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

对新生 RNA 进行直接测序，揭示遗传变异对 RNA 加工的功能影响

• 批准号: 10372582
• 负责人: Lee Stirling Churchman
• 金额: $ 45.45万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372582
• 关键词: 3' Untranslated Regions; Affect; Alleles; Alternative Splicing; Biological Assay; Cell Line; Cells; Code; Complex; DNA Polymerase II; DNA Sequence Alteration; Data; Defect; Diagnosis; Disease; Disease susceptibility; Event; Excision; Exons; Future; Genes; Genetic; Genetic Risk; Genetic Transcription; Genetic study; Goals; Grant; Half-Life; Human; Individual; Introns; Kinetics; Knowledge; Lead; Length; Light; Link; Location; Malignant Neoplasms; Maps; Measures; Messenger RNA; Mission; Multiple Sclerosis; Nuclear Export; Outcome; Poly A; Poly(A) Tail; Polyadenylation; Population; Positioning Attribute; Process; Production; Protein Isoforms; Public Health; Publishing; Quantitative Trait Loci; RNA; RNA Processing; RNA Splicing; RNA analysis; Regulation; Reporting; Research; Role; Time; Transcript; Translations; United States National Institutes of Health; Untranslated RNA; Variant; causal variant; computerized data processing; disorder risk; genetic variant; human disease; in vivo; lymphoblast; mRNA Precursor; molecular phenotype; nano; nanopore; nervous system disorder; novel therapeutic intervention; risk variant; trait; transcription termination; transcriptome; transcriptome sequencing

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.

大多数与疾病或性状相关的遗传变异并不位于编码区，这阻碍了它们的遗传变异。 解释。许多非编码变体映射到内含子，并可能影响RNA加工中的步骤，例如 内含子剪接、3 '-末端切割和多聚腺苷酸化，导致选择性剪接（AS）或多聚腺苷酸化 （阿帕）。群体范围内的转录组研究和数量性状基因座（QTL）分析揭示了一个 常见遗传变异在调节等位基因特异性RNA加工（sQTL和 apaQTL）。然而，这些遗传变异影响AS的机制以及它们如何导致疾病， 敏感性仍不清楚。因此，迫切需要了解遗传变异如何影响RNA 在RNA转录物的产生和成熟过程中进行加工，这反过来可以优先考虑变体， 功能分析，并帮助了解其在疾病易感性中的潜在作用。我们组最近 开发了共转录处理的纳米孔分析（nano-COP），它同时测定了 单个长RNA的多种分子表型，包括Pol II位置，跨多个内含子的剪接， 转录终止、3 '-末端切割和poly（A）尾长。我们将使用纳米COP来揭示 剪接动力学和3 ′末端加工在遗传变异体的情况下被改变。反过来，这些数据将 揭示了变异在新生RNA中持续多久，在此期间它们能够发挥作用。 我们的基本原理是，了解遗传变异如何影响RNA加工机制， 反之亦然，这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 遗传变异影响剪接动力学。为了确定遗传变异如何影响剪接动力学，我们 将在来自20个个体的人类LCL中使用已知的20个基因的靶向面板进行纳米COP， 在这些细胞中含有sQTL。在完成这一目标后，我们将了解遗传变异何时以及如何发生 在剪接过程中发挥作用，阐明了基因控制的可能机制， 如.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 '-末端加工相关。我们的目标是建立如何在变异 末端内含子、外显子和3 'UTR影响等位基因特异性3'-末端切割和聚腺苷酸尾。此外，我们将 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。于年底 目的是，我们将了解遗传变异如何影响主要的RNA加工步骤，以产生最终稳定的RNA。 状态异构体。这项资助的预期成果是展示纳米COP如何剖析 遗传变异在改变mRNA亚型中的作用，并确定sQTL和apaQTL是否发挥其作用。 部分通过它们在寿命较长的内含子中的位置来影响。这些结果将对未来产生积极影响。 疾病或性状的遗传学研究，通过提供一种策略来了解相关的SNP如何发挥作用， 突出那些可能是功能性的。