Genetic regulation of nascent RNA maturation revealed by direct RNA nanopore sequencing

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, these studies are generally performed with mature mRNA, so they report on the outcome rather than the process, and thus cannot elucidate how variants directly 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 to investigate allele-specific pre-mRNA maturation in 12 human lymphoblast cell lines. We found frequent splicing order differences between alleles and uncovered significant SNP-splicing order associations in 28 genes. Interestingly, the majority of lead associated SNPs were located 10 to hundreds of nucleotides away from splice sites, indicating that long-range regulation may be an important component of splicing order determination. Moreover, several genes showed allele-specific poly(A) tail lengths, many of which also had a skewed allelic abundance ratio. Due to their polymorphic nature, HLA transcripts showed the most allele-specific splicing orders, which frequently co-occurred with allele-specific AS, APA or poly(A) tail length differences. Together, our results expose new layers of genetic regulation of pre-mRNA maturation and highlight the power of long-read RNA sequencing for allele-specific analyses.