Post-transcriptional regulation of germline mRNAs in C. elegans

线虫种系 mRNA 的转录后调控

• 批准号: 10390502
• 负责人: Sean Patrick Ryder
• 金额: $ 34.51万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10390502
• 关键词: 3' Untranslated Regions; Address; Affinity; Alleles; Amino Acids; Animals; Atmosphere; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biology; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Coupled; Critical Pathways; Data; Databases; Development; Developmental Process; Disease; Elements; Embryo; Embryonic Development; Engineering; Ensure; Event; Fertilization; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; In Vitro; Individual; Instruction; Invertebrates; Lead; Mammals; Maps; Maternal Messenger RNA; Mediating; Meiosis; Messenger RNA; Molecular; Mutagenesis; Mutate; Mutation; Nature; Nematoda; Oogenesis; Outcome; Parents; Pattern; Pattern Formation; Phenotype; Physiological; Play; Positioning Attribute; Post-Transcriptional Regulation; Property; Proteins; RNA; RNA Binding; RNA interference screen; RNA-Binding Proteins; Regulation; Regulatory Element; Reporter; Reporter Genes; Reproduction; Reproductive Health; Research; Role; Series; Sexual Reproduction; Site; Specificity; Sterility; Technology; Testing; Time; Transcript; Transgenes; Transgenic Organisms; Translating; Untranslated Regions; Work; base; cell fate specification; experimental study; gene replacement; genome editing; in vivo; innovation; interdisciplinary approach; interest; mRNA Expression; medical schools; mutant; oocyte maturation; reproductive; stoichiometry; temporal measurement; tool; unpublished works; zygote

Project Summary: My lab is interested in defining how the maternal load is established during oogenesis and decoded after fertilization. We know the identity of most important maternal transcripts and maternally supplied RNA- binding proteins. We know that these factors are required for germline development, oocyte maturation, and pattern formation in early embryogenesis. But we do not yet know which regulatory events are most important for reproduction, or what mechanisms coordinate regulation in space and time. We employed a “protein-centric” approach to map the wiring diagram of maternal RNA regulation in the nematode Caenorhabidits elegans. We defined the sequence motifs recognized by a several maternal RNA- binding proteins (RBPs) and identified of functional cis-regulatory elements in 3’UTR reporter genes representing well studied maternal mRNAs. Our work made revealed that binding specificity is not sufficient to explain mRNA targeting in vivo. All proteins studied to date bind to short linear partially degenerate motifs present in at least 30-50% of all mRNAs. In some cases, the motifs have been shown to be necessary but not sufficient to drive regulatory activity. In other cases, the motifs do not lead to regulation. Putting a motif, even in multiple copies, into a transgene does not confer RBP-dependent regulation. Binding is not a great predictor of regulation, revealing that binding site context is also crucial for targeting. Moving forward, we are pursuing three major strategies. In the first, we are using CRISPR-cas9 genome editing to make mutations in the 3’UTRs of two critical maternal transcripts in order to identify which regulatory events are most important to define the pattern of expression and for reproductive health. Genome editing technology has advanced to the point where we can make targeted UTR deletions and substitutions, so now we can assess importance directly. Our second direction is aimed at defining regulatory mechanisms. We are performing AID-degron tagged experiments to define how RBPs and core regulatory machinery control the maternal mRNA expression with temporal resolution in the germline and in the embryo. Finally, we wish to understand how the biochemical properties of an RBP contribute to its mutant phenotypes. Proteins can have multiple activities, and it is not always clear that the most obvious activity is the one that underlies its mutant phenotypes. We are in position to address this question directly. We have expressed and purified several C. elegans RBPs over the course of the past ten years and have begun to dissect their biochemical properties using quantitative in vitro tools. We now have the ability to introduce mutations that effect RNA-binding properties into the endogenous locus in C. elegans to determine phenotype. Our innovative interdisciplinary approach, coupled to the strong atmosphere at UMass Medical School in RNA biology and C. elegans genetics, will ensure rapid progress in defining the maternal effect in embryogenesis at the functional level.

项目总结： 我的实验室感兴趣的是定义母体负荷是如何在卵子发生期间建立和解码的 受精后。我们知道最重要的母体转录和母体提供的RNA的身份- 结合蛋白。我们知道这些因素是生殖系发育、卵母细胞成熟和 早期胚胎发育中的模式形成。但我们还不知道哪些监管事件最重要 对于繁殖，或者什么机制协调空间和时间的调节。 我们使用了一种“以蛋白质为中心”的方法来绘制母体RNA调控的线路图。 线虫：秀丽线虫。我们定义了由几个母体RNA识别的序列基序- 结合蛋白及其3‘端非编码区报告基因功能顺式调控元件的鉴定 代表了研究充分的母体mRNAs。我们的工作揭示了结合的特异性不足以 解释体内的信使核糖核酸靶向。到目前为止，所有研究的蛋白质都与短线状部分简并基序结合 存在于至少30%-50%的mRNAs中。在某些情况下，这些主题被证明是必要的，但不是 足以推动监管活动。在其他情况下，这些主题不会导致监管。加入一个主题，甚至是 多个拷贝，进入一个转基因并不赋予RBP依赖的调节。绑定并不是一个很好的预测因子 监管，揭示了结合位点的上下文对靶向也是至关重要的。 展望未来，我们正在推行三大战略。首先，我们使用CRISPR-Cas9 基因组编辑，在两个关键的母体转录本的3‘UTRs中进行突变，以确定哪一个 监管事件对于定义表达方式和生殖健康是最重要的。基因组 编辑技术已经发展到可以进行有针对性的UTR删除和替换的地步，所以 现在我们可以直接评估重要性了。我们的第二个方向是定义监管机制。我们 正在进行艾滋病降级标记实验，以定义限制性商业惯例和核心监管机构如何控制 母体基因在生殖系和胚胎中的表达具有时间分辨率。最后，我们希望 了解RBP的生化特性如何影响其突变表型。蛋白质可以有 多个活动，而且并不总是清楚最明显的活动是其突变体背后的活动 表型。我们有能力直接解决这个问题。表达和纯化了几种C. 在过去的十年里，秀丽的限制性商业惯例已经开始剖析它们的生化特性 使用定量的体外工具。我们现在有能力引入影响RNA结合的突变 在线虫的内源基因座上确定表型。 我们创新的跨学科方法，再加上密歇根大学医学院的浓厚氛围 在RNA生物学和线虫遗传学方面，将确保在定义母体效应方面取得快速进展 在功能水平上的胚胎发育。