Alternative polydenylation and the regulation of male germ cell differentiation

项目描述（申请人提供）：该项目的长期目标是发现控制雄性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。我们已经确定了一种新的基因产物表达调控机制，该机制可能在从精原细胞增殖到精母细胞生长、减数分裂和终末分化的转变中发挥关键作用。在一项通过高通量rna -3‘端测序从分期睾丸样本中绘制转录本3’端图谱的初步研究中，我们发现许多mrna在精原细胞中以长3' utr表达，但由于精母细胞和分化的果蝇雄性生殖细胞中选择性多腺苷化（APA）位点的选择，它们的3' utr较短。哺乳动物精子发生过程中也存在类似的3' utr缩短现象，表明其调控机制非常保守。由于3‘ utr可以储存翻译抑制的重要信息，阶段特异性替代3’端选择可能提供一种新的机制来协调调节下一阶段男性生殖细胞分化的蛋白质群。在本R21中，我们拟利用果蝇强大的遗传工具，研究APA在雄性生殖细胞分化过程中对3'UTR缩短的阶段特异性蛋白表达的控制程度和作用。我们的创新战略包括三种方法。我们将使用直接的体内报告基因试验来验证一个假设，即特定转录物的延伸3'UTR序列抑制精原细胞中的翻译，但被APA去除以允许在精母细胞中翻译，首先以LolaF蛋白为例，该蛋白在精原细胞中不翻译，但在早期精母细胞中突然出现。与此同时，我们将使用高通量RNA-3'end- Seq来鉴定精原细胞分化过程中受3'UTR缩短影响的全基因组转录本，并鉴定可能提示协调调节机制的共享序列基序。我们将使用上述报告基因构建体在体内测试这些顺式作用基序的作用，并通过使用生殖细胞阶段特异性RNAi或抗mirna海绵敲低候选调节因子的表达来研究预计与它们结合的反式作用因子的作用。第三，我们将采用我们开发的一种新方法来诱导精原细胞同步分化，以确定APA是否在男性生殖细胞分化的一个离散时间内缩短了3'UTR，或者不同的mrna是否在不同的步骤中受到APA的影响。我们提出的方法在技术上是可行的，如果成功，我们的研究可能揭示一种新的开关机制，其中生殖细胞分化是通过翻译抑制在精原细胞中保持沉默的转录本的表达来启动的，直到发育调节的细胞类型特异性3'UTR缩短解除抑制，允许突然和快速的蛋白质表达，然后可能驱动男性生殖细胞分化的后续阶段。我们的研究结果将为研究哺乳动物精子发生提供范例，并可能揭示雄性避孕策略的新靶点机制。

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.