Mechanisms and functions of cell surface glycoRNAs

项目摘要/摘要 细胞表面是对细胞生物学进行物理和调控控制的平台，将其定位为关键 用于诊断目标和治疗干预的界面。而RNA是生物学中最重要的聚合物 致力于RNA生物学的思想和实验努力一直局限于细胞内空间，并被排除在外 参与细胞表面生物学。在细胞表面，碳水化合物聚合物(多糖)是至关重要的 由于生物物理和信号活动的重要性。有趣的是，尽管这两种聚合物都扮演着核心角色 在生物学中，RNA和多糖主要存在于完全不重叠的研究领域。然而，我的工作 已经提供了一种杂化分子的证据，一种RNA-葡聚糖偶合物(GRNA)；这类新的生物分子 代表了RNA和糖生物学之间的直接联系。关键的是，糖核糖核酸定位于外部 在活细胞表面，并能与免疫调节的Siglec受体结合。因此，糖核糖核酸被定位 在具有关键监管重要性的表面上，能够接触到细胞间的相互作用、病原体和信号 细胞表面的受体。然而，我们目前缺乏便捷的工具来研究这种新的细胞表面分子，我们 不了解糖核糖核酸的分子或原子组成，我们对 有多少物种生物合成糖核糖核酸。这份米拉提案的重点是制定和实施 方法揭示RNA糖基化的功能作用，并探讨其复杂的生物学 系统地研究糖核糖核酸。最初，我们将开发新的化学方法来标记多糖 RNA的上下文。我提出的选择性羧酸标记策略代表了一种创新的新 检测糖核糖核酸的方法，不需要合成代谢报告。这些工具将很容易 在细胞类型和物种中实施，使科学界的其他人能够参与其中。我们将应用这些工具 和其他分子分析，以扩大我们对细胞表面组成的理解 糖核糖核酸。将使用基于生化、生物物理和成像的策略来定义分子 糖核糖核酸的邻域以及RNA-糖链的化学性质；所有这些都提供了 更完整的哺乳动物细胞表面的图像。最后，我们将开发第一个糖RNA的证据在 非哺乳类生物。首先将重点放在两个主要的酵母菌株(酿酒酵母和庞氏葡萄球菌)上。 培养、功能和遗传工具，将允许快速剖析生物发生途径，最终 用于工程目的。扩展到其他生物，包括原核生物(致病的和非致病的)以及其他 像线虫这样的多细胞真核生物将更好地定义糖RNA生物合成的范围，并更有力地 准备好让我们产生合成的糖核糖核酸。更广泛地说，我们打算推进细胞如何 病原体和外源分子相互作用，就像没有细胞表面糖RNA一样，它们是 可能是不完整的。该提案开发了创新的方法来建立新的概念层和物理层 细胞与其环境之间的调节。

PROJECT SUMMARY/ ABSTRACT The cell surface is a platform for physical and regulatory control over cell biology, positioning it to be a key interface for diagnostic targeting and therapeutic intervention. While RNA is a central polymer in biology most thought and experimental effort devoted to RNA biology has been confined to intracellular spaces and excluded from participating in cell surface biology. On the cell surface, carbohydrate polymers (glycans) are of critical importance due to biophysical and signaling activities. Interestingly, despite both polymers playing central roles in biology, RNA and glycans have largely existed in entirely non-overlapping fields of study. However, my work has provided evidence of a hybrid molecule, an RNA-glycan conjugate (glycoRNA); this new class of biomolecule represents a direct link between RNA and glycobiology. Critically, glycoRNAs are localized to the external surface of living cells and can engage with immunomodulatory Siglec receptors. Thus, glycoRNAs are positioned on a surface of critical regulatory importance, with access to cell-cell interactions, pathogens, and signaling receptors on the cell surface. However, we currently lack facile tools to study this new cell surface molecule, we do not understand the molecular or atomic composition of glycoRNAs, and we have a poor understanding of how many species biosynthesize glycoRNAs. This MIRA proposal is focused on developing and implementing methods to uncover functional roles of RNA glycosylation and we will approach the complex biology of glycoRNAs in a systematic fashion. Initially we will develop novel chemical approaches to label glycans in the context of RNA. My proposed strategy of selective carboxylic acid labeling represents an innovative new approach to detecting glycoRNA, without the need for synthetic metabolic reporters. These tools will be easily implemented across cell types and species enabling others in the scientific community. We will apply these tools and other molecular assays to expand our understanding of the composition of the cell surface in the context of glycoRNA. Biochemical, biophysical, and imaging-based strategies will be used to define the molecular neighborhoods of glycoRNAs as well as the chemical nature of the RNA-glycan linkage; all together providing a more complete picture of the mammalian cell surface. Finally, we will develop the first evidence of glycoRNAs in non-mammalian organisms. First focusing on two major strains of yeast (S. cerevisiae and S. pombe) with robust culturing, functional, and genetic tools that will allow for rapid dissection of the biogenesis pathway for eventual engineering purposes. Expanding to other organisms including prokaryotes (pathogenic and not) as well as other multicellular eukaryotes like C. elegans will better define the scope of glycoRNA biosynthesis and more robustly equip us to generate synthetic glycoRNAs. More broadly, we intend to advance the general model of how cells interact with each other, pathogens, and exogenous molecules, as without cell surface glycoRNAs, they are likely incomplete. This proposal develops innovative methods to establish new conceptual and physical layers of regulation between a cell and its environment.