Alternative polydenylation and the regulation of male germ cell differentiation

描述（由申请人提供）：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，这对于理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，可能在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。在一项利用高通量RNA-3 '末端测序技术对分阶段睾丸组织中转录本3'末端进行定位的初步研究中，我们发现许多mRNA在精原细胞中以长的3'UTR表达，而在精母细胞和分化的果蝇雄性生殖细胞中由于选择了替代的多聚腺苷酸化（阿帕）位点而以短的3' UTR表达。类似的3 'UTR缩短发生在哺乳动物精子发生中，表明一种高度保守的调控机制。由于3 'UTR可以容纳翻译抑制的重要信息，阶段特异性替代3'末端选择可以提供一种新的机制来协调调节雄性生殖细胞分化下一阶段的蛋白质组。在这个R21，我们建议调查的程度和作用，在控制阶段特异性蛋白表达的3 'UTR缩短阿帕在雄性生殖细胞分化，利用强大的遗传工具，在果蝇。我们的创新战略结合了三种方法。我们将使用定向体内报告基因测定来检验以下假设：特定转录物的延伸3 'UTR中的序列抑制精原细胞中的翻译，但被阿帕去除以允许精母细胞中的翻译，以LolaF蛋白为例开始，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。平行地，我们将使用高通量RNA-3 '末端- Seq来鉴定基因组范围内随着精原细胞分化而经历3' UTR缩短的转录物，并鉴定可能提示协调调节机制的共享序列基序。我们将使用如上所述的报告构建体测试这种顺式作用基序在体内的作用，并研究预测通过使用生殖细胞阶段特异性RNAi或抗miRNA海绵敲低候选调控因子的表达来结合它们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，以确定阿帕是否在雄性生殖细胞分化的一个离散时间发生3 'UTR缩短，或者不同的mRNA是否在不同的步骤受到阿帕的影响。我们提出的方法在技术上是可行的，如果成功的话，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由转录本的表达引发的，这些转录本通过翻译抑制在精原细胞中保持沉默，直到发育调节的细胞类型特异性3 'UTR缩短解除抑制，允许蛋白质的突然和快速表达开始，所述蛋白质随后可驱动雄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，并可能为男性避孕策略揭示新的靶点机制。

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.