Platform for transcriptome-wide RNA modification identification in long reads

项目摘要 RNA修饰在整个人类转录组中普遍存在并影响转录物的稳定性， 定位和功能。特别地，RNA中的ADAR介导的腺苷到肌苷（A-to-I）编辑已经被广泛应用。 显示影响前mRNA剪接并改变密码子序列。由肌苷引起的氨基酸变化 与各种有害的情况有关，包括癌症和脑部疾病。然而，在这方面， 以前的文献在高通量中绘制肌苷位置只能在有限的细胞内这样做， 由短RNA-Seq读数提供的上下文。由于修饰可以是转录本特异性的，因此阐明了转录本的功能。 肌苷与完整的mRNA亚型的关联对于更严格地理解 我们身体组织中的肌苷修饰，更广泛地说，疾病。因此，我提议调查 使用全长mRNA纳米孔在患病和非患病系统的背景下进行A到I编辑 测序纳米孔能够通过转换电流的变化来对整个RNA链进行测序 由RNA通过孔移位到核苷酸序列中引起。 Aim 1利用高精度 有和没有阿达尔敲低的细胞系统的纳米孔cDNA测序以询问阿达尔 功能和A到I诱导的转录物表达变化的变化。为了实现后者，我将开发 工作流程，以从嘈杂的长读段确定同种型结构。除了测序全长 转录物，纳米孔天然RNA（nvRNA）测序告知RNA修饰，作为修饰的核苷酸 表现为电流信号相对于规范核苷酸的细微变化。因此，Aim 2采用了 为系统地了解肌苷 改变纳米孔中的电流信号。我将使用Cas 13 b-ADAR融合蛋白（REPAIRv 2）来创建位点特异性的 编辑，然后对编辑的转录组进行nvRNA测序。特定于站点的A-to-I编辑允许这样做 一种在nvRNA信号中创建标记的肌苷数据集的方法，我可以从中开发计算 算法来可靠地识别nvRNA数据中的肌苷。REPAIRv 2方法可以推广到 最终鉴定任何带有纳米孔的RNA修饰。 目标3将阐明A到I编辑的不同之处 组织之间的联系。我将用nvRNA测序对4种正常组织类型进行测序， 使用我正在开发的软件编辑与异构体的使用。合在一起， 这些目标不仅将为难以捉摸的阿达尔机制提供进一步的见解，而且还将为 纳米孔数据分析和任何修改的研究平台。当我在攻读博士学位的时候。与此 跨学科项目，我将获得宝贵的技能，实验和计算生物学，将准备 给我一份科学工作

Project Summary RNA modifications are pervasive throughout the human transcriptome and affect transcript stability, localization, and function. In particular, ADAR-mediated adenosine-to-inosine (A-to-I) edits in RNA have been shown to affect pre-mRNA splicing and alter codon sequence. Amino acid changes caused by inosines have been implicated in various deleterious conditions, which include cancer and diseases of the brain. However, previous literature mapping inosine positions in high-throughput were only able to do so inside the limited context provided by short RNA-Seq reads. As modifications can be transcript-specific, elucidating the association of inosines with full mRNA isoforms is crucial for a more rigorous understanding of the role of inosine modifications in the tissues of our body and, more broadly, disease. Therefore, I propose to investiate A-to-I editing in the context of diseased and non-diseased systems using full-length mRNA nanopore sequencing. The nanopore is able to sequence whole RNA strands by converting changes in electrical current caused by RNA translocating through the pore into nucleotide sequence. ​Aim 1 leverages high-accuracy nanopore cDNA sequencing of cellular systems with and without ADAR knockdown to interrogate ADAR function and A-to-I-induced changes to transcript expression changes. To accomplish the latter, I will develop workflows to determine isoform structure from noisy, long reads. In addition to sequencing full-length transcripts, nanopore native RNA (nvRNA) sequencing informs on RNA modifications, as modified nucleotides appear as subtle alteration in current signal with respect to canonical nucleotides. As such, ​Aim 2 ​employs a generalizable approach to producing cost-effective training data for systematically understanding how inosines alter current signals in nanopores. I will use a Cas13b-ADAR fusion protein (REPAIRv2) to create site-specific edits and then perform nvRNA sequencing on the edited transcriptome. Site-specific A-to-I editing allows this approach to create a labelled inosine dataset in nvRNA signal from which I can develop computational algorithms to reliably identify inosines in nvRNA data. The REPAIRv2 approach to can be generalized to eventually identify any RNA modification with nanopores. ​Aim 3 will elucidate how A-to-I editing differs between tissues. I will sequence 4 normal tissue types with nvRNA sequencing, generating a map of A-to-I edits in conjunction with isoform usage using the software I am developing. Taken together, the fulfillment of these aims will not only provide further insights on elusive ADAR mechanism, but also create workflows for nanopore data analysis and a platform for the study of any modification. As I work toward my Ph.D. with this interdisciplinary project, I will gain invaluable skills in experimental and computational biology that will prepare me for a career in science.