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

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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.