Alternative polydenylation and the regulation of male germ cell differentiation

描述(申请人提供)：该项目的长期目标是发现控制雄配子分化的基因调控机制，这对了解男性不育的遗传和分子基础至关重要。我们发现了一种新的调控基因产物表达的机制，该机制可能在精原细胞增殖向精母细胞生长、减数分裂和终末分化的转换中发挥关键作用。在一项通过高通量RNA-3‘末端测序从分期的睾丸样本中绘制转录本的3’端的初步研究中，我们发现许多mRNAs在精原细胞中表达长的3‘UTRs，但由于精母细胞中选择性的多聚腺苷酸化(APA)位点和分化的果蝇雄性生殖细胞而表达短的3’UTRs。哺乳动物精子发生过程中也存在类似的3‘UTRs缩短现象，表明存在一种高度保守的调控机制。由于3‘端非编码区可以包含重要的翻译抑制信息，阶段特异的替代3’端选择可能提供一种新的机制来协调调节下一阶段雄性生殖细胞分化的蛋白质队列。在这个R21中，我们建议利用果蝇中可用的强大遗传工具，研究APA在雄性生殖细胞分化过程中控制3‘UTR缩短的程度和作用。我们的创新战略结合了三种方法。我们将使用体内定向报告分析来检验这一假设，即特定转录本延伸的3‘UTR中的序列抑制精原细胞中的翻译，但被APA移除以允许在精母细胞中翻译，首先是LolaF蛋白，它不在精原细胞中翻译，但在早期精母细胞中突然出现。同时，我们将使用高通量RNA-3‘末端序列来鉴定基因组范围内精原细胞分化时3’端非编码区缩短的转录本，并鉴定可能暗示协调调控机制的共享序列基序。我们将使用上述报告结构在体内测试这些顺式作用基序的作用，并研究反式作用因子通过使用生殖细胞阶段特异性RNAi或抗miRNA海绵下调候选调控因子的表达来结合它们的作用。第三，我们将使用我们开发的一种新的方法来诱导精原细胞同步分化，以确定在雄性生殖细胞分化过程中，APA是在一个离散的时间段发生3‘UTR缩短，还是不同的mRNAs在不同的步骤受到APA的影响。我们提出的方法在技术上是可行的，如果我们的研究成功，我们的研究可能会揭示一种新的开关机制，其中生殖细胞分化是由转录抑制蛋白的表达启动的，这些转录产物在精原细胞中通过翻译抑制保持沉默，直到发育调节的细胞类型特定的3‘UTR缩短解除抑制，允许蛋白质的突然和快速开始表达，然后可能驱动雄性生殖细胞分化的后续阶段。我们的结果将为研究哺乳动物精子发生提供范例，并可能为男性避孕策略揭示新的靶向机制。

DESCRIPTION (provided by applicant): The long-term goal of this project is to discover gene regulatory mechanisms that control differentiation of male gametes, critical for understanding the genetic and molecular basis of male infertility. We have identified a novel mechanism of regulation of gene product expression that may play a key role in the switch from spermatogonial proliferation to spermatocyte growth, meiosis and terminal differentiation. In a pilot study mapping 3' ends of transcripts by high throughput RNA-3'end-Sequencing from staged testis samples, we discovered many mRNAs expressed with long 3' UTRs in spermatogonia but short 3'UTRs due to selection of alternative polyadenlyation (APA) sites in spermatocytes and differentiating Drosophila male germ cells. Similar shortening of 3'UTRs occurs in mammalian spermatogenesis, indicating a deeply conserved regulatory mechanism. Because 3'UTRs can house important information for translational repression, stage-specific alternative 3' end selection may provide a novel mechanism to coordinately regulate cohorts of proteins for the next stage of male germ cell differentiation. In this R21, we propose to investigate the extent and role in control of stage-specific protein expression of 3'UTR shortening by APA during male germ cell differentiation, taking advantage of powerful genetic tools available in Drosophila. Our innovative strategy combines three approaches. We will use directed in vivo reporter assays to test the hypothesis that sequences in the extended 3'UTR of specific transcripts repress translation in spermatogonia, but are removed by APA to allow translation in spermatocytes, starting with the example of LolaF protein, which is not translated in spermatogonia but appears abruptly in early spermatocytes. In parallel we will use high throughput RNA-3'end- Seq to identify genome wide the transcripts subject to 3'UTR shortening as spermatogonia differentiate and identify shared sequence motifs that may suggest coordinate regulatory mechanisms. We will test the role of such cis-acting motifs in vivo using reporter constructs as above and investigate the role of trans-acting factors predicted to bind them by knocking down expression of candidate regulators using germ cell stage-specific RNAi or anti-miRNA sponges. Third, we will employ a novel method we developed to induce spermatogonia to differentiate in synchrony to determine if 3'UTR shortening by APA occurs at one discrete time in male germ cell differentiation or if different mRNAs are subject to APA at different steps. The approaches we propose are technically feasible, and if successful, our study may reveal a novel switch mechanism where germ cell differentiation is primed by expression of transcripts that are kept silent in spermatogonia by translational repression, until developmentally regulated cell type specific 3'UTR shortening relieves the repression, allowing abrupt and rapid onset of expression of proteins that may then drive subsequent stages of male germ cell differentiation. Our results will provide paradigms to investigate in spermatogenesis in mammals and may uncover new target mechanisms for male contraceptive strategies.