Developing C. elegans as a model to understand tRNA-fragment biogenesis and function

开发线虫作为模型来了解 tRNA 片段的生物发生和功能

• 批准号: 10715193
• 负责人: Colin Conine
• 金额: $ 44.5万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715193
• 关键词: Address; Adult; Affect; Aging; Alleles; Animals; Biochemical; Biogenesis; Biological Assay; Biology; Caenorhabditis elegans; Cell physiology; Cells; Data Set; Disease; Dissection; Enzymes; Epigenetic Process; Fertility; Gene Expression; Genes; Genetic; Genetic Transcription; Human; Learning; Malignant Neoplasms; Modeling; Modification; Molecular; Nematoda; Neurodegenerative Disorders; Organism; Physiological; Physiology; Plant Physiology; Play; Post-Transcriptional Regulation; Publishing; RNA; RNA Stability; Regulation; Role; Small RNA; System; Techniques; Tissues; Transcriptional Regulation; Transfer RNA; Translations; Virus Diseases; Whole Organism; Work; Yeasts; forward genetics; loss of function mutation; mRNA Stability; mutant; reverse genetics; tool; transcriptome sequencing

Project Summary Small RNAs are ubiquitous regulators of eukaryotic gene expression in nearly all aspects of physiology from plants to humans. They function to regulate all facets of gene expression including transcription, RNA stability, and translation. An emerging class of small regulatory RNAs are tRNA-fragments (tRFs), produced from nucleolytic cleavage of tRNAs. tRFs have been implicated in cancer, neurodegenerative disease, viral infection, fertility, epigenetic inheritance, and aging. While the biogenesis of these RNAs is poorly understood, tRFs have been demonstrated to play roles in the regulation of transcription, posttranscriptional regulation of mRNA stability, and translation. Yet, the functions of tRFs throughout the different tissues of an organism in regulating cellular physiology are unknown. My lab is developing the roundworm C. elegans (worms) as a model to dissect the molecular mechanisms underlying the biogenesis tRF and cellular functions throughout the animal. The robust genetics, physiological assays, and molecular tools available in C. elegans provide a system ripe for the discovery of tRF biology. Importantly, we have developed small RNA-sequencing techniques to detect abundant tRFs in C. elegans, at levels much higher than previously published small RNA-seq datasets. Using these techniques, we will characterize tRFs in the different tissues of the fully developed adult using bulk and single-cell small RNA-seq. After determining the spatial and temporal expression of tRF species we will utilize both forward and reverse genetics to determine new factors required for their biogenesis. Further, we will employ biochemical enrichment strategies to determine factors that interact with and affect tRF function. Upon determining genes required for tRF biogenesis and function we will use mutant alleles of these factors to determine what roles these molecules have in regulating the physiology of the organism. Finally, we will use worm strains carrying loss-of-function mutations in RNA modifying enzymes to determine how RNA modifications affect tRF biogenesis, functions, and further regulate the physiology of the animal. The work on tRFs in C. elegans in my lab represents the first systematic dissections of all aspects of tRFs biology in a whole organism. What we learn about tRF biology in worms will be used to generate hypotheses about tRFs in other organisms. As tRFs have been implicated in many aspects of normal physiology in organisms ranging from yeast to humans, as well as in a wide range of diseases, comprehensively understanding how tRF are regulated and function represents a highly under-addressed aspect of biology.

项目概要 小RNA在生理学的几乎所有方面都是真核基因表达的普遍调节因子 从植物到人类。它们的功能是调节基因表达的各个方面，包括转录、 RNA稳定性和翻译。一类新兴的小调控 RNA 是 tRNA 片段 (tRF)， 由 tRNA 的溶核裂解产生。 tRF 与癌症、神经退行性疾病有关 疾病、病毒感染、生育能力、表观遗传和衰老。虽然这些 RNA 的生物发生是 人们对 tRF 知之甚少，但已被证明在转录调节中发挥作用， mRNA 稳定性和翻译的转录后调节。然而，tRF 的功能在整个 生物体的不同组织在调节细胞生理学中的作用尚不清楚。 我的实验室正在开发线虫（蠕虫）作为解剖分子的模型 动物体内生物发生 tRF 和细胞功能的机制。稳健的 线虫中可用的遗传学、生理测定和分子工具提供了一个成熟的系统 tRF 生物学的发现。重要的是，我们开发了小 RNA 测序技术来检测 线虫中丰富的 tRF，其水平远高于之前发布的小 RNA-seq 数据集。 使用这些技术，我们将表征完全发育的成人的不同组织中的 tRF 使用批量和单细胞小 RNA 测序。确定 tRF 的空间和时间表达后 我们将利用正向和反向遗传学来确定物种所需的新因素 生物发生。此外，我们将采用生化富集策略来确定相互作用的因素 具有并影响 tRF 功能。在确定 tRF 生物发生和功能所需的基因后，我们将 使用这些因子的突变等位基因来确定这些分子在调节 有机体的生理学。最后，我们将使用携带 RNA 功能丧失突变的蠕虫菌株 修饰酶以确定 RNA 修饰如何影响 tRF 生物发生、功能以及进一步 调节动物的生理机能。我实验室对线虫 tRF 的研究是第一个 对整个生物体中 tRF 生物学的各个方面进行系统剖析。我们对 tRF 的了解 线虫生物学将被用来产生关于其他生物体中 tRF 的假设。由于 tRF 已 涉及从酵母到人类的生物体正常生理学的许多方面，以及 在多种疾病中，全面了解 tRF 的调控和功能 代表了生物学中一个高度未被重视的方面。