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

生殖颗粒mRNA组成分析及其对生殖健康的影响

• 批准号: 10200263
• 负责人: Matthew Gene Niepielko
• 金额: $ 31.41万
• 依托单位: KEAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10200263
• 关键词: 3' Untranslated Regions; 3D virtual reality; Affect; Algorithms; Animals; Antibodies; Biological Models; CRISPR/Cas technology; Cell Count; Cell Differentiation process; Cells; Cellular Structures; Complex; Computer software; Confocal Microscopy; Cytoplasmic Granules; Data; Defect; Development; Developmental Process; Disease; Drosophila genus; Elements; Engineering; Exhibits; Fertility; Generations; Genetic Materials; Germ; Germ Cells; Goals; Human; Image Analysis; Immunofluorescence Immunologic; In Situ Hybridization; Infertility; Light; Link; Maintenance; Malignant Neoplasms; Measures; Messenger RNA; Metabolism; Molecular; Molecular Structure; Mutation; Orthologous Gene; Output; Phenotype; Play; Post-Transcriptional Regulation; Premature Ovarian Failure; Process; Production; Proteins; Regulation; Reproduction; Reproductive Health; Research; Resolution; Ribonucleoproteins; Role; Somatic Cell; Spermatogenesis; Sterility; Structure of primordial sex cell; Surveys; System; Techniques; Testing; Three-Dimensional Image; Transcript; Variant; analysis pipeline; egg; experimental study; fly; innovation; insight; nano; nanocluster; novel; recruit; reproductive; single molecule; sperm cell; virtual reality

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.

生殖系的发育和维持，这组高度特化的细胞负责 将遗传物质传递给下一代，需要将特定的mRNA包装成 核糖核蛋白（RNP）颗粒称为胚芽颗粒。在整个动物王国中，细菌颗粒发挥着作用 通过转录后基因在生殖细胞增殖、分化和维持中的重要作用 调控然而，生殖颗粒mRNA组成的定量变化与 生殖健康仍然不为人知。本提案的目标是确定 以果蝇为模型系统，研究生殖颗粒mRNA组成对动物生殖影响。在 在果蝇中，nos和pgc等基因是生殖细胞发育所必需的， 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mRNA类型的多个转录物。通过随机播种产生同型簇 以及依赖于在3′UTR中发现的顺式调节序列的自我募集过程，称为“聚类 元素”。初步数据表明，产蛋量的自然变化沿着 果蝇种间的胚芽颗粒组成。具体来说，50%的差异， 在种间发现了NOS和/或PGC同型簇中的转录物。通过开发一种算法 和新的图像分析软件，我们对胚芽颗粒mRNA组成的总体差异进行了评分， 果蝇物种之间的关系，并确定了增加胚芽颗粒分歧和 减少产蛋量。有趣的是，在11种果蝇中鉴定出的nos聚类元件， Rumpelstiltskin识别的序列基序的差异，Rumpelstiltskin是一种已知影响NOS积累的蛋白质。 我们的初步结果支持了一个假说，即聚类元素的多样性改变了胚芽中的NOS含量 颗粒，在生殖系发育和产蛋方面产生差异。为了验证我们的假设，目标1 旨在确定六种果蝇中生殖系发育的变异， 通过使用一种新的虚拟方法量化原始生殖细胞的发育和存活， 现实图像分析技术。目的2探讨胚粒mRNA含量与胚泡发育的关系。 利用单分子原位杂交和三维图像分析定量NOS和PGC的产蛋量 在六种果蝇中的同型簇。目标3旨在确定nos聚类中的多样性 元件足以通过工程改变nos同型簇、生殖系发育和卵生产 D.在其他物种中鉴定出具有聚类元素的黑腹鱼。在人类中，nos直向同源物的突变是 与卵巢早衰和精子生成缺陷有关。考虑到胚粒的保存 和NOS，完成我们的提案可能会揭示不孕症等疾病的新机制， 提供创新的技术和原则，可以广泛应用于其他研究领域。

项目成果

Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms in Drosophila.

果蝇中胚芽颗粒 mRNA 含量的进化变化是由多种机制驱动的。

• DOI: 10.1101/2023.02.21.529147
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Doyle,DominiqueA;Burian,FlorenciaN;Aharoni,Benjamin;Klinder,AnnabelleJ;Menzel,MelissaM;Nifras,GerardCarloC;Shabazz-Henry,AhadL;Palma,BiancaUlrich;Hidalgo,GisselleA;Sottolano,ChristopherJ;Ortega,BiancaM;Niepielko,MatthewG
• 通讯作者: Niepielko,MatthewG