Platform for transcriptome-wide RNA modification identification in long reads

项目摘要 RNA修饰普遍存在于整个人类转录组中，并影响转录稳定性， 本地化，本土化，功能。特别是，RNA中ADAR介导的腺苷到肌苷(A到I)的编辑已经 显示影响前mRNA剪接和改变密码子序列。肌苷引起的氨基酸变化 与各种有害疾病有牵连，包括癌症和脑部疾病。然而， 以前的文献在高通量测绘肌苷的位置上只能做到内部有限 由简短的RNA-Seq读取提供的上下文。由于修改可以是特定于转录的，因此澄清了 肌苷与完整的信使核糖核酸异构体的联系对于更严格地理解肌苷的作用至关重要。 我们身体组织中的肌苷修饰，更广泛地说，是疾病。因此，我提议授予 利用全长信使核糖核酸纳米孔在疾病和非疾病系统中的A-to-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通过毛孔转移到核苷酸序列而引起的。​AIM 1利用高精度 有无ADAR基因敲除的细胞系统的纳米孔基因测序 功能和A-to-I诱导的转录表达变化。为了实现后者，我将发展 从嘈杂的长时间阅读中确定异构体结构的工作流程。除了对全长进行测序 转录本，纳米孔天然RNA(NvRNA)测序通知RNA修饰，作为修饰的核苷酸 表现为相对于正则核苷酸的电流信号的细微变化。因此，​Aim 2​采用了 产生经济有效的训练数据以系统地了解肌苷如何 改变纳米孔中的电流信号。我将使用Cas13b-ADAR融合蛋白(REPAIRv2)来创建特定部位 编辑，然后对编辑的转录组进行nvRNA测序。站点特定的A-to-I编辑允许这样做 在nvRNA信号中创建标记的肌苷数据集的方法，我可以从该数据集开发计算 可靠地识别nvRNA数据中的肌苷的算法。的REPAIRv2方法可以推广到 最终确定任何带有纳米孔的RNA修饰。​Aim 3将阐明A到I编辑的不同之处 在纸巾之间。我将用nvRNA测序对4种正常组织类型进行测序，生成A-to-I图 编辑结合等格式使用我正在开发的软件。总而言之，实现 这些目标不仅将为难以捉摸的ADAR机制提供进一步的见解，还将为 纳米孔数据分析和研究任何修改的平台。当我拿着这个攻读博士学位时 跨学科项目，我将在实验和计算生物学方面获得宝贵的技能，这将为 我想要在科学方面的事业。

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.