The function of the RNA methylome in animals

RNA修饰控制着多种生物过程，如胚胎发育、性别决定、营养感知等。目前有超过150种不同的RNA修饰。RNA修饰的多样性及其合成、去除和识别所需的蛋白质对理解其生物学作用提出了重大挑战。本项目旨在揭示(1)RNA甲基化在动物发育中的功能；(2)RNA甲基化是如何被调控的。许多类型的RNA包含不同数量的RNA修饰。核糖体和转移rna被大量修饰。信使RNA内部RNA修饰的发现进一步扩展了RNA修饰在基因表达调控中的作用。此外，其他一些非编码rna也被修饰，如剪接体snrna和许多小的调控rna。我们使用遗传和生化工具来了解RNA修饰在种系发育过程中的作用，以及饮食如何影响RNA修饰水平。我们使用最先进的技术，包括纳米孔测序和高分辨率质谱。基因表达的调控在种系中至关重要。生殖细胞产生后代，生殖细胞基因表达的变化直接影响胚胎发育。在包括线虫和哺乳动物在内的许多生物中，生殖细胞中的基因表达变化可能会产生多代后果。我们的研究重点是RNA修饰和RNA修饰酶如何调节动物生殖细胞中的基因表达，以秀丽隐杆线虫为研究对象。在秀丽隐杆线虫的生殖系发育过程中，生殖细胞从有丝分裂增殖到减数分裂并分化为卵母细胞或精子。因此，我们可以利用秀丽隐杆线虫的种系同时研究细胞的增殖和分化。我们将首先通过RNA剪接的调节来描述RNA甲基转移酶在mRNA成熟中的作用。mRNA成熟是一个复杂的过程，包括帽甲基化、内含子剪接和多聚腺苷化。有效的RNA剪接需要协调一个包含许多非编码RNA的大型蛋白质复合体。除了去除内含子外，线虫和包括许多寄生物种在内的许多其他生物在mRNA成熟过程中也使用反式剪接。反式剪接用非编码SL-RNA取代mrna的5'端。我们将使用直接RNA测序和生化方法来了解snRNA修饰如何调节生殖细胞的剪接过程。接下来，我们将研究膳食维生素B12如何调节RNA修饰。维生素B12对蛋氨酸循环至关重要，蛋氨酸循环控制着s -腺苷基蛋氨酸的水平。细菌和古细菌可以合成维生素B12，而动物必须通过饮食获得维生素B12。使用我们的“纯素饮食”系统和同位素标记，我们将追踪存在和不存在维生素B12时RNA甲基化水平的变化。我们将使用纳米孔RNA测序来确定存在和不存在维生素B12时基因表达的异构体水平变化。我们将进一步扩大我们对秀丽隐杆线虫天然饮食中发现的细菌的分析。我们将确定秀丽隐杆线虫如何在基因表达水平上感知维生素B12。我们的研究是至关重要的，因为它将促进我们对RNA修饰在发育过程中如何起作用的理解，并通过生成工具和资源使该领域的未来研究成为可能。我们的研究解决了一个经常被忽视的话题，即饮食与RNA的化学变化之间的联系。

RNA modifications control diverse biological processes such as embryonic development, sex determination, nutrient sensing and many others. There are currently more than 150 distinct modifications on RNA. The diversity of RNA modifications alongside the proteins required for their synthesis, removal and recognition poses a significant challenge to understanding their biological role. This project aims to uncover (1) the function of RNA methylations in animal development and (2) how RNA methylations are regulated. Many types of RNA contain a diverse number of RNA modifications. Ribosomal and transfer RNAs are heavily modified. Discoveries of internal RNA modifications on messenger RNAs have further expanded the role RNA modifications play in gene expression regulation. Furthermore, several other non-coding RNAs are modified, such as spliceosomal snRNAs and many small regulatory RNAs. We use genetic and biochemical tools to understand how RNA modifications function during germline development and how diet affects RNA modification levels. We use state-of-the-art technologies, including Nanopore Sequencing and high-resolution mass spectrometry. Regulation of gene expression is crucial in the germline. Germ cells give rise to offspring, and gene expression changes in germ cells can directly affect embryonic development. In many organisms, including nematodes and mammals, gene expression changes in the germ cells can have multi-generational consequences. Our research focuses on how RNA modifications and RNA modifying enzymes regulate gene expression in animal germ cells using the nematode Caenorhabditis elegans as a discovery organism. During C. elegans germline development, germ cells proliferate from mitosis to meiosis and differentiate into oocytes or sperm. Therefore, we can use the C. elegans germline to investigate cell proliferation and differentiation at the same time. We will first characterise the role of RNA methyltransferases in mRNA maturation through the regulation of RNA splicing. mRNA maturation is a complex process that involves cap methylation, splicing of introns and poly-adenylation. Efficient RNA splicing requires orchestrating a large protein complex that contains many non-coding RNAs. In addition to removing introns, nematodes and many other organisms that include many parasitic species use trans-splicing during mRNA maturation. Trans-splicing replaces the 5' end of mRNAs with a non-coding SL-RNA. We will use direct RNA sequencing and biochemical approaches to understand how snRNA modifications regulate splicing processes in germ cells.Next, we will investigate how dietary vitamin B12 can regulate RNA modifications. Vitamin B12 is essential for the methionine cycle, which controls S-adenosylmethionine levels. Bacteria and archaea can synthesise vitamin B12, whereas animals must acquire vitamin B12 through their diet. Using our "vegan diet" system and isotope labelling, we will trace how RNA methylation levels change in the presence and absence of vitamin B12. We will use nanopore RNA sequencing to determine isoform level changes in gene expression in the presence and absence of vitamin B12. We will further expand our analysis to bacteria found in the natural diet of C. elegans. We will determine how C. elegans could sense vitamin B12 at the gene expression level. Our research is crucial because it will advance our understanding of how RNA modifications function during development and enable future research in this area by generating tools and resources. Our research addresses a much-overlooked topic connecting diet to chemical changes on RNA.