Alternative polydenylation and the regulation of male germ cell differentiation

选择性多聚腺苷酸化和雄性生殖细胞分化的调节

• 批准号: 8822709
• 负责人: MARGARET T FULLER
• 金额: $ 24.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-29 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822709
• 关键词: 3' Untranslated Regions; Binding; Biological; Biological Assay; Biological Models; Biological Process; Cell Differentiation process; Cell Lineage; Conserved Sequence; Defect; Development; Disease; Drosophila genus; Elements; Embryonic Development; Failure; Fingers; Gene Expression Regulation; Genetic; Germ Cells; Germ Lines; Goals; Growth; Homologous Gene; Housing; Maintenance; Male Contraceptive Agents; Male Infertility; Malignant Neoplasms; Mammals; Maps; Meiosis; Messenger RNA; Methods; MicroRNAs; Molecular Genetics; Mutate; Mutation; Pilot Projects; Play; Polyadenylation; Porifera; Proteins; RNA; RNA Interference; RNA Sequences; RNA-Binding Proteins; Regulation; Regulator Genes; Reporter; Repression; Research Project Grants; Role; Sampling; Seeds; Set protein; Site; Spermatids; Spermatocytes; Spermatogenesis; Spermatogonia; Staging; Structure; Tail; Technology; Testing; Testis; Time; Tissues; Trans-Activators; Transcript; Transcription Repressor/Corepressor; Transgenes; Translating; Translational Regulation; Translational Repression; Translations; Untranslated Regions; Work; adult stem cell; base; cell type; cohort; embryonic stem cell; gene discovery; genetic regulatory protein; genome-wide; in vivo; innovation; knock-down; loss of function; male; next generation; novel; polyadenylated messenger RNA; precursor cell; premature; protein expression; public health relevance; repaired; tissue repair; tool

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.

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基因产物表达调节的新机制，该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。在一项初步研究中，通过高通量 RNA-3' 末端测序对分阶段睾丸样本进行转录本的 3' 末端测序，我们发现许多 mRNA 在精原细胞中表达长 3' UTR，但由于精母细胞中选择性多腺苷酸化 (APA) 位点的选择和分化果蝇雄性生殖细胞而表达短 3' UTR。类似的 3'UTR 缩短发生在哺乳动物的精子发生中，表明存在深度保守的调节机制。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' 末端选择可能提供一种新的机制，为雄性生殖细胞分化的下一阶段协调调节蛋白质群。在此 R21 中，我们建议利用果蝇中强大的遗传工具，研究在雄性生殖细胞分化过程中 APA 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：特定转录本的延伸 3'UTR 中的序列抑制精原细胞中的翻译，但会被 APA 去除以允许在精母细胞中翻译，从 LolaF 蛋白的例子开始，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。同时，我们将使用高通量 RNA-3'end-Seq 来识别全基因组范围内精原细胞分化时受到 3'UTR 缩短的转录本，并识别可能表明协调调节机制的共享序列基序。我们将使用上述报告构建体在体内测试此类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测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 APA 导致的 3'UTR 缩短是否发生在雄性生殖细胞分化的一个离散时间，或者不同的 mRNA 是否在不同的步骤受到 APA 的影响。我们提出的方法在技术上是可行的，如果成功，我们的研究可能会揭示一种新的转换机制，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，从而允许蛋白质表达突然快速开始，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的精子发生提供范例，并可能揭示男性避孕策略的新目标机制。