Center for dynamic RNA epitranscriptomes

 描述(申请人提供)：RNA修饰在生物学中普遍存在，存在于所有类别的细胞RNA中，包括真核信使和长非编码RNA。哺乳动物的很大一部分mRNA/lncRNA修饰也被认为是可逆的，高度动态的，以细胞类型和细胞状态依赖的方式发生。特别是涉及N6-甲基腺苷(M6A)的动态RNA表位转录本，被认为调节许多细胞活动，包括mRNA剪接、输出、细胞质定位、稳定性、翻译活性、microRNA加工、免疫耐受，并影响细胞过程，包括增殖、发育、昼夜节律和胚胎干细胞分化。以m6A在mRNA/lncRNA中的表达为例，人类细胞中存在专门的写入器、擦除器和读取器，以协调复杂的转录后基因表达调控的额外一层。RNA修饰的新功能预计将随之而来，对人类健康和疾病的许多方面都有重大影响。尽管有很高的潜力和希望，但由于缺乏能够以高分辨率和高灵敏度定量绘制任何类型的mRNA/lncRNA修饰的技术，目前的表观转录组学研究受到严重阻碍。这项建议将开发针对m6A、5-甲基胞嘧啶(M5C)、N1-甲基腺苷(M1a)、伪尿苷(Ψ)和2‘O-甲基(Nm)等5种丰富的mRNA/lncRNA修饰的新方法，用于单碱基分辨率的高通量测序，并适用于仅从数百到数千个细胞中分离的低输入核糖核酸。将开发新的生物信息学工具，以促进数据分析。所提出的通用方法可广泛应用于其他RNA物种的序列RNA修饰，包括更丰富的核糖体RNA、转移RNA、SnRNA和snoRNA以及miRNA和piRNA。我们将应用新开发的方法来获得RNA修饰的基本分辨率图，以便与人类疾病联系在一起，并应用于神经生物学的原理证明研究。我们提出的研究将建立高通量、高分辨率和高灵敏度的方法，用于所有生物学领域的表位转录组学研究。

 DESCRIPTION (provided by applicant): RNA modifications are ubiquitous in biology and present in all classes of cellular RNAs including eukaryotic messenger and long non-coding RNA. A large fraction of mammalian mRNA/lncRNA modifications are also known to be reversible, highly dynamic, and occur in cell type and cell state dependent manner. The dynamic RNA epitranscriptomes, those involving N6-methyladenosine (m6A) in particular, are known to regulate many cellular activities including mRNA splicing, export, cytoplasmic localization, stability, translation activity, microRNA processing, immune tolerance, and to impact cellular processes including proliferation, development, circadian rhythm, and embryonic stem cell differentiation. Consider m6A in mRNA/lncRNA as an example, dedicated writers, erasers, and readers exist in human cells to orchestrate an additional layer of complex post-transcriptional gene expression regulation. Emerging new functions of RNA modifications are expected to follow, with significant implications on many aspects of human health and disease. Despite high potentials and promises, current epitranscriptome studies are significantly hampered by the lack of technologies that enable quantitative mapping of any type of mRNA/lncRNA modifications at high resolution and high sensitivity. This proposal will develop new methods that target five abundant mRNA/lncRNA modifications, namely m6A, 5-methylcytosine (m5C), N1-methyladenosine (m1A), pseudo uridine (Ψ), and 2'O- methyls (Nm) for high-throughput sequencing at single-base resolution and suitable for low input RNA isolated from just hundreds to thousands of cells. New bioinformatics tools will be developed in order to facilitate data analysis. The general approaches proposed can be broadly applied to sequence RNA modifications in other RNA species including more abundant ribosomal RNA, transfer RNA, snRNA, and snoRNA as well as miRNA and piRNA. We will apply the newly developed methods to obtain base-resolution maps of RNA modifications in order to associate with human diseases, and to proof-of-principle studies in neuro-biology. Our proposed research will establish high-throughput, high-resolution, and high-sensitivity methods for epitranscriptome research in all biological areas.