Transcriptome-wide RNA modification profiling via Adduct-IP

描述(由申请人提供)： RNA转录后修饰可以影响RNA分子的序列、结构和功能。到目前为止，已经在RNA中鉴定了100多个不同的修饰核苷酸，RNA修饰酶在基因组中占有相当大的比例。然而，对于某些关键的修饰，其全部范围、特定的酶和生物学作用还没有被很好地理解--部分原因是缺乏适当的转录组技术。这是尽管在许多RNA修饰酶功能丧失/获得时观察到的深刻的临床意义，这些酶可导致多种遗传综合征、智力低下和不孕不育。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-RMTs)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：DNMT2和NSUN2进行了转录组范围的靶向分析。在这里，在目标1中，我们应用aza-ip在HeLa细胞和hESCs中对所有其他八个M5C-RMT进行靶向分析。在目标2中，我们扩大了基于机制的浓缩策略的适用性--通常被称为‘加合物-IP’--用于另一类重要的RNA修饰酶--伪尿苷合成酶的靶标分析。我们基于机理的‘加合物-IP’方法涉及到酶在体内与其底物的共价连接。对于aza-ip，5-azacytidine在所有RNA中以转录方式取代胞嘧啶，导致适当的M5C-RMT共价结合在其特定的靶点上-随后是免疫沉淀、释放和cDNA测序。AZA-IP特别丰富了M5C-RMT的直接靶标，也产生了明确和穿透性的C&gT；G反式‘签名’，专门针对准确的靶标胞嘧啶。重要的是，aza-ip捕获了低拷贝的RNA靶标和罕见的甲基化事件，这是在其他技术的检测极限下，如RNA亚硫酸氢盐测序。在这里，我们的目标是扩展这项技术，以发现其余8个已知的人类M5C-RMT的靶点。除了M5C-RMT外，假尿苷合成酶是另一类重要的RNA修饰酶，它能将各种RNA物种中的特定尿氨酸异构化为假尿苷。假尿苷是RNA中含量最丰富的修饰核苷酸，对rRNAs、tRNAs和SnRNAs的结构和功能是必不可少的。虽然预计在其他RNA物种(ncRNAs和mRNAs)中存在假尿苷，但由于缺乏揭示这种修饰范围的转录组范围的工具，该领域受到阻碍。有趣的是，通过5-氮杂-C抑制M5C-RMT的机制，核苷酸类似物5-氟尿苷可以(通过共价键)不可逆地抑制伪尿苷合成酶。在这里，我们的目标是应用加合物-IP策略来对选定的伪尿苷合成酶(如DKC1)进行转录组范围的靶标分析，这些假尿苷合成酶与遗传性疾病(先天性角化不良)和癌症密切相关。

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.