Transcriptome-wide RNA modification profiling via Adduct-IP

通过 Adduct-IP 进行全转录组 RNA 修饰分析

基本信息

批准号：
8773425
负责人：
BRADLEY R. CAIRNS
金额：
$ 22.35万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8773425
关键词：
5 fluorouridine Azacitidine Biological Biological Process Brain Cancer cell line Cataloging Catalogs Cell Line Cells Clinical Complement Complementary DNA Complex Cytosine Data Defect Detection Development Disease Dyskeratosis Congenita Enzymes Event Foundations Functional RNA Gene Expression Profile Genes Genetic Genome Growth Hela Cells Hereditary Disease Human Immunoprecipitation Infertility Link Malignant Neoplasms Mental Retardation Messenger RNA Methylation Methyltransferase Modification Nucleotides Pathogenesis Pathway interactions Positioning Attribute Post-Transcriptional RNA Processing Precipitation Process Property Proteins Pseudouridine RNA Ribosomal RNA Role Site Small Nuclear Ribonucleoproteins Stress Structure Syndrome System Techniques Technology Testing Transfer RNA Uridine Validation Variant adduct base bisulfite cancer cell embryonic stem cell gain of function human embryonic stem cell improved in vivo innovation member methylome novel novel strategies nucleotide analog public health relevance research study response stable cell line tool tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Post-transcriptional RNA modifications can impact the sequence, structure and function of RNA molecules. Over 100 distinct modified nucleotides have been characterized so far in RNA, and RNA modifying enzymes comprise a marked proportion of genomes. However, for certain key modifications their full scope, specific enzymes and biological roles are not well understood - due in part to lack of appropriate transcriptome-wide technologies. This is despite the profound clinical implications observed upon loss/gain of function of many RNA modification enzymes, which can confer diverse genetic syndromes, mental retardation, and infertility. We recently developed Aza-IP, a mechanism-based novel technique that greatly enriches the precise direct targets of RNA cytosine methyltransferases (m5C-RMTs). We tested Aza-IP in HeLa cells, and conducted transcriptome-wide target profiling of two important human m5C-RMTs; DNMT2 and NSUN2. Here, in Aim 1 we apply Aza-IP for target profiling of all eight other m5C-RMTs in both HeLa cells and hESCs. In Aim 2, we expand the applicability of the mechanism-based enrichment strategy - which can be generally referred to as 'Adduct-IP' - for target profiling of another important class of RNA modifying enzymes; pseudouridine synthases. Our mechanism-based 'Adduct-IP' approaches involve the in-vivo covalent attachment of the enzyme to its substrate. For Aza-IP, 5-azacytidine is transcriptionally incorporated in place of cytosine within all RNAs, leading to covalent attachment of the proper m5C-RMT at its specific target - which is followed by immuno-precipitation, release and cDNA sequencing. Aza-IP specifically enriches the direct targets of m5C- RMTs, and also generates a clear and penetrant C>G transversion 'signature' exclusively at the exact target cytosine. Importantly Aza-IP captures low copy RNA targets and also rare methylation events, which are under the detection limit of other techniques such as RNA bisulfite sequencing. Here, we aim to expand this technique to discover the targets for the remaining eight known human m5C-RMTs. Beyond m5C-RMTs, pseudouridine synthases are another important class of RNA modifying enzymes that isomerize specific uridines in various RNA species into pseudouridine. Pseudouridine is the most abundant modified nucleotide in RNA and is essential for proper structure and function of rRNAs, tRNAs and snRNAs. Although presence of pseudouridine in other RNA species (ncRNAs and mRNAs) is expected, the field is hampered by lack of tools for transcriptome-wide profiling of this modification which would reveal the scope of this modification. Interestingly, pseudouridine synthases can become irreversibly inhibited (through covalent linkage) by the nucleotide analogue 5-flurourdine through a similar mechanism as of 5-aza-C inhibition of m5C-RMTs. Here we aim to apply the Adduct-IP strategy for transcriptome-wide target profiling of selected pseudouridine synthases (such as DKC1), with significant involvement in a genetic disease (dyskeratosis congenita) and also in cancer.

描述（由申请人提供）： RNA 转录后修饰会影响 RNA 分子的序列、结构和功能。迄今为止，RNA 中已鉴定出超过 100 种不同的修饰核苷酸，并且 RNA 修饰酶在基因组中占有显着比例。然而，对于某些关键修饰，其全部范围、特定酶和生物学作用尚不清楚——部分原因是缺乏适当的转录组技术。尽管许多RNA修饰酶的功能丧失/获得具有深远的临床意义，这可能会导致多种遗传综合征、智力低下和不孕症。我们最近开发了 Aza-IP，这是一种基于机制的新技术，极大地丰富了 RNA 胞嘧啶甲基转移酶 (m5C-RMT) 的精确直接靶标。我们在 HeLa 细胞中测试了 Aza-IP，并对两个重要的人类 m5C-RMT 进行了全转录组靶标分析； DNMT2 和 NSUN2。在这里，在目标 1 中，我们应用 Aza-IP 对 HeLa 细胞和 hESC 中的所有其他八个 m5C-RMT 进行靶标分析。在目标 2 中，我们扩展了基于机制的富集策略（通常可称为“Adduct-IP”）的适用性，用于另一类重要的 RNA 修饰酶的靶标分析；假尿苷合酶。我们基于机制的“加合物-IP”方法涉及酶与其底物的体内共价连接。对于 Aza-IP，5-氮杂胞苷在转录上取代所有 RNA 中的胞嘧啶，从而导致正确的 m5C-RMT 共价附着在其特定靶标上 - 随后进行免疫沉淀、释放和 cDNA 测序。 Aza-IP 特异性富集 m5C-RMT 的直接靶标，并且还专门在精确的靶标胞嘧啶上产生清晰且渗透的 C>G 颠换“特征”。重要的是，Aza-IP 可以捕获低拷贝 RNA 靶标以及罕见的甲基化事件，这些事件处于 RNA 亚硫酸氢盐测序等其他技术的检测极限之下。在这里，我们的目标是扩展这项技术，以发现其余八种已知的人类 m5C-RMT 的靶点。除了 m5C-RMT 之外，假尿苷合酶是另一类重要的 RNA 修饰酶，可将各种 RNA 种类中的特定尿苷异构化为假尿苷。假尿苷是 RNA 中最丰富的修饰核苷酸，对于 rRNA、tRNA 和 snRNA 的正确结构和功能至关重要。尽管预计其他 RNA 物种（ncRNA 和 mRNA）中也会存在假尿苷，但由于缺乏对这种修饰进行全转录组分析的工具（无法揭示这种修饰的范围），该领域受到了阻碍。有趣的是，假尿苷合酶可以通过与 5-aza-C 抑制 m5C-RMT 类似的机制，被核苷酸类似物 5-氟尿苷不可逆地抑制（通过共价连接）。在这里，我们的目标是应用加合物-IP 策略对选定的假尿苷合酶（例如 DKC1）进行全转录组靶标分析，这些假尿苷合酶与遗传疾病（先天性角化不良）和癌症密切相关。