the circadian transcriptome of *Drosophila melanogaster* brain from wild-type and *period*-null clock-defective animals. We identify several hundred transcripts whose abundance oscillates with 24-hour periods, including a number of non-coding RNAs (ncRNAs) that were not identified in previous microarray studies. Of particular interest are *U snoRNA host genes* (*Uhgs*), a family of cycling ncRNAs that encode the precursors of over 50 box C/D snoRNAs, key regulators of ribosomal biogenesis. Transcriptional profiling at the level of individual exons reveals alternative splice isoforms for many genes whose relative abundances are regulated by either *period* or circadian time, although the effect of circadian time is muted in comparison to that of *period*. Interestingly, *period* loss-of-function significantly alters the frequency of RNA editing at a number of editing sites, suggesting an unexpected link between a key circadian gene and RNA editing. We also identify tens of thousands of novel splicing events beyond those previously annotated by the modENCODE consortium, including several that affect key circadian genes. These studies demonstrate extensive circadian control of ncRNA expression, reveal the extent of clock control of alternative splicing and RNA editing, and provide a novel, genome-wide map of splicing in *Drosophila* brain. Overall design: RNA-Seq transcriptional profiling of Drosophila brains from wildtype and period loss-of-function (per0) flies with time points taken over two days in constant darkness. Time points at CT24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, and 68. 10-12 brains per time point.

真核生物昼夜节律钟包含转录/翻译反馈环路，可驱动24小时周期的转录节律。此类转录节律构成蛋白质丰度振荡的基础，进而介导行为、生理与代谢的昼夜节律。近十年来，诸多研究已利用微阵列（microarray）在多种生物及组织中解析昼夜节律转录节律。本研究采用RNA测序（RNA sequencing, RNA-Seq），对野生型以及*period*基因敲除的节律缺陷型黑腹果蝇（*Drosophila melanogaster*）大脑的昼夜转录组进行分析。我们鉴定出数百个丰度以24小时周期振荡的转录本，其中包含若干此前微阵列研究未发现的非编码RNA（non-coding RNA, ncRNA）。尤为值得关注的是U snoRNA宿主基因（U snoRNA host genes, Uhgs）——一类编码超过50种盒式C/D snoRNA前体的周期性非编码RNA家族，而后者是核糖体生物发生的关键调控因子。通过对单个外显子进行转录谱分析，我们发现多个基因存在可变剪接异构体，其相对丰度受*period*基因或昼夜时间调控，但相较于*period*基因的影响，昼夜时间的调控作用相对较弱。有趣的是，*period*功能丧失会显著改变多个编辑位点的RNA编辑频率，这提示关键昼夜节律基因与RNA编辑之间存在此前未被发现的关联。我们还鉴定出远超modENCODE联盟此前注释的数万种全新剪接事件，其中若干事件会影响关键昼夜节律基因。本研究揭示了昼夜节律对非编码RNA表达的广泛调控，阐明了生物钟对可变剪接与RNA编辑的调控范围，并为黑腹果蝇大脑提供了全新的全基因组范围剪接图谱。

整体实验设计：对野生型与*period*功能丧失型（per0）果蝇的大脑开展RNA-Seq转录谱分析，在持续黑暗条件下于两天内采集时间点样本，采样时间点为CT24、28、32、36、40、44、48、52、56、60、64及68，每个时间点采集10-12个大脑。