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NCM 2022: Direct RNA sequencing reveals multi-intron splicing order and poly(A) tail lengths across subcellular compartments


Newly synthesised 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 characterisation of splicing and polyadenylation across long mRNA isoforms in distinct subcellular compartments.

Authors: Karine Choquet

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