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：
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
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/10390502
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 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interest mRNA Expression medical schools mutant oocyte maturation reproductive stoichiometry temporal measurement tool unpublished works zygote

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.

项目摘要：我的实验室有兴趣定义在卵子发生过程中如何建立母体负载并解码受精后。我们知道最重要的母亲转录本和母体提供的RNA的身份 - 结合蛋白。我们知道这些因素是种系开发，卵母细胞成熟和早期胚胎发生的模式形成。但是我们还不知道哪些监管事件是最重要的用于繁殖或哪些机制协调时空调节。我们使用一种“以蛋白质为中心”的方法来绘制母体RNA调节的接线图线虫秀丽隐杆线虫。我们定义了几个主要的RNA-识别的序列基序结合蛋白（RBP），并鉴定出3'UTR报告基因中功能性顺式调节元件代表研究良好的母体mRNA。我们做出的工作表明，具有约束力的特异性不足以解释靶向体内的mRNA。迄今为止研究的所有蛋白质都与短线性部分变性基序结合至少有30-50％的所有mRNA。在某些情况下，图案已被证明是必要的，但不是足以驱动监管活动。在其他情况下，主题不会导致调节。即使在多个副本不赋予RBP依赖性调节。绑定不是一个很好的预测指标调节，揭示结合位点环境对于靶向也至关重要。向前迈进，我们正在采取三种主要策略。首先，我们使用CRISPR-CAS9 基因组编辑以在两个关键母体成绩单的3'Utrs中进行突变，以确定哪个调节事件对于定义表达方式和生殖健康最重要。基因组编辑技术已经发展到我们可以进行有针对性的UTR删除和替换的地步，因此现在我们可以直接评估重要性。我们的第二个方向旨在定义调节机制。我们是否正在执行辅助degron标记的实验，以定义RBP和核心调节机械如何控制母体mRNA表达在种系和胚胎中暂时分辨率。最后，我们希望了解RBP的生化特性如何促进其突变表型。蛋白质可以多种活动，并不总是清楚最明显的活动是其突变体的基础的活动表型。我们可以直接解决这个问题。我们已经表达并纯化了几个C。在过去十年的过程中使用定量的体外工具。我们现在有能力引入影响RNA结合的突变秀丽隐杆线虫中内源性基因座的性质以确定表型。我们创新的跨学科方法，与UMass医学院的强烈气氛相结合在RNA生物学和秀丽隐杆线虫遗传学中，将确保在定义母体效应方面的快速进步功能水平的胚胎发生。