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.

种系的发育和维持，这是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 包装成 核糖核蛋白（RNP）颗粒称为胚芽颗粒，在整个动物界中，胚芽颗粒发挥着作用。 通过转录后基因在种系增殖、分化和维持中发挥重要作用 然而，胚芽颗粒 mRNA 组成的数量变化与调控之间的联系。 该提案的目标是确定生殖健康的数量变化。 使用果蝇作为模型系统，生殖颗粒 mRNA 组成影响动物繁殖。 果蝇、纳米 (nos) 和极性颗粒成分 (pgc) 等 mRNA 对于种系至关重要 通过形成同型簇而在胚芽颗粒内发育和积累。 通过随机播种生成特定 mRNA 类型的多个转录本。 以及依赖于 3'UTR 中发现的顺式调控序列的自我招募过程，称为“聚类” 初步数据表明鸡蛋生产的自然变化以及惊人的多样性。 果蝇物种之间的胚芽颗粒组成具体而言，数量存在 50% 的差异。 通过开发算法，在物种之间发现了 nos 和/或 pgc 同型簇中的转录本。 和新颖的图像分析软件，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，并确定了胚芽颗粒分歧增加和 暗示产蛋量减少，在 11 种果蝇中发现的 nos 聚集元件已减少。 Rumpelstiltskin 识别的序列基序存在差异，Rumpelstiltskin 是一种已知影响 nos 积累的蛋白质。 我们的初步结果支持这样的假设：聚类元素多样性改变了胚芽中的 nos 含量 颗粒，在种系发育和产蛋方面产生差异 为了检验我们的假设，目标 1。 旨在识别表现出自然特征的六种果蝇种系发育的差异 通过使用一种新颖的虚拟方法量化原始生殖细胞的发育和存活来观察鸡蛋产量的差异 目标 2 探讨胚芽颗粒 mRNA 含量与现实图像分析技术之间的关系。 通过使用单分子原位杂交和 3D 图像分析来量化 nos 和 pgc 来产蛋 目标 3 旨在确定 6 个果蝇物种中的同型簇是否具有多样性。 元素足以通过工程改变 nos 同型簇、种系发育和产蛋 在其他物种中鉴定出具有聚类元件的黑腹果蝇，在人类中，nos 直系同源物发生突变。 鉴于生殖颗粒的保存，与卵巢早衰和精子发生缺陷有关。 不，我们的提案的完成可能会揭示不孕症等疾病的新机制，同时 贡献可广泛应用于其他研究领域的创新技术和原理。

项目成果

Mutational analysis of the functional motifs of the DEAD-box RNA helicase Me31B/DDX6 in Drosophila germline development.

果蝇种系发育中 DEAD-box RNA 解旋酶 Me31B/DDX6 功能基序的突变分析。

• 发表时间: 2023-07
• 影响因子: 3.5
• 作者: Kara, Evan;McCambridge, Aidan;Proffer, Megan;Dilts, Carol;Pumnea, Brooke;Eshak, John;Smith, Korey A;Fielder, Isaac;Doyle, Dominique A;Ortega, Bianca M;Mukatash, Yousif;Malik, Noor;Mohammed, Ammaar R;Govani, Deep;Niepielko, Matthew G;Gao, Mi
• 通讯作者: Gao, Mi

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。

• 发表时间: 2023-02-21
• 作者: Doyle, Dominique A;Burian, Florencia N;Aharoni, Benjamin;Klinder, Annabelle J;Menzel, Melissa M;Nifras, Gerard Carlo C;Shabazz;Palma, Bianca Ulrich;Hidalgo, Gisselle A;Sottolano, Christopher J;Ortega, Bianca M;Niepielko, Matthew G
• 通讯作者: Niepielko, Matthew G

Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development.

胚芽颗粒进化为果蝇种系发育提供了机制见解。

• 发表时间: 2023-08-03
• 影响因子: 10.7
• 作者: Doyle, Dominique A;Burian, Florencia N;Aharoni, Benjamin;Klinder, Annabelle J;Menzel, Melissa M;Nifras, Gerard Carlo C;Shabazz;Palma, Bianca Ulrich;Hidalgo, Gisselle A;Sottolano, Christopher J;Ortega, Bianca M;Niepielko, Matthew G
• 通讯作者: Niepielko, Matthew G