Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase [RNA-seq]

During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase. Overall design: These RNAseq libraries were used as control for easyCLIP analysis. We used 150 pairs of testes from 0-2 day-old wild type male flies per replicate. We chose Bam>GAL4 flies as WT samples.

在减数分裂过程中，生殖细胞及阶段特异性组分对核心细胞周期调控系统施加多层额外调控，从而建立被称为减数分裂前期的延长G2期。在雄性果蝇中，减数分裂前期持续3.5天，在此期间精母细胞会上调超过1800个基因，并发生25倍的体积增长。既往研究表明，细胞周期调控蛋白细胞周期蛋白B（Cyclin B，CycB）在未成熟精母细胞中受到翻译抑制，该抑制过程由RNA结合蛋白Rbp4及其伴侣蛋白Fest介导。本研究显示，精母细胞特异性蛋白Lut在精母细胞完全成熟前的8小时窗口内，是cycB翻译抑制所必需的。在rbp4或lut突变的雄性果蝇中，精母细胞进入并退出减数分裂的时间比野生型早6-8小时。此外，Syncrip（Syp）的精母细胞特异性剪接亚型对于成熟精母细胞中CycB蛋白的表达以及正常进入减数分裂过程是必需的。Lut与Syp可与Fest发生不依赖于RNA的相互作用。因此，一组精母细胞特异性调控因子协同调控雄性减数分裂前期中CycB蛋白的表达时序。整体实验设计：本研究的RNA测序文库被用作简易交联免疫沉淀（easyCLIP）分析的对照样本。我们每一次重复实验均使用0-2日龄野生型雄性果蝇的150对睾丸。我们选取Bam>GAL4果蝇作为野生型对照样本。