Analysis of germ granule mRNA composition and its impact on reproductive health

生殖系的发展和维护，一组高度专业化的细胞负责 将遗传物质传递给下一代，需要将特定的mRNA包装到 核糖核蛋白(RNP)颗粒称为胚芽颗粒。在整个动物界，细菌颗粒扮演着 转录后基因在生殖系增殖、分化和维持中的重要作用 监管。然而，生殖细胞颗粒mRNA组成的数量变化与 生殖健康仍是个未知数。这项提案的目标是确定 以果蝇为模型系统，研究生殖粒mRNA组成对动物繁殖的影响。在……里面 果蝇，如纳米(NOS)和极粒成分(PGC)等mRNAs是生殖系必不可少的 通过形成同型簇在胚芽颗粒内发育和积累。这些集群包含 特定信使核糖核酸类型的多个转录本。通过随机种子生成同型集群 以及依赖于在3‘非编码区中发现的顺式调控序列的自我招募过程，称为“聚类” 元素。“初步数据显示，鸡蛋产量的自然变化以及惊人的多样性 果蝇种间的生殖粒组成。具体地说，50%的数量差异 在物种间发现了NOS和/或PGC同型簇中的转录本。通过开发一种算法 和新颖的图像分析软件，我们在细菌颗粒的mRNA组成上进行了总体差异评分 在果蝇物种之间的关系，并确定了胚粒分化增加与 鸡蛋产量减少。有趣的是，在11种果蝇中发现的nos聚簇元件有 Rumpelstiltkin识别的序列基序的差异，Rumpelstiltkin是一种已知影响一氧化氮合酶积累的蛋白质。 我们的初步结果支持这样一个假设，即聚类元素多样性改变了胚芽中一氧化氮合酶的含量。 颗粒，在生殖系发育和产卵方面产生差异。为了验证我们的假设，目标1 旨在确定六种果蝇物种在生殖系发育方面的差异，这些物种表现出天然的 使用一种新的虚拟方法量化原始生殖细胞的发育和存活，以实现卵子生产的差异 实景图像分析技术。目的2探讨细菌颗粒mRNA含量与细胞周期的关系。 用单分子原位杂交和三维图像分析定量测定一氧化氮合酶和前列腺素C的产卵 六种果蝇的同型簇。目标3试图确定nos群聚中的多样性 元素足以通过工程改变nos同型群、生殖系发育和产蛋 D.具有在其他物种中发现的聚集性元素的黑腹蛇。在人类中，nos同源基因的突变是 与卵巢早衰和精子发生缺陷有关。考虑到细菌颗粒的守恒性 不，我们的建议的完成可能会揭示不孕不育等疾病的新机制 贡献可广泛应用于其他研究领域的创新技术和原则。

The development and maintenance of the germline, the set of highly-specialized cells responsible for passing on genetic material to the following generation, requires the packaging of specific mRNAs into ribonucleoprotein (RNP) granules called germ granules. Throughout the animal kingdom, germ granules play essential roles in germline proliferation, differentiation, and maintenance through post-transcriptional gene regulation. However, the connection between quantitative changes in germ granule mRNA composition and reproductive health remains unknown. The goal of this proposal is to determine how quantitative changes in germ granule mRNA composition influence animal reproduction using Drosophila as a model system. In Drosophila, mRNAs such as nanos (nos) and polar granule component (pgc) are essential for germline development and accumulate within germ granules by forming homotypic clusters. These clusters contain multiple transcripts of a specific mRNA type. Homotypic clusters are generated through a stochastic seeding and self-recruitment process that relies on cis-regulatory sequences found in the 3′UTR called “clustering elements.” Preliminary data demonstrate natural variations in egg production along with striking diversity in germ granule composition among Drosophila species. Specifically, a 50% difference in the number of transcripts in nos and/or pgc homotypic clusters was discovered between species. By developing an algorithm and novel image analysis software, we scored the overall divergence in germ granule mRNA composition between Drosophila species and identified a correlation between increased germ granule divergence and reduced egg production. Interestingly, the nos clustering element identified in eleven Drosophila species had differences in a sequence motif recognized by Rumpelstiltskin, a protein known to influence nos accumulation. Our preliminary results support a hypothesis that clustering element diversity alters the nos content in germ granules, yielding differences in germline development and egg production. To test our hypothesis, Aim 1 seeks to identify variations in germline development among six Drosophila species that exhibit natural differences in egg production by quantifying primordial germ cell development and survival using a novel virtual reality image analysis technique. Aim 2 explores the relationship between germ granule mRNA content and egg production by using single molecule in situ hybridization and 3D image analyses to quantify nos and pgc homotypic clusters in six Drosophila species. Aim 3 seeks to determine whether diversity in the nos clustering element is sufficient to alter nos homotypic clusters, germline development, and egg production by engineering D. melanogaster with clustering elements identified in other species. In humans, mutations in nos orthologs are linked to premature ovarian failure and defects in spermatogenesis. Given the conservation of germ granules and nos, completion of our proposal may reveal new mechanisms underlying diseases such as infertility while contributing innovative techniques and principles that can be broadly applied to other fields of research.