Direct sequencing of nascent RNA to uncover the functional impact of genetic variants on RNA processing

大多数与疾病或性状相关的遗传变异并不位于编码区，从而阻碍了它们的 释义。许多非编码变体映射到内含子，并可能影响RNA加工的步骤，例如 内含子剪接、3‘端切割和聚腺苷酸化，导致选择性剪接(AS)或聚腺苷酸化 (APA)。全群体转录组研究和数量性状基因座(QTL)分析揭示了 常见遗传变异在调节等位基因特异性RNA加工(sQTL和 ApaQTL)。然而，这些基因变异影响AS的机制以及它们如何导致疾病 易感性仍不清楚。因此，迫切需要了解基因变异如何影响rna。 在RNA转录本的生产和成熟过程中的处理，这反过来又可以为 功能分析，并帮助了解它们在疾病易感性中的潜在作用。我们的小组最近 开发了共转录处理的纳米孔分析(Nano-COP)，它同时检测了 单个长RNA的各种分子表型，包括Pol II位置，跨多个内含子的剪接， 转录终止、3‘端切割和Poly(A)尾长。我们将使用纳米CoP来揭示如何 剪接动力学和3‘端加工在遗传变异的背景下发生改变。反过来，这些数据将 揭示变异体在新生的RNA中持续多久，在这段时间内它们能够发挥作用。 我们的理论基础是了解遗传变异如何影响RNA加工机制和 Versa将有助于努力确定导致疾病风险的因果变异。具体目标1：分析如何 基因变异会影响剪接动力学。为了确定基因变异如何影响剪接动力学，我们 将使用一个由20个已知基因组成的靶向小组，在20个人的LCLS中进行纳米COP 在这些单元中包含sQTL。在完成这一目标后，我们将了解基因变异的时间和方式 在剪接过程中发挥它们的作用，阐明了基因控制的可能机制 就像。具体目标2：分析遗传变异对3‘端加工的影响。末端内含子的剪接是 在功能上与转录终止和3‘端加工有关。我们的目标是确定变种是如何在 末端内含子、外显子和3‘端非编码区影响等位基因特异的3’端切割和Poly(A)尾巴。此外，我们还将 研究3‘端加工步骤如何相互关联以及与末端内含子剪接的关系。在这个结束的时候 目的，我们将了解基因变异如何影响主要的RNA加工步骤，以产生最终的稳定- 状态异构体。这笔赠款的预期结果是纳米COP如何剖析 基因变异在改变mRNA异构体中的作用以及确定sQTL和apaQTL是否发挥其作用 影响部分是通过它们在寿命更长的内含子中的位置来实现的。这些结果将对未来产生积极的影响 对疾病或特征的遗传学研究，通过提供一种策略来了解相关的SNP如何起作用和 突出显示可能起作用的选项。

The majority of genetic variants associated with a disease or trait do not lie in coding regions, impeding their interpretation. Many non-coding variants map to introns and may impact steps in RNA processing, such as intron splicing, 3’-end cleavage and polyadenylation, leading to alternative splicing (AS) or polyadenylation (APA). Population-wide transcriptome studies and quantitative trait loci (QTL) analyses have revealed an unappreciated role for common genetic variants in regulating allele-specific RNA processing (sQTLs and apaQTLs). However, the mechanisms by which these genetic variants impact AS and how they lead to disease susceptibility remain unclear. Thus, there is a critical need to understand how genetic variants impact RNA processing during the production and maturation of RNA transcripts, which can in turn prioritize variants for functional analyses and help understand their potential role in disease susceptibility. Our group recently developed nanopore analysis of co-transcriptional processing (nano-COP), which simultaneously assays a variety of molecular phenotypes for single long RNAs, including Pol II position, splicing across multiple introns, transcription termination, 3’-end cleavage and poly(A) tail length. We will use nano-COP to uncover how splicing kinetics and 3’ end processing are altered in the context of genetic variants. In turn, these data will reveal how long variants persist in nascent RNA during which time they are capable of exerting an effect. Our rationale is that an understanding of how genetic variants impact RNA processing mechanisms and vice versa will help efforts to identify causal variants that contribute to disease risk. Specific Aim 1: Analyze how genetic variants influence splicing dynamics. To determine how genetic variants impact splicing dynamics, we will perform nano-COP in human LCLs from 20 individuals using a targeted panel of 20 genes known to contain sQTLs in these cells. At the completion of this Aim, we will understand when and how genetic variants exert their effect during the splicing process, illuminating possible mechanisms underlying genetic control of AS. Specific Aim 2: Analyze the impact of genetic variants on 3’-end processing. Splicing of terminal introns is functionally linked to transcription termination and 3’-end processing. We aim to establish how variants in terminal introns, exons and 3’UTRs affect allele-specific 3’-end cleavage and poly(A) tails. Additionally, we will investigate how 3’-end processing steps relate to one another and to terminal intron splicing. At the end of this Aim, we will have learned how genetic variants influence major RNA processing steps to yield the final steady- state isoforms. The expected outcomes of this grant are a demonstration of how nano-COP can dissect the role of genetic variants in altering mRNA isoforms and determine whether sQTLs and apaQTLs exert their influence in part through their locations in longer-lived introns. These results would positively impact future genetics studies of diseases or traits by providing a strategy to understand how associated SNPs may act and highlight those that may be functional.