Transcriptome-wide RNA modification profiling via Adduct-IP

描述（由申请人提供）： 转录后RNA修饰可以影响RNA分子的序列、结构和功能。到目前为止，在RNA中已经表征了超过100种不同的修饰核苷酸，并且RNA修饰酶包含显著比例的基因组。然而，对于某些关键修饰，其全部范围，特定的酶和生物学作用尚未得到很好的理解-部分原因是缺乏适当的全转录组技术。尽管在许多RNA修饰酶的功能丧失/获得后观察到了深刻的临床意义，但这可能导致不同的遗传综合征、智力迟钝和不育。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（m5 C-RMTs）的精确直接靶标。我们在HeLa细胞中测试了Aza-IP，并对两种重要的人m5 C-RMT; DNMT 2和NSUN 2进行了转录组范围的靶向分析。在这里，在目的1中，我们应用Aza-IP对HeLa细胞和hESC中的所有八种其他m5 C-RMT进行靶分析。在目标2中，我们扩展了基于机制的富集策略的适用性-其通常可以被称为“加合物-IP”-用于另一类重要的RNA修饰酶的靶向分析;假尿苷激酶。我们的基于机制的“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-氮杂胞苷在转录上掺入所有RNA中的胞嘧啶位置，导致适当的m5 C-RMT共价连接在其特异性靶标上，随后进行免疫沉淀、释放和cDNA测序。Aza-IP特异性地富集m5 C-RMT的直接靶标，并且还仅在确切的靶标胞嘧啶处产生清晰且渗透性的C>G颠换“特征”。重要的是，Aza-IP可以捕获低拷贝RNA靶点和罕见的甲基化事件，这些事件低于RNA亚硫酸氢盐测序等其他技术的检测限。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m5 C-RMT的目标。除了m5 C-RMT之外，假尿苷二磷酸酶是另一类重要的RNA修饰酶，其将各种RNA物质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，并且对于rRNA、tRNA和snRNA的适当结构和功能是必需的。尽管预期在其他RNA种类（ncRNA和mRNA）中存在假尿苷，但该领域受到缺乏用于该修饰的转录组范围谱分析的工具的阻碍，该工具将揭示该修饰的范围。有趣的是，假尿苷激酶可以通过与5-aza-C抑制m5 C-RMT相似的机制被核苷酸类似物5-flurourdine不可逆地抑制（通过共价键）。在这里，我们的目标是应用Adduct-IP策略对选定的假尿苷激酶（如DKC 1）进行全转录组靶向分析，这些酶与遗传性疾病（先天性角化不良）和癌症有重要关系。

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.