Post-transcriptional regulation of germline mRNAs in C. elegans

线虫种系 mRNA 的转录后调控

基本信息

批准号：
10610874
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
项目类别：
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/10610874
关键词：
3&apos 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 Genome In Vitro Individual Instruction Invertebrates Learning 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 candidate identification cell fate specification experimental study gene replacement genome editing in vivo innovation interdisciplinary approach interest mRNA Expression medical schools mutant oocyte maturation posttranscriptional reproductive stoichiometry temporal measurement tool unpublished works zygote

项目摘要

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.

项目概要：我的实验室有兴趣定义卵子发生过程中母体负荷是如何建立和解码的受精后，我们知道最重要的母体转录物和母体提供的RNA-的身份。我们知道这些因子是种系发育、卵母细胞成熟和发育所必需的。但我们还不知道哪些调控事件是最重要的。繁殖，或者什么机制协调空间和时间的调节。我们采用“以蛋白质为中心”的方法来绘制母体 RNA 调节的接线图我们定义了线虫 Caenorhabidits elegans 的几个母体 RNA 识别的序列基序。结合蛋白 (RBP) 并鉴定 3'UTR 报告基因中的功能性顺式调控元件我们的工作表明，结合特异性不足以代表母体 mRNA。解释迄今为止研究的所有蛋白质都与短线性部分简并基序结合。存在于至少 30-50% 的 mRNA 中。在某些情况下，这些基序已被证明是必要的，但并非如此。在其他情况下，主题不会导致监管，即使是在其中。转基因中的多个拷贝不会赋予 RBP 依赖性调节。监管，揭示结合位点背景对于靶向也至关重要。展望未来，我们正在推行三大战略：首先，我们正在使用 CRISPR-cas9。基因组编辑使两个关键母体转录本的 3’UTR 发生突变，以确定哪些调控事件对于定义表达模式和基因组健康最为重要。编辑技术已经进步到我们可以进行有针对性的UTR删除和替换，所以现在我们可以直接评估重要性。正在进行 AID-degron 标记实验，以定义 RBP 和核心调节机制如何控制最后，我们希望在种系和胚胎中具有时间分辨率的母体 mRNA 表达。了解 RBP 的生化特性如何影响其突变蛋白的表型。多种活性，并且并不总是清楚最明显的活性是其突变体的基础活性我们已经表达并纯化了几个 C. 在过去的十年里，我们对线虫的 RBP 进行了研究，并开始剖析它们的生化特性使用定量体外工具，我们现在有能力引入影响 RNA 结合的突变。特性进入秀丽隐杆线虫的内源基因座以确定表型。我们创新的跨学科方法，加上麻省大学医学院的浓厚氛围 RNA生物学和线虫遗传学方面的研究将确保在定义母体效应方面取得快速进展功能水平上的胚胎发生。