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

项目摘要 小分子RNA是真核生物基因表达的普遍调节因子，几乎在生理学的各个方面都有其存在 从植物到人类。它们的功能是调节基因表达的所有方面，包括转录， RNA稳定性和翻译。一类新兴的小调节RNA是tRNA片段（tRF）， 由tRNA的核酸裂解产生。tRF与癌症、神经退行性疾病 疾病、病毒感染、生育、表观遗传和衰老。虽然这些RNA的生物发生是 尽管对tRF了解甚少，但已证明tRF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方面是未知的。 我的实验室正在开发蛔虫C。线虫（蠕虫）作为模型来解剖分子 生物发生tRF和整个动物的细胞功能的基础机制。鲁棒 遗传学、生理学测定和分子工具，可在C.优雅的人提供了一个成熟的系统， tRF生物学的发现重要的是，我们已经开发了小RNA测序技术来检测 C. elegans，其水平远高于先前发表的小型RNA-seq数据集。 利用这些技术，我们将在发育完全的成年人的不同组织中表征tRFs 使用批量和单细胞小RNA-seq.在确定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它们所需的新因素。 生物起源此外，我们将采用生化富集策略来确定相互作用的因素， 并影响TRF功能。在确定tRF生物发生和功能所需的基因后， 使用这些因子的突变等位基因来确定这些分子在调节 生物体的生理学。最后，我们将使用携带RNA功能缺失突变的蠕虫菌株 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。在C.我实验室里的秀丽线虫代表了 对整个生物体中tRFs生物学的所有方面进行系统解剖。我们对tRF的了解 蠕虫中的生物学将用于产生关于其他生物中tRF的假设。由于tRF一直是 涉及从酵母到人类的生物体的正常生理学的许多方面， 在广泛的疾病中，全面了解tRF是如何调节和发挥作用的 代表了生物学中一个高度未得到重视的方面。

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.