Achilles-mediated and sex-specific regulation of circadian mRNA rhythms in Drosophila. Achilles-mediated and sex-specific regulation of circadian mRNA rhythms in Drosophila

The circadian clock is an evolutionarily conserved mechanism that drives rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes either directly or indirectly, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in fly heads. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is not detectable in core clock neurons using in-situ hybridization, although its expression is found in neurons throughout the fly brain. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous observations, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, a subset of circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms include many genes involved in immune function and neuronal signaling such as Prosap, Nemy and Jhl-21. Comparison of RNAseq data from control flies reveal that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome. Overall design: Achl RNAi and control flies; male and female; 96 samples with no replicates in total. 7-8 heads per sample, sampled every 2 hours for 2 days.

昼夜节律钟（circadian clock）是一类进化保守的机制，可驱动下游基因的节律性表达。核心昼夜节律钟通过直接或间接途径调控时钟控制基因（clock-controlled genes）的转录，而此类时钟控制基因在诸多节律性生理过程的执行中发挥关键作用。然而，时钟输出基因协调从大脑到外周组织的节律信号的分子机制，目前在很大程度上尚未阐明。

本研究聚焦节律基因*Achilles*，探究其在果蝇头部节律转录组调控中的作用。*Achilles*是果蝇（Drosophila）中的时钟控制基因，编码一种推定的RNA结合蛋白。通过原位杂交（in-situ hybridization）未在核心时钟神经元中检测到*Achilles*的表达，但该基因在果蝇全脑的各类神经元中均有转录。上述结果提示，*Achilles*并不参与核心昼夜节律钟的调控。

为评估其对昼夜节律mRNA节律的影响，本研究开展了RNA测序（RNA sequencing, RNAseq）实验，对比对照果蝇与全神经元*Achilles*表达被敲低（knock-down）的果蝇的节律转录组。与既往研究结果一致，我们观察到*Achilles*敲低后，免疫应答相关基因的表达显著上调。此外，在*Achilles*敲低的果蝇中，部分节律性mRNA丧失了其节律特征，这表明节律转录组中的一部分基因受到*Achilles*的直接或间接调控。此类受*Achilles*介导的节律基因涵盖诸多参与免疫功能与神经元信号传导的基因，例如*Prosap*、*Nemy*及*Jhl-21*。

通过对比分析果蝇脑部的RNA测序数据，我们发现果蝇脑部仅42.7%的时钟控制基因在雌雄个体中均表现出节律性。由于核心时钟基因的mRNA节律在雌雄个体间基本无差异，该结果提示，性别特异性调控机制是节律转录组的一类重要且此前未被充分重视的调控因素。

实验设计概况：设置*Achl* RNA干扰（RNAi）组与对照组果蝇，涵盖雌雄两性；共包含96个样本，无生物学重复。每个样本采集7-8个果蝇头部，每2小时取样1次，连续取样2天。