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男配子分化的基因调节机制，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基因产物表达调节的新型机制，该机制可能在从精子增生到精子生长，减数分裂和末端分化的转换中起关键作用。在一项试点研究中，通过静态睾丸样本的高吞吐量RNA-3'端进行了映射3'的转录，我们发现了许多在精子中用长3'UTR表示的mRNA，但由于选择了替代性多腺苷（APA）位点（APA）位点，因此在精子中的替代品（APA）中，精子细胞和细胞中有分裂的男性。哺乳动物的精子发生中发生了类似的3'UTRS，表明具有深厚保守的调节机制。由于3'UTR可以容纳重要信息以进行翻译抑制，因此特定于阶段的替代3'末端选择可以提供一种新型的机制，可以协调蛋白质的同类蛋白，以便为男性生殖细胞分化的下一个阶段。在此R21中，我们建议研究果蝇可用的强大遗传工具在男性生殖细胞分化过程中APA在男性生殖细胞分化过程中3'UTR缩短的阶段特异性蛋白表达的程度和作用。我们的创新策略结合了三种方法。我们将使用定向的体内记者测定法来测试以下假设：在延长的3'Utr中，特定转录物的序列抑制精子中的抑制翻译，但是APA被驱除以允许精子中的翻译，从LOLAF蛋白的示例开始，在Spermatogonia中不会翻译出早期的Spermaties in Prument in Prumperties in Prument spermaties。同时，我们将使用高吞吐量RNA-3'End-seq来识别基因组范围宽，因为精子分化了3'UTR缩短的转录物，并识别可能建议坐标调节机制的共享序列基序。我们将使用上述记者构建体在体内测试这种顺式作用基序的作用，并研究通过使用生殖细胞特异性RNAI或抗MIRNA Sponges击倒候选调节剂的表达来预测将其结合的反式作用因子的作用。第三，我们将采用一种新的方法来诱导精子症在同步中进行区分，以确定在男性生殖细胞分化的一个离散时间发生APA的3'UTR缩短，还是在不同步骤处进行APA。我们提出的方法在技术上是可行的，如果成功的话，我们的研究可能会揭示一种新型的切换机制，在该机制中，生殖细胞分化是通过表达转录物来表达的，这些转录物通过翻译抑制在精子中保持沉默，直到在发育中受到特定的细胞类型缩短3'UUTR会缩短抑制作用，从而使抑制作用，从而使蛋白质的表达迅速延伸，从而使蛋白质的表达能够驱动式逐渐变化。我们的结果将为哺乳动物的精子发生提供范式，并可能发现男性避孕策略的新目标机制。