Скачать или смотреть Karine Choquet: Direct RNA sequencing reveals multi-intron splicing order and poly(A) tail lengths

Newly synthesized messenger RNAs (mRNAs) undergo several processing steps prior to their export to the cytoplasm. To explore the landscape of full-length mRNA isoforms across different subcellular compartments, we performed direct RNA nanopore sequencing of poly(A)-selected RNA from whole-cell, chromatin, cytoplasm, and polysome fractions in human cells. This revealed that multi-intron pre-mRNA splicing order is not stochastic, but largely predetermined, with most genes using only a small number of splicing orders out of the many possible ones available to reach a fully spliced transcript. Furthermore, we observed that pre-mRNA splicing and polyadenylation progress in parallel on chromatin, where poly(A) tail lengths were also associated with the time that each transcript spends on chromatin. In the cytoplasm, long-lived transcripts tended to have shorter poly(A) tails than those that were rapidly degraded. Together, we describe the first transcriptome-wide characterization of splicing and polyadenylation across long mRNA isoforms in distinct subcellular compartments.