Dazl maintains proliferating germ cells through a network of polyA-proximal mRNA interactions [GFP cells RNA-Seq]

Dazl (deleted in azoospermia like) is a member of the DAZ family of germ cell-restricted RNA binding proteins required for gametogenesis from worm to human. The direct RNA targets and functions of these essential proteins are poorly understood. Here, we generated high-resolution, transcriptome-wide maps of Dazl-RNA interactions in mouse testes. These maps provide important insights into the mechanism of Dazl recruitment to mRNA and reveal Dazl binding to thousands of mRNAs predominantly through sequence-specific interactions near the polyA tail. Using transgenic mice and fluorescence activated cell sorting (FACS), we isolated DAZL knockout germ cells and used RNA-Seq to identify mRNAs sensitive to DAZL-ablation. Intersecting the RNA-Seq and Dazl-RNA interaction datasets revealed that Dazl enhances expression of a subset of directly-bound transcripts, namely mRNAs for a network of essential cell cycle regulatory genes. Collectively, our integrative analysis delineates a Dazl-dependent post-transcriptional gene regulatory program essential for mammalian germ cell maintenance. Overall design: RNA-seq libraries generated Dazl WT and Dazl KO GFP+ germ cells无精症缺失样蛋白（Dazl, deleted in azoospermia like）是DAZ家族的成员，该家族为生殖细胞限制性RNA结合蛋白，在从线虫到人类的所有物种中均为配子发生所必需。目前学界对这类核心蛋白的直接RNA靶标与具体生物学功能仍知之甚少。 本研究构建了小鼠睾丸中Dazl与RNA互作的高分辨率全转录组图谱。该图谱为解析Dazl被招募至mRNA的分子机制提供了重要线索，并揭示Dazl主要通过多聚腺苷酸尾（polyA tail）附近的序列特异性相互作用结合数千种mRNA。 本研究借助转基因小鼠与荧光激活细胞分选术（FACS, fluorescence activated cell sorting）分离得到DAZL敲除的生殖细胞，并通过RNA测序（RNA-Seq）筛选出对DAZL缺失敏感的mRNA。将RNA测序数据集与Dazl-RNA互作图谱进行整合分析后发现，Dazl可增强一部分直接结合的转录本的表达，其中包括一系列核心细胞周期调控基因的mRNA。 综上，本研究通过整合分析构建了依赖于Dazl的转录后基因调控程序，该程序对哺乳动物生殖细胞的维持至关重要。实验整体设计：构建Dazl野生型（WT）与Dazl敲除型（KO）的GFP阳性生殖细胞的RNA测序文库。