Genetic regulation of nascent RNA maturation revealed by direct RNA sequencing

Abstract Quantitative trait loci analyses have revealed an important role for genetic variants in regulating alternative splicing (AS) and alternative cleavage and polyadenylation (APA) in humans. Yet, because these studies are generally performed with mature mRNA, they report on the outcome rather than the process, and thus cannot elucidate how variants act to modulate pre-mRNA maturation. The order in which the many introns of a human gene are removed can substantially influence AS, while nascent RNA polyadenylation can affect RNA stability and decay. However, how splicing order and poly(A) tail length are regulated at the genetic level has never been explored. We used direct RNA nanopore sequencing of newly synthesized RNA to investigate allele-specific pre-mRNA maturation in 12 human lymphoblast cell lines. We found numerous instances of allele-specific splicing order and identified genetic variants associated with splicing order changes. While some of these variants were located proximal to splice sites, others were found more distally, providing novel examples of longer-range splicing regulation. Moreover, we uncovered several genes with significant differences in poly(A) tail lengths between alleles, which were enriched among genes with allele-specific nascent RNA abundance. Interestingly, HLA transcripts displayed frequent cooccurrence of allele-specific splicing order, AS, APA and/or poly(A) tail length differences. Together, our results expose new layers of genetic regulation of pre-mRNA maturation and highlight the power of longread direct RNA sequencing for allele-specific analyses. Biography Dr. Karine Choquet completed her PhD in human genetics under the supervision of Drs. Bernard Brais and Claudia Kleinman at McGill University. She then pursued a post-doctoral fellowship in genetics in the lab of Dr. Stirling Churchman at Harvard Medical School. Since July 2023, Karine has been an Assistant Professor in the Department of Biochemistry and Functional Genomics at the University of Sherbrooke. Dr. Choquet’s research focuses on the use of nanopore sequencing to analyze RNAs at all stages of their life cycle, with a particular interest in RNA splicing and isoform quantification.